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Sample records for disease virus glycoprotein

  1. Intracellular processing of the Newcastle disease virus fusion glycoprotein

    SciTech Connect

    Morrison, T.; Ward, L.J.; Semerjian, A.

    1985-03-01

    The fusion glycoprotein (Fo) of Newcastle disease virus is cleaved at an intracellular site into F1 and F2. This result was confirmed by comparing the transit time of the fusion protein to the cell surface with the time course of cleavage of Fo. The time required for cleavage of half of the pulse-labeled Fo protein is ca. 40 min faster than the half time of the transit of the fusion protein to the cell surface. To determine the cell compartment in which cleavage occurs, use was made of inhibitors which block glycoprotein migration at specific points and posttranslational modifications known to occur in specific cell membranes. Cleavage of Fo is inhibited by carbonyl cyanide m-chlorophenylhydrazone; thus, cleavage does not occur in the rough endoplasmic reticulum. Monensin blocks the incorporation of Newcastle disease virus glycoproteins into virions and blocks the cleavage of the fusion glycoprotein. However, Fo cannot be radioactively labeled with (/sup 3/H) fucose, whereas F1 is readily labeled. These results argue that cleavage occurs in the trans Golgi membranes or in a cell compartment occupied by glycoproteins quite soon after their transit through the trans Golgi membranes. The implications of the results presented for the transit times of the fusion protein between subcellular organelles are discussed.

  2. Development of Recombinant Newcastle Disease Viruses Expressing the Glycoprotein (G) of Avian Metapneumovirus as Bivalent Vaccines

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Using reverse genetics technology, Newcastle disease virus (NDV) LaSota strain-based recombinant viruses were engineered to express the glycoprotein (G) of avian metapneumovirus (aMPV), subtype A, B or C, as bivalent vaccines. These recombinant viruses were slightly attenuated in vivo, yet maintaine...

  3. Immunogenicity of a recombinant lumpy skin disease virus (neethling vaccine strain) expressing the rabies virus glycoprotein in cattle.

    PubMed

    Aspden, Kate; van Dijk, Alberdina A; Bingham, John; Cox, Dermot; Passmore, Jo-Ann; Williamson, Anna-Lise

    2002-06-21

    Rabies virus (RV) readily infects cattle and causes a fatal neurological disease. A stable vaccine, which does not require the maintenance of a cold chain and that is administered once to elicit lifelong immunity to rabies would be advantageous. The present study describes the construction of a live recombinant lumpy skin disease virus (LSDV) vaccine, expressing the glycoprotein of rabies virus (RG) and assessment of its ability to generate a humoral and cellular immune response against rabies virus in cattle. Cattle inoculated with the recombinant virus (rLSDV-RG) developed humoral immunity that was demonstrated in ELISA and neutralisation assays to RV. High titres of up to 1513IU/ml of RV neutralising antibodies were induced. In addition, peripheral blood mononuclear cells from rLSDV-RG-immunised animals demonstrated the ability to proliferate in response to stimulation with inactivated RV, whereas the animal vaccinated with wild type LSDV did not. This recombinant vaccine candidate thus has the potential to be used in ruminants as a cost-effective vaccine against both lumpy skin disease (LSD) and rabies. PMID:12034095

  4. Generation of Newcastle Disease Virus (NDV) Recombinants Expressing the Infectious Laryngotracheitis Virus (ILTV) Glycoprotein gB or gD as Dual Vaccines.

    PubMed

    Zhao, Wei; Spatz, Stephen; Zsak, Laszlo; Yu, Qingzhong

    2016-01-01

    Infectious laryngotracheitis (ILT) is a highly contagious acute respiratory disease of chickens caused by infection with infectious laryngotracheitis virus (ILTV), a member of the family Herpesviridae. The current commercial ILT vaccines are either unsafe or ineffective. Therefore, there is a pressing need to develop safer and more efficacious vaccines. Newcastle disease (ND), caused by infection with Newcastle disease virus (NDV), a member of the family Paramyxoviridae, is one of the most serious infectious diseases of poultry. The NDV LaSota strain, a naturally occurring low-virulence NDV strain, has been routinely used as a live vaccine throughout the world. This chapter describes the generation of Newcastle disease virus (NDV) LaSota vaccine strain-based recombinant viruses expressing glycoprotein B (gB) or glycoprotein D (gD) of ILTV as dual vaccines against ND and ILT using reverse genetics technology. PMID:27076292

  5. Herpes simplex type 2 virus deleted in glycoprotein D protects against vaginal, skin and neural disease

    PubMed Central

    Petro, Christopher; González, Pablo A; Cheshenko, Natalia; Jandl, Thomas; Khajoueinejad, Nazanin; Bénard, Angèle; Sengupta, Mayami; Herold, Betsy C; Jacobs, William R

    2015-01-01

    Subunit vaccines comprised of glycoprotein D (gD-2) failed to prevent HSV-2 highlighting need for novel strategies. To test the hypothesis that deletion of gD-2 unmasks protective antigens, we evaluated the efficacy and safety of an HSV-2 virus deleted in gD-2 and complemented allowing a single round of replication on cells expressing HSV-1 gD (ΔgD−/+gD−1). Subcutaneous immunization of C57BL/6 or BALB/c mice with ΔgD−/+gD1 provided 100% protection against lethal intravaginal or skin challenges and prevented latency. ΔgD−/+gD1 elicited no disease in SCID mice, whereas 1000-fold lower doses of wild-type virus were lethal. HSV-specific antibodies were detected in serum (titer 1:800,000) following immunization and in vaginal washes after intravaginal challenge. The antibodies elicited cell-mediated cytotoxicity, but little neutralizing activity. Passive transfer of immune serum completely protected wild-type, but not Fcγ-receptor or neonatal Fc-receptor knock-out mice. These studies demonstrate that non-neutralizing Fc-mediated humoral responses confer protection and support advancement of this attenuated vaccine. DOI: http://dx.doi.org/10.7554/eLife.06054.001 PMID:25756612

  6. In vitro and in vivo broad antiviral activity of peptides homologous to fusion glycoproteins of Newcastle disease virus and Marek's disease virus.

    PubMed

    Chi, Xiao-Jing; Wang, Xiao-Jun; Wang, Cheng-Yu; Cui, Xiao-Jing; Wang, Xiao-Jia

    2014-04-01

    Newcastle disease virus (NDV) of paramyxovirus and Marek's disease virus (MDV) of herpesvirus, two of the most serious threats to the poultry industry, can give rise to complex co-infections that hinder diagnosis and prevention. In the current study, two different peptides, derived from the MDV gH (gHH2L) and gB (gBH3), respectively, exhibit antiviral activity against NDV in vitro. The potent inhibitory effect of heptad repeat 2 from fusion glycoprotein of the NDV on MDV infection also has been demonstrated. Plaque formation and embryo infectivity assays confirmed these antiviral results. Furthermore, each tandem peptide consisting of two motifs from different viruses exhibits more potent antiviral activity than the constituent peptides. The current work provides a new strategy for developing novel peptides and vaccines against virus infection and co-infections. PMID:24412629

  7. Generation of recombinant newcastle disease viruses, expressing the glycoprotein (G) of avian metapneumovirus, subtype A, or B, for use as bivalent vaccines

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Using reverse genetics technology, Newcastle disease virus (NDV) LaSota strain-based recombinant viruses were engineered to express the glycoprotein (G) of avian metapneumovirus (aMPV), subtype A, or B, as bivalent vaccines. These recombinant viruses, rLS/aMPV-A G and rLS/aMPV-B G, were slightly att...

  8. Evaluation of swinepox virus as a vaccine vector in pigs using an Aujeszky's disease (pseudorabies) virus gene insert coding for glycoproteins gp50 and gp63.

    PubMed

    van der Leek, M L; Feller, J A; Sorensen, G; Isaacson, W; Adams, C L; Borde, D J; Pfeiffer, N; Tran, T; Moyer, R W; Gibbs, E P

    1994-01-01

    Pigs were vaccinated by scarification or intramuscular injection with a swinepox virus-Aujeszky's disease (pseudorabies) recombinant (rSPV-AD) constructed by inserting the linked Aujeszky's disease virus genes coding for glycoproteins gp50 and gp63, attached to a vaccinia virus p7.5 promoter, into the thymidine kinase gene of swinepox virus. By 21 days after vaccination, 90 and 100 per cent of the animals vaccinated by scarification or intramuscular injection, respectively, had developed serum neutralising antibodies to Aujeszky's disease virus. Upon challenge with virulent virus, significantly fewer vaccinated pigs developed clinical Aujeszky's disease, nasal shedding of challenge virus was markedly reduced, and the vaccinated groups of pigs maintained or gained weight during the week after challenge whereas the unvaccinated control group lost weight. No transmission of rSPV-AD to in-contact controls was detected during the three weeks before challenge. In a second experiment, serum neutralising antibodies to Aujeszky's disease virus persisted for 150 days after the pigs were vaccinated with rSPV-AD by scarification or intramuscular injection and all the pigs showed an anamnestic response when they were revaccinated. PMID:8128561

  9. Newcastle Disease Virus (NDV) Recombinants Expressing Infectious Laryngotracheitis Virus (ILTV) Glycoproteins gB and gD Protect Chickens against ILTV and NDV Challenges

    PubMed Central

    Zhao, Wei; Spatz, Stephen; Zhang, Zhenyu; Wen, Guoyuan; Garcia, Maricarmen; Zsak, Laszlo

    2014-01-01

    ABSTRACT Infectious laryngotracheitis (ILT) is a highly contagious acute respiratory disease of chickens caused by infectious laryngotracheitis virus (ILTV). The disease is controlled mainly through biosecurity and vaccination with live attenuated strains of ILTV and vectored vaccines based on turkey herpesvirus (HVT) and fowlpox virus (FPV). The current live attenuated vaccines (chicken embryo origin [CEO] and tissue culture origin [TCO]), although effective, can regain virulence, whereas HVT- and FPV-vectored ILTV vaccines are less efficacious than live attenuated vaccines. Therefore, there is a pressing need to develop safer and more efficacious ILTV vaccines. In the present study, we generated Newcastle disease virus (NDV) recombinants, based on the LaSota vaccine strain, expressing glycoproteins B (gB) and D (gD) of ILTV using reverse genetics technology. These recombinant viruses, rLS/ILTV-gB and rLS/ILTV-gD, were slightly attenuated in vivo yet retained growth dynamics, stability, and virus titers in vitro that were similar to those of the parental LaSota virus. Expression of ILTV gB and gD proteins in the recombinant virus-infected cells was detected by immunofluorescence assay. Vaccination of specific-pathogen-free chickens with these recombinant viruses conferred significant protection against virulent ILTV and velogenic NDV challenges. Immunization of commercial broilers with rLS/ILTV-gB provided a level of protection against clinical disease similar to that provided by the live attenuated commercial vaccines, with no decrease in body weight gains. The results of the study suggested that the rLS/ILTV-gB and -gD viruses are safe, stable, and effective bivalent vaccines that can be mass administered via aerosol or drinking water to large chicken populations. IMPORTANCE This paper describes the development and evaluation of novel bivalent vaccines against chicken infectious laryngotracheitis (ILT) and Newcastle disease (ND), two of the most economically

  10. Disulfide bond formation is a determinant of glycosylation site usage in the hemagglutinin-neuraminidase glycoprotein of Newcastle disease virus.

    PubMed Central

    McGinnes, L W; Morrison, T G

    1997-01-01

    Determinants of glycosylation site usage were explored by using the hemagglutinin-neuraminidase (HN) glycoprotein of the paramyxovirus Newcastle disease virus. The amino acid sequence of the HN protein, a type II glycoprotein, has six N-linked glycosylation addition sites, G1 to G6, two of which, G5 and G6, are not used for the addition of carbohydrate (L. McGinnes and T. Morrison, Virology 212:398-410, 1995). The sequence of this protein also has 13 cysteine residues in the ectodomain (C2 to C14). Mutation of either cysteine 13 or cysteine 14 resulted in the addition of another oligosaccharide chain to the protein. These cysteine residues flank the normally unused G6 glycosylation addition site, and mutation of the G6 site eliminated the extra glycosylation found in the cysteine mutants. These results suggested that failure to form an intramolecular disulfide bond resulted in the usage of a normally unused glycosylation site. This conclusion was confirmed by preventing cotranslational disulfide bond formation in cells by using dithiothreitol. Under these conditions, the wild-type protein acquired extra glycosylation, which was eliminated by mutation of the G6 site. These results suggest that localized folding events on the nascent chain, such as disulfide bond formation, which block access to the oligosaccharyl transferase are a determinant of glycosylation site usage. PMID:9060670

  11. Examination of the effect of a naturally occurring mutation in glycoprotein L on Marek's disease virus pathogenesis.

    PubMed

    Santin, Elizabeth R; Shamblin, Christine E; Prigge, Jonathan T; Arumugaswami, Vaithilingaraja; Dienglewicz, Robert L; Parcells, Mark S

    2006-03-01

    We recently reported a comparison of glycoprotein-encoding genes of different Marek's disease virus pathotypes (MDVs). One mutation found predominantly in very virulent (vv)+MDVs was a 12-bp (four-amino acid) deletion in the glycoprotein L (gL)-encoding gene in four of 23 MDV strains examined (three were vv+MDVs and one was a vvMDV). This mutation was noted in the gL of the TK (615K) strain, but not in the RL (615J) strain of MDV. These strains have identical mutations in the meq gene characteristic of vv+MDVs but can be distinguished by the mutation in the gL-encoding gene. The TK strain was originally isolated from vaccinated chickens and appeared to confer or enhance horizontal transmission of the vaccine virus, herpesvirus of turkeys (HVT). Because the molecular basis for increased virulence of MDV field strains is unknown, we hypothesized that one mechanism might be by coreplication of MDV-1 strains with HVT and that it could be mediated by the mutation of gL, an essential component of the glycoprotein H/L complex. In this study, we compared the pathogenicity of TK (615K) and RL (615J) strains of MDV in the presence and absence of simultaneous HVT coinfection. MDV infections were monitored at the levels of viremia (for both MDV-1 and HVT), clinical signs of MD, tumor incidence, and mortality in 1) inoculated chickens, 2) chickens exposed at 1 day of age, 3) chickens exposed at 2 wk of age, and 4) chickens exposed to both TK/HVT- and RL/HVT-infected chickens at 6 wk of age. We found high incidences of clinical MD signs in all inoculated treatment groups and all chickens exposed to TK and RL viruses, regardless of the presence of HVT. The median time to death of chickens exposed to TK1HVT-infected chickens, however, was lower than the other treatment groups for contact-exposed chickens. Although this difference was not considered to be statistically significant to a rigorously interpreted degree because of the removal of chickens for sampling from the test

  12. Characterization of the sites of proteolytic activation of Newcastle disease virus membrane glycoprotein precursors.

    PubMed

    Gorman, J J; Nestorowicz, A; Mitchell, S J; Corino, G L; Selleck, P W

    1988-09-01

    The F1- and F2-polypeptide components of the fusion proteins and the hemagglutinin/neuraminidase proteins of the avirulent Queensland (V4) and virulent Australia-Victoria (AuV) strains of Newcastle disease virus have been isolated and subjected to extensive primary structural analysis including amino-terminal sequence analysis and fast atom bombardment-mass spectrometry mapping. Nucleotide sequence analysis was performed on the gene which encodes the V4 hemagglutinin/neuraminidase protein. Signal peptidase cleavage was found to have occurred at the Ser31-Leu32 peptide bond of the primary translation products of the fusion protein genes. Activation cleavage of the V4 fusion protein precursor generated a sequence of -Gly-Lys-Gln-Gly84 at the carboxyl terminus of the F2-polypeptide and an amino-terminal sequence of the F1-polypeptide commencing with 86Leu-Ile-Gly-. The V4 hemagglutinin/neuraminidase protein gene was found to encode a primary translation product 45 amino acids longer at the carboxyl terminus than obtainable from the corresponding gene of the AuV strain (McGinnes, L. W., and Morrison, T. G. (1986) Virus Res. 5, 343-356). However, post-translational proteolytic processing, exclusive to the primary translation product of the V4 hemagglutinin/neuraminidase protein gene, was found to have removed the last 42 residues of this carboxyl-terminal appendage. PMID:3045120

  13. A recombinant Newcastle disease virus (NDV) expressing infectious laryngotracheitis virus (ILTV) surface glycoprotein D protects against highly virulent ILTV and NDV challenges in chickens.

    PubMed

    Kanabagatte Basavarajappa, Mallikarjuna; Kumar, Sachin; Khattar, Sunil K; Gebreluul, Girmay T; Paldurai, Anandan; Samal, Siba K

    2014-06-12

    Infectious laryngotracheitis (ILT) is a highly contagious acute respiratory disease of chickens caused by infectious laryngotracheitis virus (ILTV). Currently, modified live ILTV vaccines are used to control ILT infections. However, the live ILTV vaccines can revert to virulence after bird-to-bird passage and are capable of establishing latent infections, suggesting the need to develop safer vaccines against ILT. We have evaluated the role of three major ILTV surface glycoproteins, namely, gB, gC, and gD in protection and immunity against ILTV infection in chickens. Using reverse genetics approach, three recombinant Newcastle disease viruses (rNDVs) designated rNDV gB, rNDV gC, and rNDV gD were generated, each expressing gB, gC, and gD, respectively, of ILTV. Chickens received two immunizations with rNDVs alone (gB, gC, and gD) or in combination (gB+gC, gB+gD, gC+gD, and gB+gC+gD). Immunization with rNDV gD induced detectable levels of neutralizing antibodies with the magnitude of response greater than the rest of the experimental groups including those vaccinated with commercially available vaccines. The birds immunized with rNDV gD showed complete protection against virulent ILTV challenge. The birds immunized with rNDV gC alone or multivalent vaccines consisting of combination of rNDVs displayed partial protection with minimal disease and reduced replication of challenge virus in trachea. Immunization with rNDV gB neither reduced the severity of the disease nor the replication of challenge virus in trachea. The superior protective efficacy of rNDV gD vaccine compared to rNDV gB or rNDV gC vaccine was attributed to the higher levels of envelope incorporation and infected cell surface expression of gD than gB or gC. Our results suggest that rNDV expressing gD is a safe and effective bivalent vaccine against NDV and ILTV. PMID:24793943

  14. Vesicular Stomatitis Virus glycoprotein G carrying a tandem dimer of Foot and Mouth Disease Virus antigenic site A can be used as DNA and peptide vaccine for cattle.

    PubMed

    Capozzo, Alejandra V; Wilda, Maximiliano; Bucafusco, Danilo; de los Ángeles Lavoria, María; Franco-Mahecha, Olga L; Mansilla, Florencia C; Pérez-Filgueira, Daniel M; Grigera, Pablo R

    2011-11-01

    Effective Foot and Mouth Disease Virus (FMDV) peptide vaccines for cattle have two major constraints: resemblance of one or more of the multiple conformations of the major VP1 antigenic sites to induce neutralizing antibodies, and stimulation of T cells despite the variable bovine-MHC polymorphism. To overcome these limitations, a chimeric antigen was developed, using Vesicular Stomatitis Virus glycoprotein (VSV-G) as carrier protein of an in tandem-dimer of FMDV antigenic site A (ASA), the major epitope on the VP1 capsid protein (aa 139-149, FMDV-C3 serotype). The G-ASA construct was expressed in the Baculovirus system to produce a recombinant protein (DEL BAC) (cloned in pCDNA 3.1 plasmid) (Invitrogen Corporation, Carlsbad, CA) and was also prepared as a DNA vaccine (pC DEL). Calves vaccinated with both immunogens elicited antibodies that recognized the ASA in whole virion and were able to neutralize FMDV infectivity in vitro. After two vaccine doses, DEL BAC induced serum neutralizing titers compatible with an "expected percentage of protection" above 90%. Plasmid pC DEL stimulated FMDV specific humoral responses earlier than DEL BAC, though IgG1 to IgG2 ratios were lower than those induced by both DEL BAC and inactivated FMDV-C3 after the second dose. DEL BAC induced FMDV-specific secretion of IFN-γ in peripheral blood mononuclear cells of outbred cattle immunized with commercial FMDV vaccine, suggesting its capacity to recall anamnestic responses mediated by functional T cell epitopes. The results show that exposing FMDV-VP1 major neutralizing antigenic site in the context of N-terminal sequences of the VSV G protein can overcome the immunological limitations of FMDV-VP1 peptides as effective protein and DNA vaccines for cattle. PMID:21889542

  15. Expression of a secreted version of the hemagglutinin-neuraminidase glycoprotein of Newcastle disease virus: its evaluation as a diagnostic reagent.

    PubMed

    Zoth, Silvina Chimeno; Gómez, Evangelina; Carballeda, Juan Manuel; Carrillo, Elisa; Berinstein, Analía

    2011-05-01

    The hemagglutinin-neuraminidase (HN) glycoprotein of Newcastle disease virus (NDV) constitutes, together with the fusion glycoprotein, the main surface antigen of this avian pathogen, which causes a highly contagious disease, relevant economically worldwide. The purpose of this work was to obtain the HN glycoprotein as a soluble antigen in culture supernatants of recombinant baculovirus-infected Spodoptera frugiperda (Sf9) cells and to evaluate its application to the development of a recombinant enzyme-linked immunosorbent assay (rELISA) for the analysis of chicken sera. A transfer vector for baculovirus containing the sequence of a melittin signal peptide was constructed and the sequence coding for HN protein without its own signal peptide was cloned. The recombinant protein was secreted and recovered easily from the culture medium of Sf9-infected cells. The recombinant protein was evaluated as antigen for ELISA coating the plates with the recovered HN using 79 positive and 142 negative samples. The Cohen kappa value resulted 0.91, indicating excellent agreement between the rELISA and the hemagglutinin inhibition tests. The rELISA was also compared with a commercial ELISA, finding high levels of agreement between both assays. The present results show that the cloning strategy developed yielded the HN protein free in the cell culture supernatant and that the recombinant protein retained its reactivity with anti-NDV HN antibodies in chicken sera. PMID:21908282

  16. Newcastle disease virus (NDV) recombinants expressing infectious laryngotracheitis virus (ILTV) glycoproteins gB and gD protect chickens against ILTV and NDV challenges

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Infectious laryngotracheitis (ILT) is a highly contagious acute respiratory disease of chickens caused by infectious laryngotracheitis virus (ILTV). The disease is mainly controlled through biosecurity and vaccination with live-attenuated strains of the virus and vectored vaccines based on turkey he...

  17. Vaccination of ferrets with a recombinant G glycoprotein subunit vaccine provides protection against Nipah virus disease for over 12 months

    PubMed Central

    2013-01-01

    Background Nipah virus (NiV) is a zoonotic virus belonging to the henipavirus genus in the family Paramyxoviridae. Since NiV was first identified in 1999, outbreaks have continued to occur in humans in Bangladesh and India on an almost annual basis with case fatality rates reported between 40% and 100%. Methods Ferrets were vaccinated with 4, 20 or 100 μg HeVsG formulated with the human use approved adjuvant, CpG, in a prime-boost regime. One half of the ferrets were exposed to NiV at 20 days post boost vaccination and the other at 434 days post vaccination. The presence of virus or viral genome was assessed in ferret fluids and tissues using real-time PCR, virus isolation, histopathology, and immunohistochemistry; serology was also carried out. Non-immunised ferrets were also exposed to virus to confirm the pathogenicity of the inoculum. Results Ferrets exposed to Nipah virus 20 days post vaccination remained clinically healthy. Virus or viral genome was not detected in any tissues or fluids of the vaccinated ferrets; lesions and antigen were not identified on immunohistological examination of tissues; and there was no increase in antibody titre during the observation period, consistent with failure of virus replication. Of the ferrets challenged 434 days post vaccination, all five remained well throughout the study period; viral genome – but not virus - was recovered from nasal secretions of one ferret given 20 μg HeVsG and bronchial lymph nodes of the other. There was no increase in antibody titre during the observation period, consistent with lack of stimulation of a humoral memory response. Conclusions We have previously shown that ferrets vaccinated with 4, 20 or 100 μg HeVsG formulated with CpG adjuvant, which is currently in several human clinical trials, were protected from HeV disease. Here we show, under similar conditions of use, that the vaccine also provides protection against NiV-induced disease. Such protection persists for at least 12 months

  18. Interaction of Sindbis virus glycoproteins during morphogenesis.

    PubMed Central

    Jones, K J; Scupham, R K; Pfeil, J A; Wan, K; Sagik, B P; Bose, H R

    1977-01-01

    In cells infected with the Sindbis temperature-sensitive mutants ts-23 and ts-10 (complementation group D), which contain a defect in the envelope glycoprotein E1, the precursor polypeptide PE2 is not cleaved to the envelope glycoprotein E2 at the nonpermissive temperature. This defect is phenotypically identical to the defect observed in the complementation group E mutant, ts-20. The lesion in ts-23 is reversible upon shift to permissive temperature, whereas that of ts-10 is not. Antiserum against whole virus, E1, or E2 also prevents the cleavage of PE2 in cells infected with wild-type Sindbis virus. Because the cleavage of PE2 is inhibited by the lesion in mutants that are genotypically distinct and by anti-E1 or -E2 serum, it appears that PE2 and E1 exist as a complex in the membrane of the infected cell. Images PMID:833949

  19. Evaluation of a LaSota strain-based recombinant Newcastle disease virus (NDV) expressing the glycoprotein (G) of avian metapneumovirus (aMPV) subgroup A or B as a bivalent vaccine in turkeys

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To develop a bivalent vaccine candidate, a LaSota strain-based recombinant Newcastle disease virus (NDV) clone expressing the glycoprotein (G) of avian metapneumovirus (aMPV) subgroup A or B was generated using reverse genetics. Vaccination of turkeys with the NDV/aMPV-A G or NDV/aMPV-B G recombinan...

  20. Induction of apoptosis in MCF-7 cells by the hemagglutinin-neuraminidase glycoprotein of Newcastle disease virus Malaysian strain AF2240.

    PubMed

    Ghrici, Mohamed; El Zowalaty, Mohamed; Omar, Abdul Rahman; Ideris, Aini

    2013-09-01

    Newcastle disease virus (NDV) exerts its naturally occurring oncolysis possibly through the induction of apoptosis. We hypothesized that the binding of the virus to the cell via the hemagglutinin-neuraminidase (HN) glycoprotein may be sufficient to not only induce apoptosis but to induce a higher apoptosis level than the parental NDV AF2240 virus. NDV AF2240 induction of apoptosis in MCF-7 human breast cancer cells was analyzed and quantified. In addition, the complete HN gene of NDV strain AF2240 was amplified, sequenced and cloned into the pDisplay eukaryotic expression vector. HN gene expression was first detected at the cell surface membrane of the transfected MCF-7 cells. HN induction of apoptosis in transfected MCF-7 cells was analyzed and quantified. The expression of the HN gene alone was able to induce apoptosis in MCF-7 cells but it was a less potent apoptosis inducer compared to the parental NDV AF2240 strain. In conclusion, the NDV AF2240 strain is a more suitable antitumor candidate agent than its recombinant HN gene unless the latter is further improved by additional modifications. PMID:23807159

  1. Recoding structural glycoprotein E2 in classical swine fever virus (CSFV) produces complete virus attenuation in swine and protects infected animals against disease.

    PubMed

    Velazquez-Salinas, Lauro; Risatti, Guillermo R; Holinka, Lauren G; O'Donnell, Vivian; Carlson, Jolene; Alfano, Marialexia; Rodriguez, Luis L; Carrillo, Consuelo; Gladue, Douglas P; Borca, Manuel V

    2016-07-01

    Controlling classical swine fever (CSF) mainly involves vaccination with live attenuated vaccines (LAV). Experimental CSFV LAVs has been lately developed through reverse genetics using several different approaches. Here we present that codon de-optimization in the major CSFV structural glycoprotein E2 coding region, causes virus attenuation in swine. Four different mutated constructs (pCSFm1-pCSFm4) were designed using various mutational approaches based on the genetic background of the highly virulent strain Brescia (BICv). Three of these constructs produced infectious viruses (CSFm2v, CSFm3v, and CSFm4v). Animals infected with CSFm2v presented a reduced and extended viremia but did not display any CSF-related clinical signs. Animals that were infected with CSFm2v were protected against challenge with virulent parental BICv. This is the first report describing the development of an attenuated CSFV experimental vaccine by codon usage de-optimization, and one of the few examples of virus attenuation using this methodology that is assessed in a natural host. PMID:27110709

  2. Pseudorabies Virus Glycoprotein M Inhibits Membrane Fusion

    PubMed Central

    Klupp, Barbara G.; Nixdorf, Ralf; Mettenleiter, Thomas C.

    2000-01-01

    A transient transfection-fusion assay was established to investigate membrane fusion mediated by pseudorabies virus (PrV) glycoproteins. Plasmids expressing PrV glycoproteins under control of the immediate-early 1 promoter-enhancer of human cytomegalovirus were transfected into rabbit kidney cells, and the extent of cell fusion was quantitated 27 to 42 h after transfection. Cotransfection of plasmids encoding PrV glycoproteins B (gB), gD, gH, and gL resulted in formation of polykaryocytes, as has been shown for homologous proteins of herpes simplex virus type 1 (HSV-1) (A. Turner, B. Bruun, T. Minson, and H. Browne, J. Virol. 72:873–875, 1998). However, in contrast to HSV-1, fusion was also observed when the gD-encoding plasmid was omitted, which indicates that PrV gB, gH, and gL are sufficient to mediate fusion. Fusogenic activity was enhanced when a carboxy-terminally truncated version of gB (gB-008) lacking the C-terminal 29 amino acids was used instead of wild-type gB. With gB-008, only gH was required in addition for fusion. A very rapid and extended fusion was observed after cotransfection of plasmids encoding gB-008 and gDH, a hybrid protein consisting of the N-terminal 271 amino acids of gD fused to the 590 C-terminal amino acids of gH. This protein has been shown to substitute for gH, gD, and gL function in the respective viral mutants (B. G. Klupp and T. C. Mettenleiter, J. Virol. 73:3014–3022, 1999). Cotransfection of plasmids encoding PrV gC, gE, gI, gK, and UL20 with gB-008 and gDH had no effect on fusion. However, inclusion of a gM-expressing plasmid strongly reduced the extent of fusion. An inhibitory effect was also observed after inclusion of plasmids encoding gM homologs of equine herpesvirus 1 or infectious laryngotracheitis virus but only in conjunction with expression of the gM complex partner, the gN homolog. Inhibition by PrV gM was not limited to PrV glycoprotein-mediated fusion but also affected fusion induced by the F protein of bovine

  3. Enhanced Efficacy of a Codon-Optimized DNA Vaccine Encoding the Glycoprotein Precursor Gene of Lassa Virus in a Guinea Pig Disease Model When Delivered by Dermal Electroporation.

    PubMed

    Cashman, Kathleen A; Broderick, Kate E; Wilkinson, Eric R; Shaia, Carl I; Bell, Todd M; Shurtleff, Amy C; Spik, Kristin W; Badger, Catherine V; Guttieri, Mary C; Sardesai, Niranjan Y; Schmaljohn, Connie S

    2013-01-01

    Lassa virus (LASV) causes a severe, often fatal, hemorrhagic fever endemic to West Africa. Presently, there are no FDA-licensed medical countermeasures for this disease. In a pilot study, we constructed a DNA vaccine (pLASV-GPC) that expressed the LASV glycoprotein precursor gene (GPC). This plasmid was used to vaccinate guinea pigs (GPs) using intramuscular electroporation as the delivery platform. Vaccinated GPs were protected from lethal infection (5/6) with LASV compared to the controls. However, vaccinated GPs experienced transient viremia after challenge, although lower than the mock-vaccinated controls. In a follow-on study, we developed a new device that allowed for both the vaccine and electroporation pulse to be delivered to the dermis. We also codon-optimized the GPC sequence of the vaccine to enhance expression in GPs. Together, these innovations resulted in enhanced efficacy of the vaccine. Unlike the pilot study where neutralizing titers were not detected until after virus challenge, modest neutralizing titers were detected in guinea pigs before challenge, with escalating titers detected after challenge. The vaccinated GPs were never ill and were not viremic at any timepoint. The combination of the codon-optimized vaccine and dermal electroporation delivery is a worthy candidate for further development. PMID:26344112

  4. Enhanced Efficacy of a Codon-Optimized DNA Vaccine Encoding the Glycoprotein Precursor Gene of Lassa Virus in a Guinea Pig Disease Model When Delivered by Dermal Electroporation

    PubMed Central

    Cashman, Kathleen A.; Broderick, Kate E.; Wilkinson, Eric R.; Shaia, Carl I.; Bell, Todd M.; Shurtleff, Amy C.; Spik, Kristin W.; Badger, Catherine V.; Guttieri, Mary C.; Sardesai, Niranjan Y.; Schmaljohn, Connie S.

    2013-01-01

    Lassa virus (LASV) causes a severe, often fatal, hemorrhagic fever endemic to West Africa. Presently, there are no FDA-licensed medical countermeasures for this disease. In a pilot study, we constructed a DNA vaccine (pLASV-GPC) that expressed the LASV glycoprotein precursor gene (GPC). This plasmid was used to vaccinate guinea pigs (GPs) using intramuscular electroporation as the delivery platform. Vaccinated GPs were protected from lethal infection (5/6) with LASV compared to the controls. However, vaccinated GPs experienced transient viremia after challenge, although lower than the mock-vaccinated controls. In a follow-on study, we developed a new device that allowed for both the vaccine and electroporation pulse to be delivered to the dermis. We also codon-optimized the GPC sequence of the vaccine to enhance expression in GPs. Together, these innovations resulted in enhanced efficacy of the vaccine. Unlike the pilot study where neutralizing titers were not detected until after virus challenge, modest neutralizing titers were detected in guinea pigs before challenge, with escalating titers detected after challenge. The vaccinated GPs were never ill and were not viremic at any timepoint. The combination of the codon-optimized vaccine and dermal electroporation delivery is a worthy candidate for further development. PMID:26344112

  5. Processing of virus-specific glycoproteins of varicella zoster virus

    SciTech Connect

    Namazue, J.; Campo-Vera, H.; Kitamura, K.; Okuno, T.; Yamanishi, K.

    1985-05-01

    Monoclonal antibodies to varicella zoster virus (VZV) glycoproteins were used to study the processing of three glycoproteins with molecular weights of 83K-94K (gp 2), 64K (gp 3), and 55K (gp 5). Immunoprecipitation experiments performed with VZV-infected cells, pulse labeled with (/sup 3/H)glucosamine in the presence of tunicamycin, suggest that O-linked oligosaccharide is present on the glycoprotein of gp 2. Use of the enzyme endo-beta-N-acetylglucosaminidase H revealed that the fully processed form of gp 3 had high-mannose type and that of gp 5 had only complex type of N-linked oligosaccharides. Experiments with monensin suggest that the precursor form (116K) of gp 3 is cleaved during the processing from Golgi apparatus to cell surface membrane. The extension of O-linked oligosaccharide chain and the complex type of N-linked oligosaccharide chains also occurs during this processing.

  6. P-glycoprotein in autoimmune rheumatic diseases.

    PubMed

    García-Carrasco, M; Mendoza-Pinto, C; Macias Díaz, S; Vera-Recabarren, M; Vázquez de Lara, L; Méndez Martínez, S; Soto-Santillán, P; González-Ramírez, R; Ruiz-Arguelles, A

    2015-07-01

    P-glycoprotein (Pgp) is a transmembrane protein of 170 kD encoded by the multidrug resistance 1 (MDR-1) gene, localized on chromosome 7. More than 50 polymorphisms of the MDR-1 gene have been described; a subset of these has been shown to play a pathophysiological role in the development of inflammatory bowel disease, femoral head osteonecrosis induced by steroids, lung cancer and renal epithelial tumors. Polymorphisms that have a protective effect on the development of conditions such as Parkinson disease have also been identified. P-glycoprotein belongs to the adenosine triphosphate binding cassette transporter superfamily and its structure comprises a chain of approximately 1280 aminoacid residues with an N-C terminal structure, arranged as 2 homologous halves, each of which has 6 transmembrane segments, with a total of 12 segments with 2 cytoplasmic nucleotide binding domains. Many cytokines like interleukin 2 and tumor necrosis factor alpha increase Pgp expression and activity. Pgp functions as an efflux pump for a variety of toxins in order to protect particular organs and tissues as the central nervous system. Pgp transports a variety of substrates including glucocorticoids while other drugs such as tacrolimus and cyclosporine A act as modulators of this protein. The most widely used method to measure Pgp activity is flow cytometry using naturally fluorescent substrates such as anthracyclines or rhodamine 123. The study of drug resistance and its association to Pgp began with the study of resistance to chemotherapy in the treatment of cancer and antiretroviral therapy for human immunodeficiency virus; however, the role of Pgp in the treatment of systemic lupus erythematosus, rheumatoid arthritis and psoriatic arthritis has been a focus of study lately and has emerged as an important mechanism by which treatment failure occurs. The present review analyzes the role of Pgp in these autoimmune diseases. PMID:25712147

  7. Role of envelope glycoproteins in intracellular virus maturation

    SciTech Connect

    Matsuoka, Y.

    1988-01-01

    The possible role viral glycoproteins in intracellular maturation was studied by using two different viruses, avian infectious bronchitis virus (IBV), a coronavirus, and Punta Toro virus (PTV), a bunyavirus. Using the antibiotic tunicamycin, which inhibits glycosylation of N-linked glycoproteins, it was shown that coronavirus particles are formed in the absence of glycosylation. Analysis of the protein composition of these particles indicated that they contain an unglycosylated form of the membrane-associated E1 glycoprotein but lack the E2 spike glycoprotein. A cDNA clone derived from the PTV M RNA genome segment, which encodes the G1 and G2 glycoproteins, was cloned into vaccinia virus. Studies by indirect immunofluorescence microscopy revealed that the glycoproteins synthesized from this recombinant were found to accumulate intracellularly at the Golgi complex, where virus budding usually takes place. Surface immunoprecipitation and {sup 125}I-protein A binding assays also demonstrated that a majority of the glycoproteins are retained intracellularly and are not transported to the cellular surface. The sequences which encode the G1 and G2 glycoproteins were independently cloned into vaccinia virus as well.

  8. Solubilization of glycoproteins of envelope viruses by detergents

    SciTech Connect

    Berezin, V.E.; Zaides, V.M.; Artamsnov, A.F.; Isaeva, E.S.; Zhdanov, V.M.

    1986-11-20

    The action of a number of known ionic and nonionic detergents, as well as the new nonionic detergent MESK, on envelope viruses was investigated. It was shown that the nonionic detergents MESK, Triton X-100, and octyl-..beta..-D-glucopyranoside selectively solubilize the outer glycoproteins of the virus particles. The nonionic detergent MESK has the mildest action. Using MESK, purified glycoproteins of influenza, parainfluenza, Venezuelan equine encephalomyelitis, vesicular stomatitis, rabies, and herpes viruses were obtained. The procedure for obtaining glycoproteins includes incubation of the virus suspension with the detergent MESK, removal of subvirus structures by centrifuging, and purification of glycoproteins from detergents by dialysis. Isolated glycoproteins retain a native structure and biological activity and possess high immunogenicity. The detergent MESK is promising for laboratory tests and with respect to the production of subunit vaccines.

  9. Enhanced BBB permeability of osmotically active poly(mannitol-co-PEI) modified with rabies virus glycoprotein via selective stimulation of caveolar endocytosis for RNAi therapeutics in Alzheimer's disease.

    PubMed

    Park, Tae-Eun; Singh, Bijay; Li, Huishan; Lee, Jun-Yeong; Kang, Sang-Kee; Choi, Yun-Jaie; Cho, Chong-Su

    2015-01-01

    RNA interference (RNAi) holds one of the promising tools for Alzheimer's disease (AD) treatment by directly arresting the causative genes. For successful RNAi therapeutics for AD, limited access of therapeutic genes to the brain needs to be overcome by developing siRNA delivery system that could cross the blood-brain barrier (BBB). Here, we report a non-viral vector, rabies virus glycoprotein (RVG)-modified poly(mannitol-co-PEI) gene transporter (PMT), R-PEG-PMT. The RVG ligand directed the PMT/siRNA complexes toward the brain through binding to nicotinic acetylcholine receptors expressed on BBB. In mechanistic study using in vitro BBB model, we observed that osmotically-active PMT enhanced the receptor-mediated transcytosis by stimulating the caveolar endocytosis. The potential of RNAi therapeutics for AD using R-PEG-PMT/siBACE1 complexes was demonstrated in vitro and in vivo. Our results suggest that R-PEG-PMT is a powerful gene carrier system for brain targeted RNAi therapeutics with synergistic effect of RVG ligand and PMT on well-modulated receptor-mediated transcytosis through BBB. PMID:25457984

  10. Yellow fever 17D-vectored vaccines expressing Lassa virus GP1 and GP2 glycoproteins provide protection against fatal disease in guinea pigs.

    PubMed

    Jiang, Xiaohong; Dalebout, Tim J; Bredenbeek, Peter J; Carrion, Ricardo; Brasky, Kathleen; Patterson, Jean; Goicochea, Marco; Bryant, Joseph; Salvato, Maria S; Lukashevich, Igor S

    2011-02-01

    Yellow Fever (YF) and Lassa Fever (LF) are two prevalent hemorrhagic fevers co-circulating in West Africa and responsible for thousands of deaths annually. The YF vaccine 17D has been used as a vector for the Lassa virus glycoprotein precursor (LASV-GPC) or their subunits, GP1 (attachment glycoprotein) and GP2 (fusion glycoprotein). Cloning shorter inserts, LASV-GP1 and -GP2, between YF17D E and NS1 genes enhanced genetic stability of recombinant viruses, YF17D/LASV-GP1 and -GP2, in comparison with YF17D/LASV-GPC recombinant. The recombinant viruses were replication competent and properly processed YF proteins and LASV GP antigens in infected cells. YF17D/LASV-GP1 and -GP2 induced specific CD8+ T cell responses in mice and protected strain 13 guinea pigs against fatal LF. Unlike immunization with live attenuated reassortant vaccine ML29, immunization with YF17D/LASV-GP1 and -GP2 did not provide sterilizing immunity. This study demonstrates the feasibility of YF17D-based vaccine to control LF in West Africa. PMID:21145373

  11. Yellow fever 17D-vectored vaccines expressing Lassa virus GP1 and GP2 glycoproteins provide protection against fatal disease in guinea pigs

    PubMed Central

    Jiang, Xiaohong; Dalebout, Tim J.; Bredenbeek, Peter J.; Carrion, Ricardo; Brasky, Kathleen; Patterson, Jean; Goicochea, Marco; Bryant, Joseph; Salvato, Maria S.; Lukashevich, Igor S.

    2010-01-01

    Yellow Fever (YF) and Lassa Fever (LF) are two prevalent hemorrhagic fevers co-circulating in West Africa and responsible for thousands of deaths annually. The YF vaccine 17D has been used as a vector for the Lassa virus glycoprotein precursor (LASV-GPC) or their subunits, GP1 (attachment glycoprotein) and GP2 (fusion glycoprotein). Cloning shorter inserts, LASV GP1 and GP2, between YF17D E and NS1 genes enhanced genetic stability of recombinant viruses, YF17D/LASV-GP1 and –GP2, in comparison with YF17D/LASV-GPC recombinant. The recombinant viruses were replication competent and properly processed YF and LASV GP proteins in infected cells. YF17D/LASV-GP1&GP2 induced specific CD8+ T cell responses in mice and protected strain 13 guinea pigs against fatal LF. Unlike immunization with live attenuated reassortant vaccine ML29, immunization with YF17D/LASV-GP1&GP2 did not provide sterilizing immunity. This study demonstrates the feasibility of YF17D-based vaccine to control LF in West Africa. PMID:21145373

  12. EXPRESSION OF THE MAIZE MOSAIC VIRUS GLYCOPROTEIN IN INSECT CELLS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Maize mosaic virus (genus Nucleorhabdovirus, family Rhabdoviridae) is transmitted in a persistent-propagative manner by Peregrinus maidis, the corn planthopper. Like other rhabdoviruses, the MMV genome encodes a surface glycoprotein that is likely involved in virus attachment and entry into host ce...

  13. Identification of a Novel Virulence Determinant Within the E2 Structural Glycoprotein of Classical Swine Fever Virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Classical Swine Fever Virus (CSFV) E2 glycoprotein contains a discrete epitope (TAVSPTTLR, residues 829-837 of CSFV polyprotein) recognized by monoclonal antibody (mAb) WH303, used to differentiate CSFV from related ruminant Pestiviruses, Bovine Viral Diarrhea Virus (BVDV) and Border Disease Virus ...

  14. Generation and evaluation of a recombinant Newcastle disease virus expressing the glycoprotein (G) of avian metapneumovirus subgroup C as a bivalent vaccine in turkeys

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Virulent strains of Newcastle disease virus (NDV) and avian metapneumovirus (aMPV) can cause serious respiratory diseases in poultry. Vaccination combined with strict biosecurity practices has been the recommendation for controlling both NDV and aMPV diseases in the field. In the present study, an N...

  15. Vaccinia recombinant virus expressing the rabies virus glycoprotein: safety and efficacy trials in Canadian wildlife.

    PubMed Central

    Artois, M; Charlton, K M; Tolson, N D; Casey, G A; Knowles, M K; Campbell, J B

    1990-01-01

    Twenty-six meadow voles (Microtus pennsylvanicus), ten woodchucks (Marmota monax), thirteen grey squirrels (Sciurus carolinensis), thirteen ring-billed gulls (Larus delawarensis), six red-tailed hawks (Buteo jamaicensis) and eight great horned owls (Bubo virginianus) received vaccinia virus recombinant expressing the rabies virus glycoprotein (V-RG) by direct instillation into the oral cavity. Each of ten coyotes (Canis latrans) received the virus in two vaccine-laden baits. Several voles and most of the gulls died from diseases unrelated to vaccination during the observation period, but all other animals remained healthy and survived. These deaths from causes other than vaccination and the absence of any lesions suggestive of vaccinia infection indicate that it is unlikely that any animal suffered or died as a result of V-RG administration. In addition several animals showed an unexpected high level of rabies neutralizing antibodies. PMID:2249183

  16. Vaccinia recombinant virus expressing the rabies virus glycoprotein: safety and efficacy trials in Canadian wildlife.

    PubMed

    Artois, M; Charlton, K M; Tolson, N D; Casey, G A; Knowles, M K; Campbell, J B

    1990-10-01

    Twenty-six meadow voles (Microtus pennsylvanicus), ten woodchucks (Marmota monax), thirteen grey squirrels (Sciurus carolinensis), thirteen ring-billed gulls (Larus delawarensis), six red-tailed hawks (Buteo jamaicensis) and eight great horned owls (Bubo virginianus) received vaccinia virus recombinant expressing the rabies virus glycoprotein (V-RG) by direct instillation into the oral cavity. Each of ten coyotes (Canis latrans) received the virus in two vaccine-laden baits. Several voles and most of the gulls died from diseases unrelated to vaccination during the observation period, but all other animals remained healthy and survived. These deaths from causes other than vaccination and the absence of any lesions suggestive of vaccinia infection indicate that it is unlikely that any animal suffered or died as a result of V-RG administration. In addition several animals showed an unexpected high level of rabies neutralizing antibodies. PMID:2249183

  17. A Recombinant Hendra Virus G Glycoprotein Subunit Vaccine Protects Nonhuman Primates against Hendra Virus Challenge

    PubMed Central

    Mire, Chad E.; Geisbert, Joan B.; Agans, Krystle N.; Feng, Yan-Ru; Fenton, Karla A.; Bossart, Katharine N.; Yan, Lianying; Chan, Yee-Peng; Geisbert, Thomas W.

    2014-01-01

    ABSTRACT Hendra virus (HeV) is a zoonotic emerging virus belonging to the family Paramyxoviridae. HeV causes severe and often fatal respiratory and/or neurologic disease in both animals and humans. Currently, there are no licensed vaccines or antiviral drugs approved for human use. A number of animal models have been developed for studying HeV infection, with the African green monkey (AGM) appearing to most faithfully reproduce the human disease. Here, we assessed the utility of a newly developed recombinant subunit vaccine based on the HeV attachment (G) glycoprotein in the AGM model. Four AGMs were vaccinated with two doses of the HeV vaccine (sGHeV) containing Alhydrogel, four AGMs received the sGHeV with Alhydrogel and CpG, and four control animals did not receive the sGHeV vaccine. Animals were challenged with a high dose of infectious HeV 21 days after the boost vaccination. None of the eight specifically vaccinated animals showed any evidence of clinical illness and survived the challenge. All four controls became severely ill with symptoms consistent with HeV infection, and three of the four animals succumbed 8 days after exposure. Success of the recombinant subunit vaccine in AGMs provides pivotal data in supporting its further preclinical development for potential human use. IMPORTANCE A Hendra virus attachment (G) glycoprotein subunit vaccine was tested in nonhuman primates to assess its ability to protect them from a lethal infection with Hendra virus. It was found that all vaccinated African green monkeys were completely protected against subsequent Hendra virus infection and disease. The success of this new subunit vaccine in nonhuman primates provides critical data in support of its further development for future human use. PMID:24522928

  18. Diseases Caused by Viruses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The symptoms, causal agents, epidemiology and management of important virus diseases in chickpea and lentil crops were reviewed in depth. The virus diseases include.Alflafa mosaic virus, Cucumber mosaiv virus, Faba bean necrotic yellows virus, Pea enation mosaic virus, Pea seed-borne mosaci virus,...

  19. Reversible conformational changes and fusion activity of rabies virus glycoprotein.

    PubMed Central

    Gaudin, Y; Tuffereau, C; Segretain, D; Knossow, M; Flamand, A

    1991-01-01

    In an attempt to understand the implication of the rabies virus glycoprotein (G) in the first steps of the viral cycle, we studied the pH dependence of virus-induced fusion and hemagglutination, as well as modifications of the structure and properties of the viral glycoprotein following pH acidification. Our results suggest that the G protein adopts at least three distinct configurations, each associated with different properties. At neutral pH, G did not fuse membranes or hemagglutinate erythrocytes. It was insensitive to digestion with bromelain and trypsin. At pH 6.4, the glycoprotein became sensitive to proteases. Hemagglutination was at its maximum and then sharply decreased with the pH. No fusion was detected. Aggregation of virus was also observed. The third configuration, at below pH 6.1, was associated with the appearance of fusion. Some neutralizing monoclonal antibodies were able to differentiate these three configurations. Preincubation of the virus at below pH 6 inhibited fusion, but this inhibition, like the structural modifications of the glycoprotein, was reversible when G was reincubated at neutral pH. Images PMID:1870204

  20. Synonymous codon usage pattern in glycoprotein gene of rabies virus.

    PubMed

    Morla, Sudhir; Makhija, Aditi; Kumar, Sachin

    2016-06-10

    Rabies virus (RABV) is the causative agent of a fatal nervous system ailment. The disease is zoonotic and prevalent in many developing countries. The glycoprotein (G) of RABV is the major antigenic determinant of the virus and plays a pivotal role in its neurovirulence. Various aspects of 'G' protein biology have been explored, but the factors affecting the nucleotide choice and synonymous codon usage have never been reported. In the present study, we have analyzed the relative synonymous codon usage and effective number of codons (Nc) using 132 'G' protein genes of RABV. Corresponding analysis was used to calculate major trends in codon usage. The correlation between base composition and codon usage as well as the plot between Nc and GC3 suggest that mutational pressure is the major factor that influences the codon usage in the G gene of RABV. In addition, factors like aromaticity, aliphatic index and hydropathy have shown slight correlation suggesting that natural selection also contributes to the codon usage variations of the 'G' gene. In conclusion, codon usage bias in 'G' gene of RABV is mainly by mutational pressure and natural selection. PMID:26945626

  1. Recombinant Measles Virus Vaccine Expressing the Nipah Virus Glycoprotein Protects against Lethal Nipah Virus Challenge

    PubMed Central

    Yoneda, Misako; Georges-Courbot, Marie-Claude; Ikeda, Fusako; Ishii, Miho; Nagata, Noriyo; Jacquot, Frederic; Raoul, Hervé; Sato, Hiroki; Kai, Chieko

    2013-01-01

    Nipah virus (NiV) is a member of the genus Henipavirus, which emerged in Malaysia in 1998. In pigs, infection resulted in a predominantly non-lethal respiratory disease; however, infection in humans resulted in over 100 deaths. Nipah virus has continued to re-emerge in Bangladesh and India, and person-to-person transmission appeared in the outbreak. Although a number of NiV vaccine studies have been reported, there are currently no vaccines or treatments licensed for human use. In this study, we have developed a recombinant measles virus (rMV) vaccine expressing NiV envelope glycoproteins (rMV-HL-G and rMV-Ed-G). Vaccinated hamsters were completely protected against NiV challenge, while the mortality of unvaccinated control hamsters was 90%. We trialed our vaccine in a non-human primate model, African green monkeys. Upon intraperitoneal infection with NiV, monkeys showed several clinical signs of disease including severe depression, reduced ability to move and decreased food ingestion and died at 7 days post infection (dpi). Intranasal and oral inoculation induced similar clinical illness in monkeys, evident around 9 dpi, and resulted in a moribund stage around 14 dpi. Two monkeys immunized subcutaneously with rMV-Ed-G showed no clinical illness prior to euthanasia after challenge with NiV. Viral RNA was not detected in any organ samples collected from vaccinated monkeys, and no pathological changes were found upon histopathological examination. From our findings, we propose that rMV-NiV-G is an appropriate NiV vaccine candidate for use in humans. PMID:23516477

  2. Recombinant measles virus vaccine expressing the Nipah virus glycoprotein protects against lethal Nipah virus challenge.

    PubMed

    Yoneda, Misako; Georges-Courbot, Marie-Claude; Ikeda, Fusako; Ishii, Miho; Nagata, Noriyo; Jacquot, Frederic; Raoul, Hervé; Sato, Hiroki; Kai, Chieko

    2013-01-01

    Nipah virus (NiV) is a member of the genus Henipavirus, which emerged in Malaysia in 1998. In pigs, infection resulted in a predominantly non-lethal respiratory disease; however, infection in humans resulted in over 100 deaths. Nipah virus has continued to re-emerge in Bangladesh and India, and person-to-person transmission appeared in the outbreak. Although a number of NiV vaccine studies have been reported, there are currently no vaccines or treatments licensed for human use. In this study, we have developed a recombinant measles virus (rMV) vaccine expressing NiV envelope glycoproteins (rMV-HL-G and rMV-Ed-G). Vaccinated hamsters were completely protected against NiV challenge, while the mortality of unvaccinated control hamsters was 90%. We trialed our vaccine in a non-human primate model, African green monkeys. Upon intraperitoneal infection with NiV, monkeys showed several clinical signs of disease including severe depression, reduced ability to move and decreased food ingestion and died at 7 days post infection (dpi). Intranasal and oral inoculation induced similar clinical illness in monkeys, evident around 9 dpi, and resulted in a moribund stage around 14 dpi. Two monkeys immunized subcutaneously with rMV-Ed-G showed no clinical illness prior to euthanasia after challenge with NiV. Viral RNA was not detected in any organ samples collected from vaccinated monkeys, and no pathological changes were found upon histopathological examination. From our findings, we propose that rMV-NiV-G is an appropriate NiV vaccine candidate for use in humans. PMID:23516477

  3. A Hendra virus G glycoprotein subunit vaccine protects African green monkeys from Nipah virus challenge.

    PubMed

    Bossart, Katharine N; Rockx, Barry; Feldmann, Friederike; Brining, Doug; Scott, Dana; LaCasse, Rachel; Geisbert, Joan B; Feng, Yan-Ru; Chan, Yee-Peng; Hickey, Andrew C; Broder, Christopher C; Feldmann, Heinz; Geisbert, Thomas W

    2012-08-01

    In the 1990s, Hendra virus and Nipah virus (NiV), two closely related and previously unrecognized paramyxoviruses that cause severe disease and death in humans and a variety of animals, were discovered in Australia and Malaysia, respectively. Outbreaks of disease have occurred nearly every year since NiV was first discovered, with case fatality ranging from 10 to 100%. In the African green monkey (AGM), NiV causes a severe lethal respiratory and/or neurological disease that essentially mirrors fatal human disease. Thus, the AGM represents a reliable disease model for vaccine and therapeutic efficacy testing. We show that vaccination of AGMs with a recombinant subunit vaccine based on the henipavirus attachment G glycoprotein affords complete protection against subsequent NiV infection with no evidence of clinical disease, virus replication, or pathology observed in any challenged subjects. Success of the recombinant subunit vaccine in nonhuman primates provides crucial data in supporting its further preclinical development for potential human use. PMID:22875827

  4. Horizontal transmission of Marek’s disease virus requires both the unique-long (UL) 13 protein kinase (UL13) and the UL44 glycoprotein C

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Marek’s disease virus (MDV), an Alphaherpesvirus, causes a general malaise in chickens that is mostly characterized by the development of lymphoblastoid tumors in multiple organs. The use of bacterial artificial chromosomes (BACs) for cloning and manipulation of the MDV genome has facilitated chara...

  5. Ebola Virus Disease

    MedlinePlus

    ... 2014 Fact sheets Features Commentaries 2014 Multimedia Contacts Ebola virus disease Fact sheet Updated January 2016 Key ... for survivors of Ebola virus disease Symptoms of Ebola virus disease The incubation period, that is, the ...

  6. Characterization and mapping of a nonessential pseudorabies virus glycoprotein

    SciTech Connect

    Wathen, M.W.; Wathen, L.M.K.

    1986-04-01

    Antigenic variants of pseudorabies virus (PRV) containing mutations in a viral glycoprotein with a molecular weight of 82,000 (gIII) were isolated by selecting for resistance to a complement-dependent neutralizing monoclonal antibody (MCA82-2) directed against gIII. These mutants were completely resistant to neutralization with MCA82-2 in the presence of complement. Two mutants selected for further studies either did not express gIII or expressed an improperly processed form of the glycoproteins. The mutations were also associated with an altered plaque morphology (syncytium formation). The gIII gene was mapped by the marker rescue of a gIII/sup -/ mutant with cloned restriction enzyme fragments to the long unique region of the PRV genome between 0.376 and 0.383 map units. This corresponds to the map location of a glycoprotein described by Robbins et al. Since gIII is nonessential for viral replication in cell culture and has several other characteristics in common with the herpes simplex virus glycoprotein gC, gIII may represent the PRV equivalent to herpes simplex virus gC.

  7. Mokola virus glycoprotein and chimeric proteins can replace rabies virus glycoprotein in the rescue of infectious defective rabies virus particles.

    PubMed Central

    Mebatsion, T; Schnell, M J; Conzelmann, K K

    1995-01-01

    A reverse genetics approach which allows the generation of infectious defective rabies virus (RV) particles entirely from plasmid-encoded genomes and proteins (K.-K. Conzelmann and M. Schnell, J. Virol. 68:713-719, 1994) was used to investigate the ability of a heterologous lyssavirus glycoprotein (G) and chimeric G constructs to function in the formation of infectious RV-like particles. Virions containing a chloramphenicol acetyltransferase (CAT) reporter gene (SDI-CAT) were generated in cells simultaneously expressing the genomic RNA analog, the RV N, P, M, and L proteins, and engineered G constructs from transfected plasmids. The infectivity of particles was determined by a CAT assay after passage to helper virus-infected cells. The heterologous G protein from Eth-16 virus (Mokola virus, lyssavirus serotype 3) as well as a construct in which the ectodomain of RV G was fused to the cytoplasmic and transmembrane domains of the Eth-16 virus G rescued infectious SDI-CAT particles. In contrast, a chimeric protein composed of the amino-terminal half of the Eth-16 virus G and the carboxy-terminal half of RV G failed to produce infectious particles. Site-directed mutagenesis was used to convert the antigenic site III of RV G to the corresponding sequence of Eth-16 G. This chimeric protein rescued infectious SDI-CAT particles as efficiently as RV G. Virions containing the chimeric protein were specifically neutralized by an anti-Eth-16 virus serum and escaped neutralization by a monoclonal antibody directed against RV antigenic site III. The results show that entire structural domains as well as short surface epitopes of lyssavirus G proteins may be exchanged without affecting the structure required to mediate infection of cells. PMID:7853476

  8. Decrease in Formalin-Inactivated Respiratory Syncytial Virus (FI-RSV) Enhanced Disease with RSV G Glycoprotein Peptide Immunization in BALB/c Mice

    PubMed Central

    Rey, Gertrud U.; Miao, Congrong; Caidi, Hayat; Trivedi, Suvang U.; Harcourt, Jennifer L.; Tripp, Ralph A.; Anderson, Larry J.; Haynes, Lia M.

    2013-01-01

    Respiratory syncytial virus (RSV) is a high priority target for vaccine development. One concern in RSV vaccine development is that a non-live virus vaccine would predispose for enhanced disease similar to that seen with the formalin inactivated RSV (FI-RSV) vaccine. Since a mAb specific to RSV G protein can reduce pulmonary inflammation and eosinophilia seen after RSV infection of FI-RSV vaccinated mice, we hypothesized that RSV G peptides that induce antibodies with similar reactivity may limit enhanced disease after subunit or other non-live RSV vaccines. In support of this hypothesis, we show that FI-RSV vaccinated mice administered RSV G peptide vaccines had a significant reduction in enhanced disease after RSV challenge. These data support the importance of RSV G during infection to RSV disease pathogenesis and suggest that use of appropriately designed G peptide vaccines to reduce the risk of enhanced disease with non-live RSV vaccines merits further study. PMID:24376637

  9. Rabies virus glycoprotein as a carrier for anthrax protective antigen

    SciTech Connect

    Smith, Mary Ellen; Koser, Martin; Xiao Sa; Siler, Catherine; McGettigan, James P.; Calkins, Catherine; Pomerantz, Roger J.; Dietzschold, Bernhard; Schnell, Matthias J. . E-mail: matthias.schnell@jefferson.edu

    2006-09-30

    Live viral vectors expressing foreign antigens have shown great promise as vaccines against viral diseases. However, safety concerns remain a major problem regarding the use of even highly attenuated viral vectors. Using the rabies virus (RV) envelope protein as a carrier molecule, we show here that inactivated RV particles can be utilized to present Bacillus anthracis protective antigen (PA) domain-4 in the viral membrane. In addition to the RV glycoprotein (G) transmembrane and cytoplasmic domains, a portion of the RV G ectodomain was required to express the chimeric RV G anthrax PA on the cell surface. The novel antigen was also efficiently incorporated into RV virions. Mice immunized with the inactivated recombinant RV virions exhibited seroconversion against both RV G and anthrax PA, and a second inoculation greatly increased these responses. These data demonstrate that a viral envelope protein can carry a bacterial protein and that a viral carrier can display whole polypeptides compared to the limited epitope presentation of previous viral systems.

  10. Immunization of cattle with recombinant Newcastle disease virus expressing bovine herpesvirus-1 (BHV-1) glycoprotein D induces mucosal and serum antibody responses and provides partial protection against BHV-1

    PubMed Central

    Khattar, Sunil K.; Collins, Peter L.; Samal, Siba K.

    2012-01-01

    Bovine herpesvirus-1 (BHV-1) is a major cause of respiratory tract diseases in cattle. Vaccination of cattle against BHV-1 is a high priority. A major concern of currently modified live BHV-1 vaccines is their ability to cause latent infection and subsequent reactivation resulting in many outbreaks. Thus, there is a need for alternative strategies. We generated two recombinant Newcastle disease viruses (NDVs) expressing the glycoprotein D (gD) of BHV-1 from an added gene. One recombinant, rLaSota/gDFL, expressed gD without any modification. The other recombinant, rLaSota/gDF, expressed a chimeric gD in which the ectodomain of gD was fused with the transmembrane domain and cytoplasmic tail of the NDV fusion F glycoprotein. Remarkably, the native gD expressed by rLaSota/gDFL virus was incorporated into the NDV virion 2.5-fold more efficiently than the native NDV proteins, whereas the chimeric gD was not detectably incorporated even though it was abundantly expressed on the infected cell surface. The expression of gD did not increase the virulence of the rNDV vectors in chickens. A single intranasal and intratracheal inoculation of calves with either recombinant NDV elicited mucosal and systemic antibodies specific to BHV-1, with the responses to rLaSota/gDFL being higher than those to rLaSota/gDF. Following challenge with BHV-1, calves immunized with the recombinant NDVs had lower titers and earlier clearance of challenge virus compared to the empty vector control, and reduced disease was observed with rLaSota/gDFL. Following challenge, the titers of serum antibodies specific to BHV-1 were higher in the animals immunized with the rNDV vaccines compared to the rNDV parent virus, indicating that the vaccines primed for secondary responses. Our data suggest that NDV can be used as a vaccine vector in bovines and that BHV-1 gD may be useful in mucosal vaccine against BHV-1 infection, but might require augmentation by a second dose or the inclusion of additional BHV-1

  11. Toremifene interacts with and destabilizes the Ebola virus glycoprotein.

    PubMed

    Zhao, Yuguang; Ren, Jingshan; Harlos, Karl; Jones, Daniel M; Zeltina, Antra; Bowden, Thomas A; Padilla-Parra, Sergi; Fry, Elizabeth E; Stuart, David I

    2016-07-01

    Ebola viruses (EBOVs) are responsible for repeated outbreaks of fatal infections, including the recent deadly epidemic in West Africa. There are currently no approved therapeutic drugs or vaccines for the disease. EBOV has a membrane envelope decorated by trimers of a glycoprotein (GP, cleaved by furin to form GP1 and GP2 subunits), which is solely responsible for host cell attachment, endosomal entry and membrane fusion. GP is thus a primary target for the development of antiviral drugs. Here we report the first, to our knowledge, unliganded structure of EBOV GP, and high-resolution complexes of GP with the anticancer drug toremifene and the painkiller ibuprofen. The high-resolution apo structure gives a more complete and accurate picture of the molecule, and allows conformational changes introduced by antibody and receptor binding to be deciphered. Unexpectedly, both toremifene and ibuprofen bind in a cavity between the attachment (GP1) and fusion (GP2) subunits at the entrance to a large tunnel that links with equivalent tunnels from the other monomers of the trimer at the three-fold axis. Protein–drug interactions with both GP1 and GP2 are predominately hydrophobic. Residues lining the binding site are highly conserved among filoviruses except Marburg virus (MARV), suggesting that MARV may not bind these drugs. Thermal shift assays show up to a 14 °C decrease in the protein melting temperature after toremifene binding, while ibuprofen has only a marginal effect and is a less potent inhibitor. These results suggest that inhibitor binding destabilizes GP and triggers premature release of GP2, thereby preventing fusion between the viral and endosome membranes. Thus, these complex structures reveal the mechanism of inhibition and may guide the development of more powerful anti-EBOV drugs. PMID:27362232

  12. Envelope glycoproteins of human immunodeficiency virus type 1: profound influences on immune functions.

    PubMed Central

    Chirmule, N; Pahwa, S

    1996-01-01

    Infection by human immunodeficiency virus type 1 (HIV-1) leads to progressive destruction of the CD4+ T-cell subset, resulting in immune deficiency and AIDS. The specific binding of the viral external envelope glycoprotein of HIV-1, gp120, to the CD4 molecules initiates viral entry. In the past few years, several studies have indicated that the interaction of HIV-1 envelope glycoprotein with cells and molecules of the immune system leads to pleiotropic biological effects on immune functions, which include effects on differentiation of CD34+ lymphoid progenitor cells and thymocytes, aberrant activation and cytokine secretion patterns of mature T cells, induction of apoptosis, B-cell hyperactivity, inhibition of T-cell dependent B-cell differentiation, modulation of macrophage functions, interactions with components of complement, and effects on neuronal cells. The amino acid sequence homologies of the envelope glycoproteins with several cellular proteins have suggested that molecular mimicry may play a role in the pathogenesis of the disease. This review summarizes work done by several investigators demonstrating the profound biological effects of envelope glycoproteins of HIV-1 on immune system cells. Extensive studies have also been done on interactions of the viral envelope proteins with components of the immune system which may be important for eliciting a "protective immune response." Understanding the influences of HIV-1 envelope glycoproteins on the immune system may provide valuable insights into HIV-1 disease pathogenesis and carries implications for the trials of HIV-1 envelope protein vaccines and immunotherapeutics. PMID:8801439

  13. A recombinant rabies virus expressing vesicular stomatitis virus glycoprotein fails to protect against rabies virus infection

    PubMed Central

    Foley, Heather D.; McGettigan, James P.; Siler, Catherine A.; Dietzschold, Bernhard; Schnell, Matthias J.

    2000-01-01

    To investigate the importance of the rabies virus (RV) glycoprotein (G) in protection against rabies, we constructed a recombinant RV (rRV) in which the RV G ecto- and transmembrane domains were replaced with the corresponding regions of vesicular stomatitis virus (VSV) glycoprotein (rRV-VSV-G). We were able to recover rRV-VSV-G and found that particle production was equal to rRV. However, the budding of the chimeric virus was delayed and infectious titers were reduced 10-fold compared with the parental rRV strain containing RV G. Biochemical analysis showed equal replication rates of both viruses, and similar amounts of wild-type and chimeric G were present in the respective viral particles. Additional studies were performed to determine whether the immune response against rRV-VSV-G was sufficient to protect against rabies. Mice were primed with rRV or rRV-VSV-G and challenged with a pathogenic strain of RV 12 days later. Similar immune responses against the internal viral proteins of both viruses indicated successful infection. All mice receiving the rRV vaccine survived the challenge, whereas immunization with rRV-VSV-G did not induce protection. The results confirm the crucial role of RV G in an RV vaccine. PMID:11114165

  14. Raccoon poxvirus rabies virus glycoprotein recombinant vaccine in sheep.

    PubMed

    DeMartini, J C; Bickle, H M; Brodie, S J; He, B X; Esposito, J J

    1993-01-01

    Twenty sheep were divided into groups and inoculated by various routes with recombinant raccoon poxvirus expressing the CVS rabies virus glycoprotein (rRCNV-G) or with raccoon poxvirus (RCNV). The apparent innocuous pathologic responses to each virus coupled with development of high levels of rabies virus neutralizing antibodies in animals vaccinated with rRCNV-G intradermally or intramuscularly suggested that the recombinant is effective and that RCNV would be a suitable substrate for further development of sheep vaccines. Poor antibody response to rRCNV-G given orally implied that it would be relatively harmless if inadvertently ingested by sheep. Virus transmission between vaccinated and sentinel sheep was not observed or detected serologically. PMID:8240013

  15. Dimeric Architecture of the Hendra Virus Attachment Glycoprotein: Evidence for a Conserved Mode of Assembly▿ †

    PubMed Central

    Bowden, Thomas A.; Crispin, Max; Harvey, David J.; Jones, E. Yvonne; Stuart, David I.

    2010-01-01

    Hendra virus is a negative-sense single-stranded RNA virus within the Paramyxoviridae family which, together with Nipah virus, forms the Henipavirus genus. Infection with bat-borne Hendra virus leads to a disease with high mortality rates in humans. We determined the crystal structure of the unliganded six-bladed β-propeller domain and compared it to the previously reported structure of Hendra virus attachment glycoprotein (HeV-G) in complex with its cellular receptor, ephrin-B2. As observed for the related unliganded Nipah virus structure, there is plasticity in the Glu579-Pro590 and Lys236-Ala245 ephrin-binding loops prior to receptor engagement. These data reveal that henipaviral attachment glycoproteins undergo common structural transitions upon receptor binding and further define the structural template for antihenipaviral drug design. Our analysis also provides experimental evidence for a dimeric arrangement of HeV-G that exhibits striking similarity to those observed in crystal structures of related paramyxovirus receptor-binding glycoproteins. The biological relevance of this dimer is further supported by the positional analysis of glycosylation sites from across the paramyxoviruses. In HeV-G, the sites lie away from the putative dimer interface and remain accessible to α-mannosidase processing on oligomerization. We therefore propose that the overall mode of dimer assembly is conserved for all paramyxoviruses; however, while the geometry of dimerization is rather closely similar for those viruses that bind flexible glycan receptors, significant (up to 60°) and different reconfigurations of the subunit packing (associated with a significant decrease in the size of the dimer interface) have accompanied the independent switching to high-affinity protein receptor binding in Hendra and measles viruses. PMID:20375167

  16. Genetic Changes at the Glycoprotein Editing Site Associated With Serial Passage of Sudan Virus.

    PubMed

    Alfson, Kendra J; Avena, Laura E; Beadles, Michael W; Menzie, Heather; Patterson, Jean L; Carrion, Ricardo; Griffiths, Anthony

    2015-10-01

    Sudan virus (SUDV), like the closely related Ebola virus (EBOV), is a filovirus that causes severe hemorrhagic disease. They both contain an RNA editing site in the glycoprotein gene that controls expression of soluble and full-length protein. We tested the consequences of cell culture passage on the genome sequence at the SUDV editing site locus and determined whether this affected virulence. Passage resulted in expansion of the SUDV editing site, similar to that observed with EBOV. We compared viruses possessing either the wild-type or expanded editing site, using a nonhuman primate model of disease. Despite differences in virus serum titer at one time point, there were no significant differences in time to death or any other measured parameter. These data imply that changes at this locus were not important for SUDV lethality. PMID:25920319

  17. Protective Efficacy of Recombinant Modified Vaccinia Virus Ankara Delivering Middle East Respiratory Syndrome Coronavirus Spike Glycoprotein

    PubMed Central

    Volz, Asisa; Kupke, Alexandra; Song, Fei; Jany, Sylvia; Fux, Robert; Shams-Eldin, Hosam; Schmidt, Jörg; Becker, Christin; Eickmann, Markus; Becker, Stephan

    2015-01-01

    Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe respiratory disease in humans. We tested a recombinant modified vaccinia virus Ankara (MVA) vaccine expressing full-length MERS-CoV spike (S) glycoprotein by immunizing BALB/c mice with either intramuscular or subcutaneous regimens. In all cases, MVA-MERS-S induced MERS-CoV-specific CD8+ T cells and virus-neutralizing antibodies. Vaccinated mice were protected against MERS-CoV challenge infection after transduction with the human dipeptidyl peptidase 4 receptor. This MERS-CoV infection model demonstrates the safety and efficacy of the candidate vaccine. PMID:26018172

  18. Infectious salmon anaemia virus (ISAV) isolated from the ISA disease outbreaks in Chile diverged from ISAV isolates from Norway around 1996 and was disseminated around 2005, based on surface glycoprotein gene sequences

    PubMed Central

    Kibenge, Frederick SB; Godoy, Marcos G; Wang, Yingwei; Kibenge, Molly JT; Gherardelli, Valentina; Mansilla, Soledad; Lisperger, Angelica; Jarpa, Miguel; Larroquete, Geraldine; Avendaño, Fernando; Lara, Marcela; Gallardo, Alicia

    2009-01-01

    Background Infectious salmon anaemia (ISA) virus (ISAV) is a pathogen of marine-farmed Atlantic salmon (Salmo salar); a disease first diagnosed in Norway in 1984. For over 25 years ISAV has caused major disease outbreaks in the Northern hemisphere, and remains an emerging fish pathogen because of the asymptomatic infections in marine wild fish and the potential for emergence of new epidemic strains. ISAV belongs to the family Orthomyxoviridae, together with influenza viruses but is sufficiently different to be assigned to its own genus, Isavirus. The Isavirus genome consists of eight single-stranded RNA species, and the virions have two surface glycoproteins; fusion (F) protein encoded on segment 5 and haemagglutinin-esterase (HE) protein encoded on segment 6. However, comparision between different ISAV isolates is complicated because there is presently no universally accepted nomenclature system for designation of genetic relatedness between ISAV isolates. The first outbreak of ISA in marine-farmed Atlantic salmon in the Southern hemisphere occurred in Chile starting in June 2007. In order to describe the molecular characteristics of the virus so as to understand its origins, how ISAV isolates are maintained and spread, and their virulence characteristics, we conducted a study where the viral sequences were directly amplified, cloned and sequenced from tissue samples collected from several ISA-affected fish on the different fish farms with confirmed or suspected ISA outbreaks in Chile. This paper describes the genetic characterization of a large number of ISAV strains associated with extensive outbreaks in Chile starting in June 2007, and their phylogenetic relationships with selected European and North American isolates that are representative of the genetic diversity of ISAV. Results RT-PCR for ISAV F and HE glycoprotein genes was performed directly on tissue samples collected from ISA-affected fish on different farms among 14 fish companies in Chile during the

  19. Characterization of the glycoproteins of bat-derived influenza viruses.

    PubMed

    Maruyama, Junki; Nao, Naganori; Miyamoto, Hiroko; Maeda, Ken; Ogawa, Hirohito; Yoshida, Reiko; Igarashi, Manabu; Takada, Ayato

    2016-01-15

    Recently found bat-derived influenza viruses (BatIVs) have hemagglutinin (HA) and neuraminidase (NA) gene segments distinct from those of previously known influenza A viruses. However, pathogenicities of these BatIVs remain unknown since infectious virus strains have not been isolated yet. To gain insight into the biological properties of BatIVs, we generated vesicular stomatitis viruses (VSVs) pseudotyped with the BatIV HA and NA. We found that VSVs pseudotyped with BatIV HAs and NAs efficiently infected particular bat cell lines but not those derived from primates, and that proteolytic cleavage with a trypsin-like protease was necessary for HA-mediated virus entry. Treatment of the susceptible bat cells with some enzymes and inhibitors revealed that BatIV HAs might recognize some cellular glycoproteins as receptors rather than the sialic acids used for the other known influenza viruses. These data provide fundamental information on the mechanisms underlying the cellular entry and host restriction of BatIVs. PMID:26605499

  20. Chimeric rabies viruses for trans-species comparison of lyssavirus glycoprotein ectodomain functions in virus replication and pathogenesis.

    PubMed

    Genz, Berit; Nolden, Tobias; Negatsch, Alexandra; Teifke, Jens-Peter; Conzelmann, Karl-Klaus; Finke, Stefan

    2012-01-01

    The glycoprotein G of lyssaviruses is the major determinant of virus pathogenicity and serves as a target for immunological responses to virus infections. However, assessment of the exact contribution of lyssavirus G proteins to observed differences in the pathogenicity of lyssavirus species is challenging, since the direct comparison of natural lyssaviruses does not allow specific ascription to individual virus proteins or domains. Here we describe the generation and characterization of recombinant rabies viruses (RABV) that express chimeric G proteins comprising of a RABV cytoplasma domain fused to transmembrane and ectodomain G sequences of a virulent RABV (challenge virus standard; CVS-11) or two European bat lyssaviruses (EBLV- and EBLV-2). These "envelope-switched" recombinant viruses were recovered from cDNAs. Similar growth kinetics and protein expression in neuroblastoma cell cultures and successful targeting of primary neurons showed that the chimeric G proteins were able to replace the authentic G protein in a RABV based virus vector. Inoculation of six week old CD-1 mice by the intracranial (i. c.) route of infection further demonstrated that all recombinant viruses were able to spread in the brain and to induce disease. The "envelope-switched" RABV therefore represent an important tool to further investigate the influence of lyssavirus ectodomains on virus tropism, and pathogenicity. PMID:22712419

  1. Virion envelope glycoproteins as epidermiological markers of Venezuelan encephalitis virus isolates.

    PubMed Central

    Wiebe, M E; Scherer, W F

    1980-01-01

    Virion polypeptide compositions of 26 isolates of Venezuelan encephalitis virus were analyzed by a reproducible and comparative technique of discontinuous sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. Although the molecular weight of the core polypeptide for each isolate was 36,000, numbers and molecular weights of envelope glycoproteins were heterogeneous. Isolates associated with human, but not equine, disease usually had two glycoproteins of 50,000 to 51,000 and 51,000 to 55,000 molecular weight, whereas isolates associated with both human and equine disease usually had an additional, third polypeptide band of either 45,000 to 46,000 or 56,000 to 58,000 molecular weight. The former isolates were in hemagglutination inhibition subtypes I-D, I-E, III, or IV, and the latter were in subtypes I-A, I-B, I-C, or II. Thus virion envelope glycoproteins should be useful markers of Venezuelan encephalitis virus isolates in epidemiological investigations. PMID:7372798

  2. [Immune efficacy of rabies virus glycoprotein expressed by baculovirus vector].

    PubMed

    Chen, Qi; Zhang, Shou-Feng; Liu, Ye; Fu, Yun-Hong; Sun, Cheng-Long; Yang, Yang; Gong, Ting; Song, Fei-Fei; Hu, Rong-Liang

    2012-09-01

    To construct a recombinant baculovirus expressing glycoprotein (GP) of RV SRV9 strain and test the immunological efficacy in mice, open reading frame of rabies virus GP gene of SRV9 strain was cloned into the shuttle vector Bacmid to construct the recombinant shuttle plasmid Bacmid-G and transfection was performed into S f9 cells with the recombinant shuttle plasmid. CPE appeared in cell cultures was identified by electronmicroscopy. Western-blot, IFA and immunity tests in mice were performed to identify the immunoreactivity and immunogenicity of the expression products. Our results showed a recombinant baculovirus expressing GP protein of rabies virus SRV9 was obtained. The expression products possessed a favorable immunogenicity and fall immunized mice could develop 100% protective level of anti-rabies neutralizing antibody. In conclusion, The SRV9 glycoprotein expressed by the recombinant baculovirus in this study had good immunogenicity and could induce anti-rabies neutralizing antibody, which laid the foundation of further development of rabies subunit vaccine. PMID:23233923

  3. Glycoprotein 2 antibodies in Crohn's disease.

    PubMed

    Roggenbuck, Dirk; Reinhold, Dirk; Werner, Lael; Schierack, Peter; Bogdanos, Dimitrios P; Conrad, Karsten

    2013-01-01

    The pathogenesis of Crohn's disease (CrD) and ulcerative colitis (UC), the two major inflammatory bowel diseases (IBD), remains poorly understood. Autoimmunity is considered to be involved in the triggering and perpetuation of inflammatory processes leading to overt disease. Approximately 30% of CrD patients and less than 8% of UC patients show evidence of humoral autoimmunity to exocrine pancreas, detected by indirect immunofluorescence. Pancreatic autoantibodies (PAB) were described for the first time in 1984, but the autoantigenic target(s) of PABs were identified only in 2009. Utilizing immunoblotting and matrix-assisted laser desorption ionization time-of-flight mass spectrometry, the major zymogen granule membrane glycoprotein 2 (GP2) has been discovered as the main PAB autoantigen. The expression of GP2 has been demonstrated at the site of intestinal inflammation, explaining the previously unaddressed contradiction of pancreatic autoimmunity and intestinal inflammation. Recent data demonstrate GP2 to be a specific receptor on microfold (M) cells of intestinal Peyer's patches, which are considered to be the original site of inflammation in CrD. Novel ELISAs, employing recombinant GP2 as the solid phase antigen, have confirmed the presence of IgA and IgG anti-GP2 PABs in CrD patients and revealed an association of anti-GP2 IgA as well as IgG levels with a specific clinical phenotype in CrD. Also, GP2 plays an important role in modulating innate and acquired intestinal immunity. Its urinary homologue, Tamm-Horsfall protein or uromodulin, has a similar effect in the urinary tract, further indicating that GP2 is not just an epiphenomenon of intestinal destruction. This review discusses the role of anti-GP2 autoantibodies as novel CrD-specific markers, the quantification of which provides the basis for further stratification of IBD patients. Given the association with a disease phenotype and the immunomodulating properties of GP2 itself, an important role for GP2

  4. Antibodies Targeting Novel Neutralizing Epitopes of Hepatitis C Virus Glycoprotein Preclude Genotype 2 Virus Infection.

    PubMed

    Deng, Kai; Liu, Ruyu; Rao, Huiying; Jiang, Dong; Wang, Jianghua; Xie, Xingwang; Wei, Lai

    2015-01-01

    Currently, there is no effective vaccine to prevent hepatitis C virus (HCV) infection, partly due to our insufficient understanding of the virus glycoprotein immunology. Most neutralizing antibodies (nAbs) were identified using glycoprotein immunogens, such as recombinant E1E2, HCV pseudoparticles or cell culture derived HCV. However, the fact that in the HCV acute infection phase, only a small proportion of patients are self-resolved accompanied with the emergence of nAbs, indicates the limited immunogenicity of glycoprotein itself to induce effective antibodies against a highly evolved virus. Secondly, in previous reports, the immunogen sequence was mostly the genotype of the 1a H77 strain. Rarely, other genotypes/subtypes have been studied, although theoretically one genotype/subtype immunogen is able to induce cross-genotype neutralizing antibodies. To overcome these drawbacks and find potential novel neutralizing epitopes, 57 overlapping peptides encompassing the full-length glycoprotein E1E2 of subtype 1b were synthesized to immunize BALB/c mice, and the neutralizing reactive of the induced antisera against HCVpp genotypes 1-6 was determined. We defined a domain comprising amino acids (aa) 192-221, 232-251, 262-281 and 292-331 of E1, and 421-543, 564-583, 594-618 and 634-673 of E2, as the neutralizing regions of HCV glycoprotein. Peptides PUHI26 (aa 444-463) and PUHI45 (aa 604-618)-induced antisera displayed the most potent broad neutralizing reactive. Two monoclonal antibodies recognizing the PUHI26 and PUHI45 epitopes efficiently precluded genotype 2 viral (HCVcc JFH and J6 strains) infection, but they did not neutralize other genotypes. Our study mapped a neutralizing epitope region of HCV glycoprotein using a novel immunization strategy, and identified two monoclonal antibodies effective in preventing genotype 2 virus infection. PMID:26406225

  5. Antibodies Targeting Novel Neutralizing Epitopes of Hepatitis C Virus Glycoprotein Preclude Genotype 2 Virus Infection

    PubMed Central

    Rao, Huiying; Jiang, Dong; Wang, Jianghua; Xie, Xingwang; Wei, Lai

    2015-01-01

    Currently, there is no effective vaccine to prevent hepatitis C virus (HCV) infection, partly due to our insufficient understanding of the virus glycoprotein immunology. Most neutralizing antibodies (nAbs) were identified using glycoprotein immunogens, such as recombinant E1E2, HCV pseudoparticles or cell culture derived HCV. However, the fact that in the HCV acute infection phase, only a small proportion of patients are self-resolved accompanied with the emergence of nAbs, indicates the limited immunogenicity of glycoprotein itself to induce effective antibodies against a highly evolved virus. Secondly, in previous reports, the immunogen sequence was mostly the genotype of the 1a H77 strain. Rarely, other genotypes/subtypes have been studied, although theoretically one genotype/subtype immunogen is able to induce cross-genotype neutralizing antibodies. To overcome these drawbacks and find potential novel neutralizing epitopes, 57 overlapping peptides encompassing the full-length glycoprotein E1E2 of subtype 1b were synthesized to immunize BALB/c mice, and the neutralizing reactive of the induced antisera against HCVpp genotypes 1–6 was determined. We defined a domain comprising amino acids (aa) 192–221, 232–251, 262–281 and 292–331 of E1, and 421–543, 564–583, 594–618 and 634–673 of E2, as the neutralizing regions of HCV glycoprotein. Peptides PUHI26 (aa 444–463) and PUHI45 (aa 604–618)-induced antisera displayed the most potent broad neutralizing reactive. Two monoclonal antibodies recognizing the PUHI26 and PUHI45 epitopes efficiently precluded genotype 2 viral (HCVcc JFH and J6 strains) infection, but they did not neutralize other genotypes. Our study mapped a neutralizing epitope region of HCV glycoprotein using a novel immunization strategy, and identified two monoclonal antibodies effective in preventing genotype 2 virus infection. PMID:26406225

  6. A recombinant canine distemper virus expressing a modified rabies virus glycoprotein induces immune responses in mice.

    PubMed

    Li, Zhili; Wang, Jigui; Yuan, Daoli; Wang, Shuang; Sun, Jiazeng; Yi, Bao; Hou, Qiang; Mao, Yaping; Liu, Weiquan

    2015-06-01

    Canine distemper virus (CDV) and rabies virus (RV) are two important pathogens of the dog. CDV, a member of the morbillivirus genus, has shown promise as an expression vector. The glycoprotein from RV is a main contributor to protective immunity and capable of eliciting the production of virus-neutralizing antibodies. In this study, we recovered an attenuated strain of canine distemper virus and constructed a recombinant virus, rCDV-RV-G, expressing a modified (R333Q) rabies virus glycoprotein (RV-G) of RV Flury strain LEP. RV-G expression by the recombinant viruses was confirmed. Furthermore, G was proved to be incorporated into the surface of CDV particles. While replication of the recombinant virus was slightly reduced compared with the parental CDV, it stably expressed the RV-G over ten serial passages. Inoculation of mice induced specific neutralizing antibodies against both RV-G and CDV. Therefore, the rCDV-RV-G has the potential as a vaccine that may be used to control rabies virus infection in dogs and other animals. PMID:25764477

  7. Glycoprotein G deficient infectious laryngotracheitis virus is a candidate attenuated vaccine.

    PubMed

    Devlin, Joanne M; Browning, Glenn F; Hartley, Carol A; Gilkerson, James R

    2007-05-01

    Infectious laryngotracheitis virus (ILTV), an alphaherpesvirus, causes respiratory disease in chickens and is currently controlled by vaccination with conventionally attenuated virus strains. These vaccines have limitations because of residual pathogenicity and reversion to virulence, suggesting that a novel vaccine strain that lacks virulence gene(s) may enhance disease control. Glycoprotein G (gG) has recently been identified as a virulence factor in ILTV. In this study the immunogenicity and relative pathogenicity of gG deficient ILTV was investigated in SPF chickens. Birds vaccinated with gG deficient ILTV were protected against clinical signs of disease following challenge with virulent ILTV and gG deficient ILTV was also shown to be less pathogenic than currently available commercial vaccine strains. Thus gG deficient ILTV appears to have potential as a vaccine candidate. PMID:17316926

  8. Most neutralizing human monoclonal antibodies target novel epitopes requiring both Lassa virus glycoprotein subunits

    PubMed Central

    Robinson, James E.; Hastie, Kathryn M.; Cross, Robert W.; Yenni, Rachael E.; Elliott, Deborah H.; Rouelle, Julie A.; Kannadka, Chandrika B.; Smira, Ashley A.; Garry, Courtney E.; Bradley, Benjamin T.; Yu, Haini; Shaffer, Jeffrey G.; Boisen, Matt L.; Hartnett, Jessica N.; Zandonatti, Michelle A.; Rowland, Megan M.; Heinrich, Megan L.; Martínez-Sobrido, Luis; Cheng, Benson; de la Torre, Juan C.; Andersen, Kristian G.; Goba, Augustine; Momoh, Mambu; Fullah, Mohamed; Gbakie, Michael; Kanneh, Lansana; Koroma, Veronica J.; Fonnie, Richard; Jalloh, Simbirie C.; Kargbo, Brima; Vandi, Mohamed A.; Gbetuwa, Momoh; Ikponmwosa, Odia; Asogun, Danny A.; Okokhere, Peter O.; Follarin, Onikepe A.; Schieffelin, John S.; Pitts, Kelly R.; Geisbert, Joan B.; Kulakoski, Peter C.; Wilson, Russell B.; Happi, Christian T.; Sabeti, Pardis C.; Gevao, Sahr M.; Khan, S. Humarr; Grant, Donald S.; Geisbert, Thomas W.; Saphire, Erica Ollmann; Branco, Luis M.; Garry, Robert F.

    2016-01-01

    Lassa fever is a severe multisystem disease that often has haemorrhagic manifestations. The epitopes of the Lassa virus (LASV) surface glycoproteins recognized by naturally infected human hosts have not been identified or characterized. Here we have cloned 113 human monoclonal antibodies (mAbs) specific for LASV glycoproteins from memory B cells of Lassa fever survivors from West Africa. One-half bind the GP2 fusion subunit, one-fourth recognize the GP1 receptor-binding subunit and the remaining fourth are specific for the assembled glycoprotein complex, requiring both GP1 and GP2 subunits for recognition. Notably, of the 16 mAbs that neutralize LASV, 13 require the assembled glycoprotein complex for binding, while the remaining 3 require GP1 only. Compared with non-neutralizing mAbs, neutralizing mAbs have higher binding affinities and greater divergence from germline progenitors. Some mAbs potently neutralize all four LASV lineages. These insights from LASV human mAb characterization will guide strategies for immunotherapeutic development and vaccine design. PMID:27161536

  9. Most neutralizing human monoclonal antibodies target novel epitopes requiring both Lassa virus glycoprotein subunits.

    PubMed

    Robinson, James E; Hastie, Kathryn M; Cross, Robert W; Yenni, Rachael E; Elliott, Deborah H; Rouelle, Julie A; Kannadka, Chandrika B; Smira, Ashley A; Garry, Courtney E; Bradley, Benjamin T; Yu, Haini; Shaffer, Jeffrey G; Boisen, Matt L; Hartnett, Jessica N; Zandonatti, Michelle A; Rowland, Megan M; Heinrich, Megan L; Martínez-Sobrido, Luis; Cheng, Benson; de la Torre, Juan C; Andersen, Kristian G; Goba, Augustine; Momoh, Mambu; Fullah, Mohamed; Gbakie, Michael; Kanneh, Lansana; Koroma, Veronica J; Fonnie, Richard; Jalloh, Simbirie C; Kargbo, Brima; Vandi, Mohamed A; Gbetuwa, Momoh; Ikponmwosa, Odia; Asogun, Danny A; Okokhere, Peter O; Follarin, Onikepe A; Schieffelin, John S; Pitts, Kelly R; Geisbert, Joan B; Kulakoski, Peter C; Wilson, Russell B; Happi, Christian T; Sabeti, Pardis C; Gevao, Sahr M; Khan, S Humarr; Grant, Donald S; Geisbert, Thomas W; Saphire, Erica Ollmann; Branco, Luis M; Garry, Robert F

    2016-01-01

    Lassa fever is a severe multisystem disease that often has haemorrhagic manifestations. The epitopes of the Lassa virus (LASV) surface glycoproteins recognized by naturally infected human hosts have not been identified or characterized. Here we have cloned 113 human monoclonal antibodies (mAbs) specific for LASV glycoproteins from memory B cells of Lassa fever survivors from West Africa. One-half bind the GP2 fusion subunit, one-fourth recognize the GP1 receptor-binding subunit and the remaining fourth are specific for the assembled glycoprotein complex, requiring both GP1 and GP2 subunits for recognition. Notably, of the 16 mAbs that neutralize LASV, 13 require the assembled glycoprotein complex for binding, while the remaining 3 require GP1 only. Compared with non-neutralizing mAbs, neutralizing mAbs have higher binding affinities and greater divergence from germline progenitors. Some mAbs potently neutralize all four LASV lineages. These insights from LASV human mAb characterization will guide strategies for immunotherapeutic development and vaccine design. PMID:27161536

  10. Virion Glycoprotein-Mediated Immune Evasion by Human Cytomegalovirus: a Sticky Virus Makes a Slick Getaway.

    PubMed

    Gardner, Thomas J; Tortorella, Domenico

    2016-09-01

    The prototypic herpesvirus human cytomegalovirus (CMV) exhibits the extraordinary ability to establish latency and maintain a chronic infection throughout the life of its human host. This is even more remarkable considering the robust adaptive immune response elicited by infection and reactivation from latency. In addition to the ability of CMV to exist in a quiescent latent state, its persistence is enabled by a large repertoire of viral proteins that subvert immune defense mechanisms, such as NK cell activation and major histocompatibility complex antigen presentation, within the cell. However, dissemination outside the cell presents a unique existential challenge to the CMV virion, which is studded with antigenic glycoprotein complexes targeted by a potent neutralizing antibody response. The CMV virion envelope proteins, which are critical mediators of cell attachment and entry, possess various characteristics that can mitigate the humoral immune response and prevent viral clearance. Here we review the CMV glycoprotein complexes crucial for cell attachment and entry and propose inherent properties of these proteins involved in evading the CMV humoral immune response. These include viral glycoprotein polymorphism, epitope competition, Fc receptor-mediated endocytosis, glycan shielding, and cell-to-cell spread. The consequences of CMV virion glycoprotein-mediated immune evasion have a major impact on persistence of the virus in the population, and a comprehensive understanding of these evasion strategies will assist in designing effective CMV biologics and vaccines to limit CMV-associated disease. PMID:27307580

  11. Herpes Simplex Virus Type 1 Glycoprotein gC Mediates Immune Evasion In Vivo

    PubMed Central

    Lubinski, John M.; Wang, Liyang; Soulika, Athena M.; Burger, Reinhard; Wetsel, Rick A.; Colten, Harvey; Cohen, Gary H.; Eisenberg, Roselyn J.; Lambris, John D.; Friedman, Harvey M.

    1998-01-01

    Many microorganisms encode proteins that interact with molecules involved in host immunity; however, few of these molecules have been proven to promote immune evasion in vivo. Herpes simplex virus type 1 (HSV-1) glycoprotein C (gC) binds complement component C3 and inhibits complement-mediated virus neutralization and lysis of infected cells in vitro. To investigate the importance of the interaction between gC and C3 in vivo, we studied the virulence of a gC-null strain in complement-intact and C3-deficient animals. Using a vaginal infection model in complement-intact guinea pigs, we showed that gC-null virus grows to lower titers and produces less severe vaginitis than wild-type or gC rescued virus, indicating a role for gC in virulence. To determine the importance of complement, studies were performed with C3-deficient guinea pigs; the results demonstrated significant increases in vaginal titers of gC-null virus, while wild-type and gC rescued viruses showed nonsignificant changes in titers. Similar findings were observed for mice where gC null virus produced significantly less disease than gC rescued virus at the skin inoculation site. Proof that C3 is important was provided by studies of C3 knockout mice, where disease scores of gC-null virus were significantly higher than in complement-intact mice. The results indicate that gC-null virus is approximately 100-fold (2 log10) less virulent that wild-type virus in animals and that gC-C3 interactions are involved in pathogenesis. PMID:9733869

  12. Evaluation of immunological responses to a glycoprotein G deficient candidate vaccine strain of infectious laryngotracheitis virus.

    PubMed

    Devlin, Joanne M; Viejo-Borbolla, Abel; Browning, Glenn F; Noormohammadi, Amir H; Gilkerson, James R; Alcami, Antonio; Hartley, Carol A

    2010-02-01

    Infectious laryngotracheitis virus (ILTV), an alphaherpesvirus, causes severe respiratory disease in poultry. Glycoprotein G (gG) is a virulence factor in ILTV. Recent studies have shown that gG-deficient ILTV is an effective attenuated vaccine however the function of ILTV gG is unknown. This study examined the function and in vivo relevance of ILTV gG. The results showed that ILTV gG binds to chemokines with high affinity and inhibits leukocyte chemotaxis. Specific-pathogen-free (SPF) chickens infected with gG-deficient virus had altered tracheal leukocyte populations and lower serum antibody levels compared with those infected with the parent virus. The findings suggest that the absence of chemokine-binding activity during infection with gG-deficient ILTV results in altered host immune responses. PMID:19932672

  13. Importance of the intracytoplasmic domain of the simian immunodeficiency virus (SIV) envelope glycoprotein for pathogenesis.

    PubMed

    Luciw, P A; Shaw, K E; Shacklett, B L; Marthas, M L

    1998-12-01

    SIVmac1A11 and SIVmac239 are nonpathogenic and pathogenic molecular clones in rhesus macaques, respectively. Although these viruses exhibit approximately 98% nucleotide and amino acid sequence homology, differences are found in the length of the translation frames for several genes. SIVmac239 has a premature stop codon in nef, whereas SIVmac1A11 has a premature stop codon in vpr and two premature stop codons in the intracytoplasmic domain of the env-transmembrane (TM) subunit. Recombinant viruses, constructed through reciprocal exchange of large DNA restriction enzyme fragments between SIVmac1A11 and SIVmac239, were evaluated in adult rhesus macaques. This in vivo analysis revealed that two or more regions of the SIVmac genome were essential for high virus load and disease progression (Marthas et al., 1993. J. Virol. 67, 6047-6055). An important gap in knowledge remaining from this study was whether the premature stop codons in env-TM of recombinant virus SIV1A11/239gag-env/1A11 (Full-length vpr and nef, two stop codons in env-TM) reverted to coding triplets in vivo. Here, we report that viral sequences in macaques, which succumbed to an AIDS-like disease after infection with SIV1A11/239gag-env/1A11, exhibited reversion of both env-TM stop codons. In addition, antibodies to the intracytoplasmic domain of env-TM were detected in macaques containing revertant virus and showing disease; this finding indicates that this domain of the env glycoprotein was expressed in vivo. Thus selection for viral variants with full-length env-TM demonstrated that the cytoplasmic domain of the SIVmac env glycoprotein plays a role in viral persistence and immunodeficiency in primates. PMID:9875311

  14. Characterization of pseudorabies virus glycoprotein B expressed by canine herpesvirus.

    PubMed

    Nishikawa, Y; Xuan, X; Kimura, M; Otsuka, H

    1999-10-01

    A recombinant canine herpesvirus (CHV) which expressed glycoprotein B (gB) of pseudorabies virus (PrV) was constructed. The antigenicity of the PrV gB expressed by the recombinant CHV is similar to that of the native PrV. The expressed PrV gB was shown to be transported to the surface of infected cells as judged by an indirected immunofluorescence test. Antibodies raised in mice immunized with the recombinant CHV neutralized the infectivity of PrV in vitro. It is known that the authentic PrV gB exists as a glycoprotein complex, which consists of gBa, gBb and gBc. In MDCK cells, PrV gB expressed by the recombinant CHV was processed like authentic PrV gB, suggesting that the cleavage mechanism of PrV gB depends on a functional cleavage domain from PrV gB gene and protease from infected cells. PMID:10563288

  15. Hepatitis C Virus E2 Envelope Glycoprotein Core Structure

    SciTech Connect

    Kong, Leopold; Giang, Erick; Nieusma, Travis; Kadam, Rameshwar U.; Cogburn, Kristin E.; Hua, Yuanzi; Dai, Xiaoping; Stanfield, Robyn L.; Burton, Dennis R.; Ward, Andrew B.; Wilson, Ian A.; Law, Mansun

    2014-08-26

    Hepatitis C virus (HCV), a Hepacivirus, is a major cause of viral hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV envelope glycoproteins E1 and E2 mediate fusion and entry into host cells and are the primary targets of the humoral immune response. The crystal structure of the E2 core bound to broadly neutralizing antibody AR3C at 2.65 angstroms reveals a compact architecture composed of a central immunoglobulin-fold β sandwich flanked by two additional protein layers. The CD81 receptor binding site was identified by electron microscopy and site-directed mutagenesis and overlaps with the AR3C epitope. The x-ray and electron microscopy E2 structures differ markedly from predictions of an extended, three-domain, class II fusion protein fold and therefore provide valuable information for HCV drug and vaccine design.

  16. Vaccinia Virus Recombinant Expressing Herpes Simplex Virus Type 1 Glycoprotein D Prevents Latent Herpes in Mice

    NASA Astrophysics Data System (ADS)

    Cremer, Kenneth J.; Mackett, Michael; Wohlenberg, Charles; Notkins, Abner Louis; Moss, Bernard

    1985-05-01

    In humans, herpes simplex virus causes a primary infection and then often a latent ganglionic infection that persists for life. Because these latent infections can recur periodically, vaccines are needed that can protect against both primary and latent herpes simplex infections. Infectious vaccinia virus recombinants that contain the herpes simplex virus type 1 (HSV-1) glycoprotein D gene under control of defined early or late vaccinia virus promoters were constructed. Tissue culture cells infected with these recombinant viruses synthesized a glycosylated protein that had the same mass (60,000 daltons) as the glycoprotein D produced by HSV-1. Immunization of mice with one of these recombinant viruses by intradermal, subcutaneous, or intraperitoneal routes resulted in the production of antibodies that neutralized HSV-1 and protected the mice against subsequent lethal challenge with HSV-1 or HSV-2. Immunization with the recombinant virus also protected the majority of the mice against the development of a latent HSV-1 infection of the trigeminal ganglia. This is the first demonstration that a genetically engineered vaccine can prevent the development of latency.

  17. Anti-glycoprotein D monoclonal antibody protects against herpes simplex virus type 1-induced diseases in mice functionally depleted of selected T-cell subsets or asialo GM1+ cells.

    PubMed Central

    Staats, H F; Oakes, J E; Lausch, R N

    1991-01-01

    Passive transfer of a monoclonal antibody (MAb) specific for glycoprotein D (gD) is highly effective in preventing the development of herpes simplex virus type 1-induced stromal keratitis. In the present study, we investigated whether animals which had been functionally depleted of T-cell subsets or asialo GM1+ cells would continue to be responsive to MAb therapy. BALB/c mice were depleted of CD4+, CD8+, or asialo GM1+ cells by treatment with anti-L3T4, anti-Lyt 2.2, or anti-asialo GM1 antibodies, respectively. Functional depletion of CD4+ cells was documented by the loss of delayed-type hypersensitivity responsiveness, while CD8+ cell depletion was accompanied by abrogation of cytotoxic lymphocyte activity. Anti-asialo GM1 treatment led to the loss of natural killer cell lytic activity. Mice depleted of the desired cell population and infected on the scarified cornea with herpes simplex virus type 1 uniformly developed necrotizing stromal keratitis by 3 weeks postinfection. A single inoculation of anti-gD MAb (55 micrograms) given intraperitoneally 24 h postinfection strongly protected hosts depleted of CD4+ cells against stromal keratitis. Likewise, antibody treatment in CD8+ or asialo GM1+ cell-depleted hosts was as therapeutically effective as that seen in non-cell-depleted mice. We also observed that in cell-depleted mice, the virus spread into the central nervous system and caused encephalitis. The CD4+ cell-depleted mice were the most severely affected, as 100% developed fatal disease. Anti-gD MAb treatment successfully protected all (32 of 32) CD4+-, CD8+-, or asialo GM1(+)-depleted hosts against encephalitis. We therefore conclude that antibody-mediated prevention of stromal keratitis and encephalitis does not require the obligatory participation of CD4+, CD8+, or asialo GM1+ cells. However, when mice were simultaneously depleted of both CD4+ and CD8+ T-cell subsets, antibody treatment could not prevent fatal encephalitis. Thus, antibody can compensate for

  18. Protection of rhesus monkeys from fatal Lassa fever by vaccination with a recombinant vaccinia virus containing the Lassa virus glycoprotein gene.

    PubMed Central

    Fisher-Hoch, S P; McCormick, J B; Auperin, D; Brown, B G; Castor, M; Perez, G; Ruo, S; Conaty, A; Brammer, L; Bauer, S

    1989-01-01

    Lassa fever is an acute febrile disease of West Africa, where there are as many as 300,000 infections a year and an estimated 3000 deaths. As control of the rodent host is impracticable at present, the best immediate prospect is vaccination. We tested as potential vaccines in rhesus monkeys a closely related virus, Mopeia virus (two monkeys), and a recombinant vaccinia virus containing the Lassa virus glycoprotein gene, V-LSGPC (four monkeys). Two monkeys vaccinated with the New York Board of Health strain of vaccinia virus as controls died after challenge with Lassa virus. The two monkeys vaccinated with Mopeia virus developed antibodies measurable by radioimmunoprecipitation prior to challenge, and they survived challenge by Lassa virus with minimal physical or physiologic disturbances. However, both showed a transient, low-titer Lassa viremia. Two of the four animals vaccinated with V-LSGPC had antibodies to both Lassa glycoproteins, as determined by radioimmunoprecipitation. All four animals survived a challenge of Lassa virus but experienced a transient febrile illness and moderate physiologic changes following challenge. Virus was recoverable from each of these animals, but at low titer and only during a brief period, as observed for the Mopeia-protected animals. We conclude that V-LSGPC can protect rhesus monkeys against death from Lassa fever. PMID:2911575

  19. Antibody Derived Peptides for Detection of Ebola Virus Glycoprotein

    PubMed Central

    López-Pacheco, Felipe; Pérez-Chavarría, Roberto; González-Vázquez, Juan Carlos; González-González, Everardo; Trujillo-de Santiago, Grissel; Ponce-Ponce de León, César Alejandro; Zhang, Yu Shrike; Dokmeci, Mehmet Remzi; Khademhosseini, Ali; Alvarez, Mario Moisés

    2015-01-01

    Background Current Ebola virus (EBOV) detection methods are costly and impractical for epidemic scenarios. Different immune-based assays have been reported for the detection and quantification of Ebola virus (EBOV) proteins. In particular, several monoclonal antibodies (mAbs) have been described that bind the capsid glycoprotein (GP) of EBOV GP. However, the currently available platforms for the design and production of full-length mAbs are cumbersome and costly. The use of antibody fragments, rather than full-length antibodies, might represent a cost-effective alternative for the development of diagnostic and possibly even therapeutic alternatives for EBOV. Methods/Principal Findings We report the design and expression of three recombinant anti-GP mAb fragments in Escherichia coli cultures. These fragments contained the heavy and light variable portions of the three well-studied anti-GP full-length mAbs 13C6, 13F6, and KZ52, and are consequently named scFv-13C6, scFv-13F6, and Fab-KZ52, respectively. All three fragments exhibited specific anti-GP binding activity in ELISA experiments comparable to that of full-length anti-GP antibodies (i.e., the same order of magnitude) and they are easily and economically produced in bacterial cultures. Conclusion/Significance Antibody fragments might represent a useful, effective, and low cost alternative to full-length antibodies in Ebola related capture and diagnostics applications. PMID:26489048

  20. Low temperature-dependent salmonid alphavirus glycoprotein processing and recombinant virus-like particle formation.

    PubMed

    Metz, Stefan W; Feenstra, Femke; Villoing, Stephane; van Hulten, Marielle C; van Lent, Jan W; Koumans, Joseph; Vlak, Just M; Pijlman, Gorben P

    2011-01-01

    Pancreas disease (PD) and sleeping disease (SD) are important viral scourges in aquaculture of Atlantic salmon and rainbow trout. The etiological agent of PD and SD is salmonid alphavirus (SAV), an unusual member of the Togaviridae (genus Alphavirus). SAV replicates at lower temperatures in fish. Outbreaks of SAV are associated with large economic losses of ~17 to 50 million $/year. Current control strategies rely on vaccination with inactivated virus formulations that are cumbersome to obtain and have intrinsic safety risks. In this research we were able to obtain non-infectious virus-like particles (VLPs) of SAV via expression of recombinant baculoviruses encoding SAV capsid protein and two major immunodominant viral glycoproteins, E1 and E2 in Spodoptera frugiperda Sf9 insect cells. However, this was only achieved when a temperature shift from 27°C to lower temperatures was applied. At 27°C, precursor E2 (PE2) was misfolded and not processed by host furin into mature E2. Hence, E2 was detected neither on the surface of infected cells nor as VLPs in the culture fluid. However, when temperatures during protein expression were lowered, PE2 was processed into mature E2 in a temperature-dependent manner and VLPs were abundantly produced. So, temperature shift-down during synthesis is a prerequisite for correct SAV glycoprotein processing and recombinant VLP production. PMID:21991361

  1. Low Temperature-Dependent Salmonid Alphavirus Glycoprotein Processing and Recombinant Virus-Like Particle Formation

    PubMed Central

    Villoing, Stephane; van Hulten, Marielle C.; van Lent, Jan W.; Koumans, Joseph; Vlak, Just M.; Pijlman, Gorben P.

    2011-01-01

    Pancreas disease (PD) and sleeping disease (SD) are important viral scourges in aquaculture of Atlantic salmon and rainbow trout. The etiological agent of PD and SD is salmonid alphavirus (SAV), an unusual member of the Togaviridae (genus Alphavirus). SAV replicates at lower temperatures in fish. Outbreaks of SAV are associated with large economic losses of ∼17 to 50 million $/year. Current control strategies rely on vaccination with inactivated virus formulations that are cumbersome to obtain and have intrinsic safety risks. In this research we were able to obtain non-infectious virus-like particles (VLPs) of SAV via expression of recombinant baculoviruses encoding SAV capsid protein and two major immunodominant viral glycoproteins, E1 and E2 in Spodoptera frugiperda Sf9 insect cells. However, this was only achieved when a temperature shift from 27°C to lower temperatures was applied. At 27°C, precursor E2 (PE2) was misfolded and not processed by host furin into mature E2. Hence, E2 was detected neither on the surface of infected cells nor as VLPs in the culture fluid. However, when temperatures during protein expression were lowered, PE2 was processed into mature E2 in a temperature-dependent manner and VLPs were abundantly produced. So, temperature shift-down during synthesis is a prerequisite for correct SAV glycoprotein processing and recombinant VLP production. PMID:21991361

  2. Resolution of two surface glycoproteins from human parainfluenza-3 virus by crossed immunoelectrophoresis.

    PubMed

    Holling, R A; Guskey, L E

    1984-07-01

    The technique of two-dimensional crossed immunoelectrophoresis (CIE) was used to resolve two glycoproteins from purified human parainfluenza type 3 virus. Virus preparations were extracted with Triton X-100 and fractionated by centrifugation in a Beckman airfuge. Two immunoprecipitates were detected by CIE in the supernatant fractions, but were not found in the pellets from extracted virus. Viral glycoproteins labeled with [35S]methionine were isolated by affinity chromatography on concanavalin A (Con A) agarose columns, resolved by CIE and detected by autoradiography. Resolution of two glycoprotein peaks from as little as 4.5 micrograms of protein from extracted virus is consistent with results from polyacrylamide gel patterns showing two unique glycoproteins with molecular weights of 48 kd and 65 kd. PMID:6088566

  3. Efficacy of the Herpes Simplex Virus 2 (HSV-2) Glycoprotein D/AS04 Vaccine against Genital HSV-2 and HSV-1 Infection and Disease in the Cotton Rat Sigmodon hispidus Model

    PubMed Central

    McKay, Jamall; Mbaye, Aissatou; Sanford-Crane, Hannah; Blanco, Jorge C. G.; Huber, Ashley; Herold, Betsy C.

    2015-01-01

    ABSTRACT Subunit vaccines based on the herpes simplex virus 2 (HSV-2) glycoprotein D (gD-2) have been the major focus of HSV-2 vaccine development for the past 2 decades. Based on the promising data generated in the guinea pig model, a formulation containing truncated gD-2, aluminum salt, and MPL (gD/AS04) advanced to clinical trials. The results of these trials, however, were unexpected, as the vaccine protected against HSV-1 infection but not against HSV-2. To address this discrepancy, we developed a Depot medroxyprogesterone acetate (DMPA)-treated cotton rat Sigmodon hispidus model of HSV-2 and HSV-1 genital infection. The severity of HSV-1 genital herpes was less than that of HSV-2 genital herpes in cotton rats, and yet the model allowed for comparative evaluation of gD/AS04 immunogenicity and efficacy. Cotton rats were intramuscularly vaccinated using a prime boost strategy with gD/AS04 (Simplirix vaccine) or control vaccine formulation (hepatitis B vaccine FENDrix) and subsequently challenged intravaginally with HSV-2 or HSV-1. The gD/AS04 vaccine was immunogenic in cotton rats and induced serum IgG directed against gD-2 and serum HSV-2 neutralizing antibodies but failed to efficiently protect against HSV-2 disease or to decrease the HSV-2 viral load. However, gD/AS04 significantly reduced vaginal titers of HSV-1 and better protected animals against HSV-1 compared to HSV-2 genital disease. The latter finding is generally consistent with the clinical outcome of the Herpevac trial of Simplirix. Passive transfer of serum from gD/AS04-immunized cotton rats conferred stronger protection against HSV-1 genital disease. These findings suggest the need for alternative vaccine strategies and the identification of new correlates of protection. IMPORTANCE In spite of the high health burden of genital herpes, there is still no effective intervention against the disease. The significant gap in knowledge on genital herpes pathogenesis has been further highlighted by the

  4. Identification and antigenicity of the major envelope glycoprotein of lymphadenopathy-associated virus

    SciTech Connect

    Montagnier, L.; Clavel, F.; Krust, B.; Chamaret, S.; Rey, F.; Barre-Sinoussi, F.; Chermann, J.C.

    1985-07-15

    The major envelope glycoprotein of the causative agent of Acquired Immune Deficiency Syndrome (AIDS) lymphadenopathy-associated virus (LAV) has been identified and characterized. The glycoprotein has an apparent molecular weight of 110,000-120,000 under denaturing conditions in polyacrylamide gel electrophoresis. Upon deglycosylation by a specific endoglycosydase, its size is reduced to 80,000. Cellular precursors of this glycoprotein have been detected with apparent molecular weight of 150,000 and 135,000. Nearly all AIDS and pre-AIDS patients have detectable antibodies against this viral glycoprotein.

  5. Inflammatory glycoproteins in cardiometabolic disorders, autoimmune diseases and cancer.

    PubMed

    Connelly, Margery A; Gruppen, Eke G; Otvos, James D; Dullaart, Robin P F

    2016-08-01

    The physiological function initially attributed to the oligosaccharide moieties or glycans on inflammatory glycoproteins was to improve protein stability. However, it is now clear that glycans play a prominent role in glycoprotein structure and function and in some cases contribute to disease states. In fact, glycan processing contributes to pathogenicity not only in autoimmune disorders but also in atherosclerotic cardiovascular disease, diabetes and malignancy. While most clinical laboratory tests measure circulating levels of inflammatory proteins, newly developed diagnostic and prognostic tests are harvesting the information that can be gleaned by measuring the amount or structure of the attached glycans, which may be unique to individuals as well as various diseases. As such, these newer glycan-based tests may provide future means for more personalized approaches to patient stratification and improved patient care. Here we will discuss recent progress in high-throughput laboratory methods for glycomics (i.e. the study of glycan structures) and glycoprotein quantification by methods such as mass spectrometry and nuclear magnetic resonance spectroscopy. We will also review the clinical utility of glycoprotein and glycan measurements in the prediction of common low-grade inflammatory disorders including cardiovascular disease, diabetes and cancer, as well as for monitoring autoimmune disease activity. PMID:27312321

  6. Use of lambdagt11 to isolate genes for two pseudorabies virus glycoproteins with homology to herpes simplex virus and varicella-zoster virus glycoproteins

    SciTech Connect

    Petrovskis, E.A.; Timmins, J.G.; Post, L.E.

    1986-10-01

    A library of pseudorabies virus (PRV) DNA fragments was constructed in the expression cloning vector lambdagt11. The library was screened with antisera which reacted with mixtures of PRV proteins to isolate recombinant bacteriophages expressing PRV proteins. By the nature of the lambdagt11 vector, the cloned proteins were expressed in Escherichia coli as ..beta..-galactosidase fusion proteins. The fusion proteins from 35 of these phages were purified and injected into mice to raise antisera. The antisera were screened by several different assays, including immunoprecipitation of (/sup 14/C)glucosamine-labeled PRV proteins. This method identified phages expressing three different PRV glycoproteins: the secreted glycoprotein, gX; gI; and a glycoprotein that had not been previously identified, which we designate gp63. The gp63 and gI genes map adjacent to each other in the small unique region of the PRV genome. The DNA sequence was determined for the region of the genome encoding gp63 and gI. It was found that gp63 has a region of homology with a herpes simplex virus type 1 (HSV-1) protein, encoded by US7, and also with varicella-zoster virus (VZV) gpIV. The gI protein sequence has a region of homology with HSV-1 gE and VZV gpI. It is concluded that PRV, HSV, and VZV all have a cluster of homologous glycoprotein genes in the small unique components of their genomes and that the organization of these genes is conserved.

  7. Positive evolution of the glycoprotein (GP) gene is related to transmission of the Ebola virus.

    PubMed

    Jing, Y X; Wang, L N; Wu, X M; Song, C X

    2016-01-01

    Ebola hemorrhagic fever is a fatal disease caused by the negative-strand RNA of the Ebola virus. A high-intensity outbreak of this fever was reported in West Africa last year; however, there is currently no definitive treatment strategy available for this disease. In this study, we analyzed the molecular evolutionary history and attempted to determine the positive selection sites in the Ebola genes using multiple-genomic sequences of the various Ebola virus subtypes, in order to gain greater clarity into the evolution of the virus and its various subtypes. Only the glycoprotein (GP) gene was positively selected among the 8 Ebola genes, with the other genes remaining in the purification stage. The positive selection sites in the GP gene were identified by a random-site model; these sites were found to be located in the mucin-like region, which is associated with transmembrane protein binding. Additionally, different branches of the phylogenetic tree displayed different positive sites, which in turn was responsible for differences in the cell adhesion ability of the virus. In conclusion, the pattern of positive sites in the GP gene is associated with the epidemiology and prevalence of Ebola in different areas. PMID:27051001

  8. Vesicular stomatitis virus with the rabies virus glycoprotein directs retrograde transsynaptic transport among neurons in vivo

    PubMed Central

    Beier, Kevin T.; Saunders, Arpiar B.; Oldenburg, Ian A.; Sabatini, Bernardo L.; Cepko, Constance L.

    2012-01-01

    Defining the connections among neurons is critical to our understanding of the structure and function of the nervous system. Recombinant viruses engineered to transmit across synapses provide a powerful approach for the dissection of neuronal circuitry in vivo. We recently demonstrated that recombinant vesicular stomatitis virus (VSV) can be endowed with anterograde or retrograde transsynaptic tracing ability by providing the virus with different glycoproteins. Here we extend the characterization of the transmission and gene expression of recombinant VSV (rVSV) with the rabies virus glycoprotein (RABV-G), and provide examples of its activity relative to the anterograde transsynaptic tracer form of rVSV. rVSV with RABV-G was found to drive strong expression of transgenes and to spread rapidly from neuron to neuron in only a retrograde manner. Depending upon how the RABV-G was delivered, VSV served as a polysynaptic or monosynaptic tracer, or was able to define projections through axonal uptake and retrograde transport. In animals co-infected with rVSV in its anterograde form, rVSV with RABV-G could be used to begin to characterize the similarities and differences in connections to different areas. rVSV with RABV-G provides a flexible, rapid, and versatile tracing tool that complements the previously described VSV-based anterograde transsynaptic tracer. PMID:23403489

  9. A reassessment of the evolutionary timescale of bat rabies viruses based upon glycoprotein gene sequences.

    PubMed

    Kuzmina, Natalia A; Kuzmin, Ivan V; Ellison, James A; Taylor, Steven T; Bergman, David L; Dew, Beverly; Rupprecht, Charles E

    2013-10-01

    Rabies, an acute progressive encephalomyelitis caused by viruses in the genus Lyssavirus, is one of the oldest known infectious diseases. Although dogs and other carnivores represent the greatest threat to public health as rabies reservoirs, it is commonly accepted that bats are the primary evolutionary hosts of lyssaviruses. Despite early historical documentation of rabies, molecular clock analyses indicate a quite young age of lyssaviruses, which is confusing. For example, the results obtained for partial and complete nucleoprotein gene sequences of rabies viruses (RABV), or for a limited number of glycoprotein gene sequences, indicated that the time of the most recent common ancestor (TMRCA) for current bat RABV diversity in the Americas lies in the seventeenth to eighteenth centuries and might be directly or indirectly associated with the European colonization. Conversely, several other reports demonstrated high genetic similarity between lyssavirus isolates, including RABV, obtained within a time interval of 25-50 years. In the present study, we attempted to re-estimate the age of several North American bat RABV lineages based on the largest set of complete and partial glycoprotein gene sequences compiled to date (n = 201) employing a codon substitution model. Although our results overlap with previous estimates in marginal areas of the 95 % high probability density (HPD), they suggest a longer evolutionary history of American bat RABV lineages (TMRCA at least 732 years, with a 95 % HPD 436-1107 years). PMID:23839669

  10. Interactome analysis of herpes simplex virus 1 envelope glycoprotein H.

    PubMed

    Hirohata, Yoshitaka; Kato, Akihisa; Oyama, Masaaki; Kozuka-Hata, Hiroko; Koyanagi, Naoto; Arii, Jun; Kawaguchi, Yasushi

    2015-06-01

    Herpes simplex virus 1 (HSV-1) envelope glycoprotein H (gH) is important for viral entry into cells and nuclear egress of nucleocapsids. To clarify additional novel roles of gH during HSV-1 replication, host cell proteins that interact with gH were screened for by tandem affinity purification coupled with mass spectrometry-based proteomics in 293T cells transiently expressing gH. This screen identified 123 host cell proteins as potential gH interactors. Of these proteins, general control nonderepressive-1 (GCN1), a trans-acting positive effector of GCN2 kinase that regulates phosphorylation of the α subunit of translation initiation factor 2 (eIF2α), was subsequently confirmed to interact with gH in HSV-1-infected cells. eIF2α phosphorylation is known to downregulate protein synthesis, and various viruses have evolved mechanisms to prevent the accumulation of phosphorylated eIF2α in infected cells. Here, it was shown that GCN1 knockdown reduces phosphorylation of eIF2α in HSV-1-infected cells and that the gH-null mutation increases eIF2α in HSV-1-infected cells, whereas gH overexpression in the absence of other HSV-1 proteins reduces eIF2α phosphorylation. These findings suggest that GCN1 can regulate eIF2α phosphorylation in HSV-1-infected cells and that the GCN1-binding viral partner gH is necessary and sufficient to prevent the accumulation of phosphorylated eIF2α. Our database of 123 host cell proteins potentially interacting with gH will be useful for future studies aimed at unveiling further novel functions of gH and the roles of cellular proteins in HSV-1-infected cells. PMID:25808324

  11. Purification and structural characterization of herpes simplex virus glycoprotein C

    SciTech Connect

    Kikuchi, G.E.; Baker, S.A.; Merajver, S.D.; Coligan, J.E.; Levine, M.; Glorioso, J.C.; Nairn, R.

    1987-01-27

    Purification of herpes simplex virus glycoprotein C (gC) in microgram amounts yielded sufficient material for an analysis of its secondary structure. Purification was facilitated by using the mutant virus gC-3, which bears a point mutation that interrupts the putative hydrophobic membrane anchor sequence, causing the secretion of gC-3 protein into the cell culture medium. gC-3 protein was purified by size fractionation of concentrated culture medium from infected cells on a gel filtration column of Sephacryl S-200, followed by immunoaffinity chromatography on a column constructed of gC-specific monoclonal antibodies cross-linked to a protein A-Sepharose CL-4B matrix. Purified gC-3 had a molecular weight of 130,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the size expected for gC, was reactive with gC-specific monoclonal antibodies in protein immunoblots, and contained amino acid sequences characteristic of gC as determined by radiochemical amino acid microsequence analyses. Polyclonal antisera obtained from a rabbit immunized with gC-3 reacted with wild-type gC in immunoprecipitation, enzyme immunoassay, and immunoelectroblot (western blot) assays. Deglycosylation by treatment with trifluoromethanesulfonic acid reduced the molecular weight of gC-3 by approximately 35%. Analyses of both native and deglycosylated gC-3 by Raman spectroscopy showed that the native molecule consists of about 17%..cap alpha..-helix, 24% ..beta..-sheet, and 60% disordered secondary structures, whereas deglycosylated gC-3 consists of about 8% ..cap alpha..-helix, 10% ..beta..-sheet, 81% disordered structures. These data were in good agreement with the 11% ..cap alpha..-helix, 18% ..beta..-sheet, 61% ..beta..-turn, and 9% disordered structures calculated from Chou-Fasman analysis of the primary sequence of gC-3.

  12. Immunogenicity of varicella zoster virus glycoprotein E DNA vaccine

    PubMed Central

    BAO, LIDAO; WEI, GUOMIN; GAN, HONGMEI; REN, XIANHUA; MA, RUILIAN; WANG, YI; LV, HAIJUN

    2016-01-01

    In the present study a eukaryotic expression vector of varicella zoster virus (VZV) glycoprotein E (gE) was constructed and enabled to express in COS7 cells. Furthermore, a specific immune response against the VZV gE eukaryotic expression plasmid was induced in BALB/c mice. The VZV gE gene was amplified using polymerase chain reaction (PCR) and cloned into a eukaryotic expression vector, pcDNA3.1. The recombinant vector was subsequently transfected into COS7 cells using a liposome transfection reagent. The recombinant protein was instantaneously expressed by the transfected cells, as detected by immunohistochemistry, and the recombinant pcDNA-VZV gE plasmid was subsequently used to immunize mice. Tissue expression levels were analyzed by reverse transcription-PCR. In addition, the levels of serum antibodies and spleen lymphocyte proliferation activity were investigated. The amplified target gene included the full-length gE gene (~2.7 kb), and the recombinant expression vector induced gE expression in COS7 cells. In addition, the expression plasmid induced sustained expression in vivo following immunization of mice. Furthermore, the plasmid was capable of inducing specific antibody production and effectively stimulating T cell proliferation. Effective humoral and cellular immunity was triggered in the mice immunized with the VZV gE eukaryotic expression vector. The results of the present study laid the foundation for future research into a VZV DNA vaccine. PMID:27168804

  13. Molecular optimization of rabies virus glycoprotein expression in Pichia pastoris.

    PubMed

    Ben Azoun, Safa; Belhaj, Aicha Eya; Göngrich, Rebecca; Gasser, Brigitte; Kallel, Héla

    2016-05-01

    In this work, different approaches were investigated to enhance the expression rabies virus glycoprotein (RABV-G) in the yeast Pichia pastoris; this membrane protein is responsible for the synthesis of rabies neutralizing antibodies. First, the impact of synonymous codon usage bias was examined and an optimized RABV-G gene was synthesized. Nevertheless, data showed that the secretion of the optimized RABV-G gene was not tremendously increased as compared with the non-optimized one. In addition, similar levels of RABV-G were obtained when α-factor mating factor from Saccharomyces cerevisiae or the acid phosphatase PHO1 was used as a secretion signal. Therefore, sequence optimization and secretion signal were not the major bottlenecks for high-level expression of RABV-G in P. pastoris. Unfolded protein response (UPR) was induced in clones containing high copy number of RABV-G expression cassette indicating that folding was the limiting step for RABV-G secretion. To circumvent this limitation, co-overexpression of five factors involved in oxidative protein folding was investigated. Among these factors only PDI1, ERO1 and GPX1 proved their benefit to enhance the expression. The highest expression level of RABV-G reached 1230 ng ml(-1) . Competitive neutralizing assay confirmed that the recombinant protein was produced in the correct conformational form in this host. PMID:26880068

  14. Seroreactive recombinant herpes simplex virus type 2-specific glycoprotein G.

    PubMed Central

    Parkes, D L; Smith, C M; Rose, J M; Brandis, J; Coates, S R

    1991-01-01

    The herpes simplex virus type 2 (HSV-2) genome codes for an envelope protein, glycoprotein G (gG), which contains predominantly type 2-specific epitopes. A portion of this gG gene has been expressed as a fusion protein in Escherichia coli. Expression was regulated by a lambda phage pL promoter. The 60,000-molecular-weight recombinant protein was purified by ion-exchange chromatography. Amino acid sequence analysis confirmed the N terminus of the purified protein. Mice immunized with recombinant gG developed antibodies reactive with native HSV-2 protein, but not with HSV-1 protein, in an indirect immunofluorescence assay. The serological activity of this purified recombinant gG protein was evaluated by immunoblot assay. This protein was reactive with an HSV-2 gG monoclonal antibody. It was also reactive with HSV-2 rabbit antiserum but not with HSV-1 rabbit antiserum. Of 15 patient serum samples known to have antibody to HSV-2, 14 were reactive with this recombinant type 2-specific gG protein, and none of 15 HSV antibody-negative patient serum samples showed reactivity. In agreement with the expected prevalence of HSV-2 infection, 27.6% of 134 serum samples from random normal individuals had antibodies reactive with recombinant gG. This recombinant gG protein may be of value in detecting HSV-2-specific antibody responses in patients infected with HSV-2. Images PMID:1653787

  15. Crystal Structure and Carbohydrate Analysis of Nipah Virus Attachment Glycoprotein: a Template for Antiviral and Vaccine Design▿ †

    PubMed Central

    Bowden, Thomas A.; Crispin, Max; Harvey, David J.; Aricescu, A. Radu; Grimes, Jonathan M.; Jones, E. Yvonne; Stuart, David I.

    2008-01-01

    Two members of the paramyxovirus family, Nipah virus (NiV) and Hendra virus (HeV), are recent additions to a growing number of agents of emergent diseases which use bats as a natural host. Identification of ephrin-B2 and ephrin-B3 as cellular receptors for these viruses has enabled the development of immunotherapeutic reagents which prevent virus attachment and subsequent fusion. Here we present the structural analysis of the protein and carbohydrate components of the unbound viral attachment glycoprotein of NiV glycoprotein (NiV-G) at a 2.2-Å resolution. Comparison with its ephrin-B2-bound form reveals that conformational changes within the envelope glycoprotein are required to achieve viral attachment. Structural differences are particularly pronounced in the 579-590 loop, a major component of the ephrin binding surface. In addition, the 236-245 loop is rather disordered in the unbound structure. We extend our structural characterization of NiV-G with mass spectrometric analysis of the carbohydrate moieties. We demonstrate that NiV-G is largely devoid of the oligomannose-type glycans that in viruses such as human immunodeficiency virus type 1 and Ebola virus influence viral tropism and the host immune response. Nevertheless, we find putative ligands for the endothelial cell lectin, LSECtin. Finally, by mapping structural conservation and glycosylation site positions from other members of the paramyxovirus family, we suggest the molecular surface involved in oligomerization. These results suggest possible pathways of virus-host interaction and strategies for the optimization of recombinant vaccines. PMID:18815311

  16. Expression and characterization of glycophospholipid-anchored human immunodeficiency virus type 1 envelope glycoproteins.

    PubMed Central

    Salzwedel, K; Johnston, P B; Roberts, S J; Dubay, J W; Hunter, E

    1993-01-01

    Four chimeric human immunodeficiency virus type 1 (HIV-1) env genes were constructed which encoded the extracellular domain of either the wild-type or a cleavage-defective HIV-1 envelope glycoprotein (gp160) fused at one of two different positions in env to a C-terminal glycosyl-phosphatidylinositol (GPI) attachment signal from the mouse Thy-1.1 glycoprotein. All four of the constructs encoded glycoproteins that were efficiently expressed when Rev was supplied in trans, and the two cleavable forms were processed normally to gp120 and a chimeric "gp41." The chimeric glycoproteins, in contrast to the wild-type glycoprotein, could be cleaved from the surface of transfected cells by treatment with phosphatidylinositol-specific phospholipase C, indicating that they were anchored in the plasma membrane by a GPI moiety. These GPI-anchored glycoproteins were transported intracellularly at a rate only slightly lower than that of the full-length HIV-1 glycoprotein and were present on the cell surface in equivalent amounts. Nevertheless, all four glycoproteins were defective in mediating both cell-cell and virus-cell fusion as determined by syncytium formation in COS-1-HeLa-T4 cell mixtures and trans complementation of an env-defective HIV-1 genome. Images PMID:8102410

  17. Zika Virus Disease.

    PubMed

    Slenczka, Werner

    2016-06-01

    The history of Zika virus disease serves as a paradigm of a typical emerging viral infection. Zika virus disease, a mosquito-borne flavivirus, was first isolated in 1947 in the Zika forest of Uganda. The same virus was also isolated from jungle-dwelling mosquitoes (Aedes [Stegomyia] africanus). In many areas of Africa and South Asia human infections with Zika virus were detected by both serology and virus isolation. About 80% of infections are asymptomatic, and in 20% a mostly mild disease with fever, rash, arthralgia, and conjunctivitis may occur. Fetal infections with malformations were not recorded in Africa or Asia. Zika virus was imported to northern Brazil possibly during the world soccer championship that was hosted by Brazil in June through July 2014. A cluster of severe fetal malformations with microcephaly and ocular defects was noted in 2015 in the northeast of Brazil, and intrauterine infections with Zika virus were confirmed. The dramatic change in Zika virus pathogenicity upon its introduction to Brazil has remained an enigma. PMID:27337468

  18. A Single gD Glycoprotein Can Mediate Infection by Herpes simplex Virus

    PubMed Central

    2013-01-01

    Herpes simplex viruses display hundreds of gD glycoproteins, and yet their neutralization requires tens of thousands of antibodies per virion, leading us to ask whether a wild-type virion with just a single free gD is still infective. By quantitative analysis of fluorescently labeled virus particles and virus neutralization assays, we show that entry of a wild-type HSV virion to a cell does indeed require just one or two of the approximately 300 gD glycoproteins to be left unbound by monoclonal antibody. This indicates that HSV entry is an extraordinarily efficient process, functioning at the level of single molecular complexes. PMID:23837576

  19. The rabies virus glycoprotein determines the distribution of different rabies virus strains in the brain.

    PubMed

    Yan, Xiuzhen; Mohankumar, Puliyur S; Dietzschold, Bernhard; Schnell, Matthies J; Fu, Zhen F

    2002-08-01

    The contribution of rabies virus (RV) glycoprotein (G) in viral distribution in the brain was examined by immunohistochemistry following stereotaxic inoculation into the rat hippocampus. Viruses used in this study include the highly neuroinvasive challenge virus standard strains (CVS-N2C and CVS-B2C) and the nonneuroinvasive attenuated SN-10 strain, as well as SN-10-derived recombinant viruses expressing the G gene from CVS-N2C (RN2C) or CVS-B2C (RB2C). The distribution of recombinant viruses in the brain was similar to those of the parental viruses from which the G was derived. For example, while CVS-B2C- and RB2C-infected neurons were seen preferentially in the hippocampus, cortex, and hypothalamus, CVS-N2C- and RN2C-infected neurons were preferentially found in the hippocampus, cortex, and thalamus. SN-10 infected efficiently almost all the brain regions. To further study the role of the RV G in virus spreading, we examined the distribution of RV antigen in brains infected with a recombinant RV in which the SN-10 G was replaced with vesicular stomatitis virus (VSV) G (SN-10-VG) was examined. The spreading of SN-10-VG to the cortex and the thalamus was drastically reduced, but the number of infected neurons in hippocampus and hypothalamus, particularly the paraventricular nucleus, was similar to the SN-10 virus. This pattern of spreading resembles that of VSV. Together, our data demonstrate that it is the G protein that determines the distribution pattern of RV in the brain. PMID:12161819

  20. Structural Characterization of the glycoprotein GP2 Core Domain from the CAS Virus, a Novel Arenavirus-like Species

    PubMed Central

    Koellhoffer, Jayne F.; Dai, Zhou; Malashkevich, Vladimir N.; Stenglein, Mark D.; Liu, Yanyun; Toro, Rafael; Harrison, Joseph; Chandran, Kartik; DeRisi, Joseph L.; Almo, Steven C.; Lai, Jonathan R.

    2014-01-01

    Fusion of the viral and host cell membranes is a necessary first step for infection by enveloped viruses, and is mediated by the envelope glycoprotein. The transmembrane subunits from the structurally defined “class I” glycoproteins adopt an α-helical “trimer- of-hairpins” conformation during the fusion pathway. Here we present our studies on the envelope glycoprotein transmembrane subunit, GP2, of the CAS virus (CASV). CASV was recently identified from annulated tree boas (Corallus annulatus) with inclusion body disease and is implicated in the disease etiology. We have generated and characterized two protein constructs consisting of the predicted CASV GP2 core domain. The crystal structure of the CASV GP2 post-fusion conformation indicates a trimeric α-helical bundle that is highly similar to those of Ebola Virus (EBOV) and Marburg Virus (MARV) GP2, despite CASV genome homology to arenaviruses. Denaturation studies demonstrate that the stability of CASV GP2 is pH-dependent with higher stability at lower pH; we propose that this behavior is due to a network of interactions among acidic residues that would destabilize the α-helical bundle under conditions where the side chains are deprotonated. The pH-dependent stability of the post-fusion structure has been observed in EBOV and MARV GP2, as well as other viruses that enter via the endosome. Infection experiments with CASV and the related Golden Gate Virus (GGV) support a mechanism of entry that requires endosomal acidification. Our results suggest that despite being primarily arenavirus-like, the transmembrane subunit of CASV is extremely similar to the filoviruses. PMID:24333483

  1. HSV-1 Glycoproteins Are Delivered to Virus Assembly Sites Through Dynamin-Dependent Endocytosis.

    PubMed

    Albecka, Anna; Laine, Romain F; Janssen, Anne F J; Kaminski, Clemens F; Crump, Colin M

    2016-01-01

    Herpes simplex virus-1 (HSV-1) is a large enveloped DNA virus that belongs to the family of Herpesviridae. It has been recently shown that the cytoplasmic membranes that wrap the newly assembled capsids are endocytic compartments derived from the plasma membrane. Here, we show that dynamin-dependent endocytosis plays a major role in this process. Dominant-negative dynamin and clathrin adaptor AP180 significantly decrease virus production. Moreover, inhibitors targeting dynamin and clathrin lead to a decreased transport of glycoproteins to cytoplasmic capsids, confirming that glycoproteins are delivered to assembly sites via endocytosis. We also show that certain combinations of glycoproteins colocalize with each other and with the components of clathrin-dependent and -independent endocytosis pathways. Importantly, we demonstrate that the uptake of neutralizing antibodies that bind to glycoproteins when they become exposed on the cell surface during virus particle assembly leads to the production of non-infectious HSV-1. Our results demonstrate that transport of viral glycoproteins to the plasma membrane prior to endocytosis is the major route by which these proteins are localized to the cytoplasmic virus assembly compartments. This highlights the importance of endocytosis as a major protein-sorting event during HSV-1 envelopment. PMID:26459807

  2. Mutating Conserved Cysteines in the Alphavirus E2 Glycoprotein Causes Virus-Specific Assembly Defects

    PubMed Central

    Snyder, Anthony J.; Sokoloski, Kevin J.

    2012-01-01

    There are 80 trimeric, glycoprotein spikes that cover the surface of an alphavirus particle. The spikes, which are composed of three E2 and E1 glycoprotein heterodimers, are responsible for receptor binding and mediating fusion between the viral and host-cell membranes during entry. In addition, the cytoplasmic domain of E2 interacts with the nucleocapsid core during the last stages of particle assembly, possibly to aid in particle stability. During assembly, the spikes are nonfusogenic until the E3 glycoprotein is cleaved from E2 in the trans-Golgi network. Thus, a mutation in E2 potentially has effects on virus entry, spike assembly, or spike maturation. E2 is a highly conserved, cysteine-rich transmembrane glycoprotein. We made single cysteine-to-serine mutations within two distinct regions of the E2 ectodomain in both Sindbis virus and Ross River virus. Each of the E2 Cys mutants produced fewer infectious particles than wild-type virus. Further characterization of the mutant viruses revealed differences in particle morphology, fusion activity, and polyprotein cleavage between Sindbis and Ross River virus mutants, despite the mutations being made at corresponding positions in E2. The nonconserved assembly defects suggest that E2 folding and function is species dependent, possibly due to interactions with a virus-specific chaperone. PMID:22238319

  3. Rabies virus (RV) glycoprotein expression levels are not critical for pathogenicity of RV.

    PubMed

    Wirblich, Christoph; Schnell, Matthias J

    2011-01-01

    Previous comparisons of different rabies virus (RV) strains suggested an inverse relationship between pathogenicity and the amount of glycoprotein produced in infected cells. In order to provide more insight into this relationship, we pursued an experimental approach that allowed us to alter the glycoprotein expression level without altering the glycoprotein sequence, thereby eliminating the contribution of amino acid changes to differences in viral virulence. To this end, we constructed an infectious clone of the highly pathogenic rabies virus strain CVS-N2c and replaced its cognate glycoprotein gene with synthetic versions in which silent mutations were introduced to replace wild-type codons with the most or least frequently used synonymous codons. A recombinant N2c variant containing the fully codon-optimized G gene and three variants carrying a partially codon-deoptimized G gene were recovered on mouse neuroblastoma cells and shown to express 2- to 3-fold more and less glycoprotein, respectively, than wild-type N2c. Pathogenicity studies in mice revealed the WT-N2c virus to be the most pathogenic strain. Variants containing partially codon-deoptimized glycoprotein genes or the codon-optimized gene were less pathogenic than WT-N2c but still caused significant mortality. We conclude that the expression level of the glycoprotein gene does have an impact on pathogenicity but is not a dominant factor that determines pathogenicity. Thus, strategies such as changes in codon usage that aim solely at altering the expression level of the glycoprotein gene do not suffice to render a pathogenic rabies virus apathogenic and are not a viable and safe approach for attenuation of a pathogenic strain. PMID:21068252

  4. Rabies Virus (RV) Glycoprotein Expression Levels Are Not Critical for Pathogenicity of RV▿

    PubMed Central

    Wirblich, Christoph; Schnell, Matthias J.

    2011-01-01

    Previous comparisons of different rabies virus (RV) strains suggested an inverse relationship between pathogenicity and the amount of glycoprotein produced in infected cells. In order to provide more insight into this relationship, we pursued an experimental approach that allowed us to alter the glycoprotein expression level without altering the glycoprotein sequence, thereby eliminating the contribution of amino acid changes to differences in viral virulence. To this end, we constructed an infectious clone of the highly pathogenic rabies virus strain CVS-N2c and replaced its cognate glycoprotein gene with synthetic versions in which silent mutations were introduced to replace wild-type codons with the most or least frequently used synonymous codons. A recombinant N2c variant containing the fully codon-optimized G gene and three variants carrying a partially codon-deoptimized G gene were recovered on mouse neuroblastoma cells and shown to express 2- to 3-fold more and less glycoprotein, respectively, than wild-type N2c. Pathogenicity studies in mice revealed the WT-N2c virus to be the most pathogenic strain. Variants containing partially codon-deoptimized glycoprotein genes or the codon-optimized gene were less pathogenic than WT-N2c but still caused significant mortality. We conclude that the expression level of the glycoprotein gene does have an impact on pathogenicity but is not a dominant factor that determines pathogenicity. Thus, strategies such as changes in codon usage that aim solely at altering the expression level of the glycoprotein gene do not suffice to render a pathogenic rabies virus apathogenic and are not a viable and safe approach for attenuation of a pathogenic strain. PMID:21068252

  5. Expression of Herpes Simplex Virus 1 Glycoprotein B by a Recombinant Vaccinia Virus and Protection of Mice against Lethal Herpes Simplex Virus 1 Infection

    NASA Astrophysics Data System (ADS)

    Cantin, Edouard M.; Eberle, Richard; Baldick, Joseph L.; Moss, Bernard; Willey, Dru E.; Notkins, Abner L.; Openshaw, Harry

    1987-08-01

    The herpes simplex virus 1 (HSV-1) strain F gene encoding glycoprotein gB was isolated and modified at the 5' end by in vitro oligonucleotide-directed mutagenesis. The modified gB gene was inserted into the vaccinia virus genome and expressed under the control of a vaccinia virus promoter. The mature gB glycoprotein produced by the vaccinia virus recombinant was glycosylated, was expressed at the cell surface, and was indistinguishable from authentic HSV-1 gB in terms of electrophoretic mobility. Mice immunized intradermally with the recombinant vaccinia virus produced gB-specific neutralizing antibodies and were resistant to a lethal HSV-1 challenge.

  6. Differential transcription patterns in wild-type and glycoprotein G-deleted infectious laryngotracheitis viruses.

    PubMed

    Mahmoudian, Alireza; Markham, Philip F; Noormohammadi, Amir H; Devlin, Joanne M; Browning, Glenn F

    2013-01-01

    Infectious laryngotracheitis virus (ILTV) causes severe respiratory disease in poultry throughout the world. Recently the role of glycoprotein G (gG) in ILTV pathogenesis has been investigated and it has been shown to have chemokine-binding activity. An ILTV vaccine candidate deficient in gG has been developed and the deletion has been shown to alter the host's immune response to the virus. To understand the effect of the gG gene on transcription of other viral genes, the global expression profile of 72 ILTV genes in gG-deleted and wild-type ILTVs were investigated both in vivo and in vitro using quantitative reverse transcription-polymerase chain reaction. Several genes were differentially expressed in the different viruses in LMH cell cultures or in the tracheas of infected birds, and the expression of a number of genes, including ICP27, gC, gJ, Ul7 and UL40, differed significantly both in vivo and in vitro, suggesting that they had direct or indirect roles in virulence. This study has provided insights into the interactions between gG and other ILTV genes that may have a role in virulence. PMID:23611157

  7. Crystallization and preliminary X-ray analysis of Chandipura virus glycoprotein G

    PubMed Central

    Baquero, Eduard; Buonocore, Linda; Rose, John K.; Bressanelli, Stéphane; Gaudin, Yves; Albertini, Aurélie A.

    2012-01-01

    Fusion in members of the Rhabdoviridae virus family is mediated by the G glycoprotein. At low pH, the G glycoprotein catalyzes fusion between viral and endosomal membranes by undergoing a major conformational change from a pre-fusion trimer to a post-fusion trimer. The structure of the G glycoprotein from vesicular stomatitis virus (VSV G), the prototype of Vesiculovirus, has recently been solved in its trimeric pre-fusion and post-fusion conformations; however, little is known about the structural details of the transition. In this work, a soluble form of the ectodomain of Chandipura virus G glycoprotein (CHAV Gth) was purified using limited proteolysis of purified virus; this soluble ectodomain was also crystallized. This protein shares 41% amino-acid identity with VSV G and thus its structure could provide further clues about the structural transition of rhabdoviral glycoproteins induced by low pH. Crystals of CHAV Gth obtained at pH 7.5 diffracted X-rays to 3.1 Å resolution. These crystals belonged to the orthorhombic space group P21212, with unit-cell parameters a = 150.3, b = 228.2, c = 78.8 Å. Preliminary analysis of the data based on the space group and the self-rotation function indicated that there was no trimeric association of the protomers. This unusual oligomeric status could result from the presence of fusion intermediates in the crystal. PMID:22949203

  8. Effect of the ionophore monensin on herpes simplex virus type 1-induced cell fusion, glycoprotein synthesis, and virion infectivity.

    PubMed

    Kousoulas, K G; Bzik, D J; Person, S

    1983-01-01

    The ionophore monensin inhibited the formation of mature, fully glycosylated glycoproteins gB, gC, and gD during herpes simplex virus type 1 infection of human embryonic lung cells. Underglycosylated forms, including the apparent high-mannose precursor forms of the major glycoproteins, appeared. Monensin inhibited virus-induced cell fusion. Infectious virions produced in the presence of monensin appeared to contain predominantly underglycosylated glycoproteins. PMID:6307921

  9. A replication-deficient rabies virus vaccine expressing Ebola virus glycoprotein is highly attenuated for neurovirulence

    SciTech Connect

    Papaneri, Amy B.; Wirblich, Christoph; Cann, Jennifer A.; Cooper, Kurt; Jahrling, Peter B.; Schnell, Matthias J.; Blaney, Joseph E.

    2012-12-05

    We are developing inactivated and live-attenuated rabies virus (RABV) vaccines expressing Ebola virus (EBOV) glycoprotein for use in humans and endangered wildlife, respectively. Here, we further characterize the pathogenesis of the live-attenuated RABV/EBOV vaccine candidates in mice in an effort to define their growth properties and potential for safety. RABV vaccines expressing GP (RV-GP) or a replication-deficient derivative with a deletion of the RABV G gene (RV{Delta}G-GP) are both avirulent after intracerebral inoculation of adult mice. Furthermore, RV{Delta}G-GP is completely avirulent upon intracerebral inoculation of suckling mice unlike parental RABV vaccine or RV-GP. Analysis of RV{Delta}G-GP in the brain by quantitative PCR, determination of virus titer, and immunohistochemistry indicated greatly restricted virus replication. In summary, our findings indicate that RV-GP retains the attenuation phenotype of the live-attenuated RABV vaccine, and RV{Delta}G-GP would appear to be an even safer alternative for use in wildlife or consideration for human use.

  10. A new rabies vaccine based on a recombinant ORF virus (parapoxvirus) expressing the rabies virus glycoprotein.

    PubMed

    Amann, Ralf; Rohde, Jörg; Wulle, Ulrich; Conlee, Douglas; Raue, Rudiger; Martinon, Olivier; Rziha, Hanns-Joachim

    2013-02-01

    The present study describes the generation of a new Orf virus (ORFV) recombinant, D1701-V-RabG, expressing the rabies virus (RABV) glycoprotein that is correctly presented on the surface of infected cells without the need of replication or production of infectious recombinant virus. One single immunization with recombinant ORFV can stimulate high RABV-specific virus-neutralizing antibody (VNA) titers in mice, cats, and dogs, representing all nonpermissive hosts for the ORFV vector. The protective immune response against severe lethal challenge infection was analyzed in detail in mice using different dosages, numbers, and routes for immunization with the ORFV recombinant. Long-term levels of VNA could be elicited that remained greater than 0.5 IU per ml serum, indicative for the protective status. Single applications of higher doses (10(7) PFU) can be sufficient to confer complete protection against intracranial (i.c.) challenge, whereas booster immunization was needed for protection by the application of lower dosages. Anamnestic immune responses were achieved by each of the seven tested routes of inoculation, including oral application. Finally, in vivo antibody-mediated depletion of CD4-positive and/or CD8-posititve T cell subpopulations during immunization and/or challenge infection attested the importance of CD4 T cells for the induction of protective immunity by D1701-V-RabG. This report demonstrates another example of the potential of the ORFV vector and also indicates the capability of the new recombinant for vaccination of animals. PMID:23175365

  11. Cross-linking of glycoprotein oligomers during herpes simplex virus type 1 entry.

    PubMed

    Handler, C G; Cohen, G H; Eisenberg, R J

    1996-09-01

    Herpes simplex virus (HSV) has 10 glycoproteins in its envelope. Glycoprotein B (gB), gC, gD, gH, and gL have been implicated in virus entry. We previously used chemical cross-linking to show that these five glycoproteins were close enough to each other to be cross-linked into homodimeric and hetero-oligomeric forms; hetero-oligomers of gB-gC, gC-gD, gD-gB, gH-gL, gC-gL and gD-gL were found in purified virions. To better understand the roles of these glycoproteins in viral entry, we have modified a standard HSV penetration assay to include cross-linkers. This allowed us to examine changes in associations of viral glycoproteins during the entry process. HSV-1(KOS) was adsorbed at 4 degrees C to human neuroblastoma cells (SY5Y). The temperature was raised to 37 degrees C and cells were treated with cross-linker at various times after the temperature shift. Cytoplasmic extracts were examined by Western blotting (immunoblotting) for viral glycoproteins. We found that (i) as in virus alone, the length and concentration of the cross-linking agent affected the number of specific complexes isolated; (ii) the same glycoprotein patterns found in purified virions were also present after attachment of virions to cells; and (iii) the ability to cross-link HSV glycoproteins changed as virus penetration proceeded, e.g., gB and gD complexes which were present during attachment disappeared with increasing time, and their disappearance paralleled the kinetics of penetration. However, this phenomenon appeared to be selective since it was not observed with gC oligomers. In addition, we examined the cross-linking patterns of gB and gD in null viruses K082 and KOSgD beta. Neither of these mutants, which attach but cannot penetrate, showed changes in glycoprotein cross-linking over time. We speculate that these changes are due to conformational changes which preclude cross-linking or spatial alterations which dissociate the glycoprotein interactions during the penetration events. PMID

  12. Viruses and Virus Diseases of Rubus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Rubus species are propagated vegetatively and are subject to infection by viruses during development, propagation and fruit production stages. Reports of initial detection and symptoms of more than 30 viruses, virus-like diseases and phytoplasmas affecting Rubus spp. have been reviewed more than 20 ...

  13. Early Activation of Primary Brain Microvascular Endothelial Cells by Nipah Virus Glycoprotein-Containing Particles.

    PubMed

    Freitag, Tanja C; Maisner, Andrea

    2016-03-01

    Nipah virus (NiV) is a highly pathogenic paramyxovirus that causes pronounced infection of brain endothelia and central nervous system (CNS) inflammation. Using primary porcine brain microvascular endothelial cells, we showed that upregulation of E-selectin precedes cytokine induction and is induced not only by infectious NiV but also by NiV-glycoprotein-containing virus-like particles. This demonstrates that very early events in NiV brain endothelial infection do not depend on NiV replication but can be triggered by the NiV glycoproteins alone. PMID:26676791

  14. Early Activation of Primary Brain Microvascular Endothelial Cells by Nipah Virus Glycoprotein-Containing Particles

    PubMed Central

    Freitag, Tanja C.

    2015-01-01

    Nipah virus (NiV) is a highly pathogenic paramyxovirus that causes pronounced infection of brain endothelia and central nervous system (CNS) inflammation. Using primary porcine brain microvascular endothelial cells, we showed that upregulation of E-selectin precedes cytokine induction and is induced not only by infectious NiV but also by NiV-glycoprotein-containing virus-like particles. This demonstrates that very early events in NiV brain endothelial infection do not depend on NiV replication but can be triggered by the NiV glycoproteins alone. PMID:26676791

  15. P-glycoprotein activity in the blood-brain barrier is affected by virus-induced neuroinflammation and antipsychotic treatment.

    PubMed

    Doorduin, Janine; de Vries, Erik F J; Dierckx, Rudi A; Klein, Hans C

    2014-10-01

    A large percentage of schizophrenic patients respond poorly to antipsychotic treatment. This could be explained by inefficient drug transport across the blood-brain barrier due to P-glycoprotein mediated efflux. P-glycoprotein activity and expression in the blood-brain barrier can be affected by inflammation and pharmacotherapy. We therefore investigated the effect of herpes simplex virus type-1 (HSV-1) induced neuroinflammation and antipsychotic treatment on P-glycoprotein activity. Rats were inoculated with HSV-1 or PBS (control) on day 0 and treated with saline, clozapine or risperidone from day 0 up until day 4 post-inoculation. Positron emission tomography with the P-glycoprotein substrate [11C]verapamil was used to assess P-glycoprotein activity at day 6 post-inoculation. Disease symptoms in HSV-1 inoculated rats increased over time and were not significantly affected by treatment. The volume of distribution (VT) of [11C]verapamil was significantly lower (10-22%) in HSV-1 inoculated rats than in control rats. In addition, antipsychotic treatment significantly affected the VT of [11C]verapamil in all brain regions, although this effect was drug dependent. In fact, VT of [11C]verapamil was significantly increased (22-39%) in risperidone treated rats in most brain regions when compared to clozapine treated rats and in midbrain when compared to saline treated rats. No interaction between HSV-1 inoculation and antipsychotic treatment on VT of [11C]verapamil was found. In this study we demonstrated that HSV-1 induced neuroinflammation increased and risperidone treatment decreased P-glycoprotein activity. This finding is of importance for the understanding of treatment resistance in schizophrenia, and warrants further investigation of the underlying mechanism and the importance in clinical practice. PMID:24973705

  16. Ebola (Ebola Virus Disease): Prevention

    MedlinePlus

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  17. Ebola (Ebola Virus Disease): Transmission

    MedlinePlus

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  18. Ebola (Ebola Virus Disease): Treatment

    MedlinePlus

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  19. Ebola (Ebola Virus Disease): Diagnosis

    MedlinePlus

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  20. Newcastle disease virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Newcastle disease virus (NDV), a member of the Avulavirus genus in the Paramyxoviridae family, has a ribonucleic acid (RNA) genome that is negative sense, non-segmented, and single-stranded. The genome codes for six structural proteins: nucleocapsid, phosphoprotein, matrix, fusion, hemagglutinin-neu...

  1. Cryo-electron Microscopy Structure of the Native Prototype Foamy Virus Glycoprotein and Virus Architecture.

    PubMed

    Effantin, Grégory; Estrozi, Leandro F; Aschman, Nick; Renesto, Patricia; Stanke, Nicole; Lindemann, Dirk; Schoehn, Guy; Weissenhorn, Winfried

    2016-07-01

    Foamy viruses (FV) belong to the genus Spumavirus, which forms a distinct lineage in the Retroviridae family. Although the infection in natural hosts and zoonotic transmission to humans is asymptomatic, FVs can replicate well in human cells making it an attractive gene therapy vector candidate. Here we present cryo-electron microscopy and (cryo-)electron tomography ultrastructural data on purified prototype FV (PFV) and PFV infected cells. Mature PFV particles have a distinct morphology with a capsid of constant dimension as well as a less ordered shell of density between the capsid and the membrane likely formed by the Gag N-terminal domain and the cytoplasmic part of the Env leader peptide gp18LP. The viral membrane contains trimeric Env glycoproteins partly arranged in interlocked hexagonal assemblies. In situ 3D reconstruction by subtomogram averaging of wild type Env and of a Env gp48TM- gp80SU cleavage site mutant showed a similar spike architecture as well as stabilization of the hexagonal lattice by clear connections between lower densities of neighboring trimers. Cryo-EM was employed to obtain a 9 Å resolution map of the glycoprotein in its pre-fusion state, which revealed extensive trimer interactions by the receptor binding subunit gp80SU at the top of the spike and three central helices derived from the fusion protein subunit gp48TM. The lower part of Env, presumably composed of interlaced parts of gp48TM, gp80SU and gp18LP anchors the spike at the membrane. We propose that the gp48TM density continues into three central transmembrane helices, which interact with three outer transmembrane helices derived from gp18LP. Our ultrastructural data and 9 Å resolution glycoprotein structure provide important new insights into the molecular architecture of PFV and its distinct evolutionary relationship with other members of the Retroviridae. PMID:27399201

  2. Cryo-electron Microscopy Structure of the Native Prototype Foamy Virus Glycoprotein and Virus Architecture

    PubMed Central

    Effantin, Grégory; Estrozi, Leandro F.; Aschman, Nick; Renesto, Patricia; Stanke, Nicole; Lindemann, Dirk; Schoehn, Guy; Weissenhorn, Winfried

    2016-01-01

    Foamy viruses (FV) belong to the genus Spumavirus, which forms a distinct lineage in the Retroviridae family. Although the infection in natural hosts and zoonotic transmission to humans is asymptomatic, FVs can replicate well in human cells making it an attractive gene therapy vector candidate. Here we present cryo-electron microscopy and (cryo-)electron tomography ultrastructural data on purified prototype FV (PFV) and PFV infected cells. Mature PFV particles have a distinct morphology with a capsid of constant dimension as well as a less ordered shell of density between the capsid and the membrane likely formed by the Gag N-terminal domain and the cytoplasmic part of the Env leader peptide gp18LP. The viral membrane contains trimeric Env glycoproteins partly arranged in interlocked hexagonal assemblies. In situ 3D reconstruction by subtomogram averaging of wild type Env and of a Env gp48TM- gp80SU cleavage site mutant showed a similar spike architecture as well as stabilization of the hexagonal lattice by clear connections between lower densities of neighboring trimers. Cryo-EM was employed to obtain a 9 Å resolution map of the glycoprotein in its pre-fusion state, which revealed extensive trimer interactions by the receptor binding subunit gp80SU at the top of the spike and three central helices derived from the fusion protein subunit gp48TM. The lower part of Env, presumably composed of interlaced parts of gp48TM, gp80SU and gp18LP anchors the spike at the membrane. We propose that the gp48TM density continues into three central transmembrane helices, which interact with three outer transmembrane helices derived from gp18LP. Our ultrastructural data and 9 Å resolution glycoprotein structure provide important new insights into the molecular architecture of PFV and its distinct evolutionary relationship with other members of the Retroviridae. PMID:27399201

  3. 1-Cinnamoyl-3,11-dihydroxymeliacarpin delays glycoprotein transport restraining virus multiplication without cytotoxicity.

    PubMed

    Bueno, Carlos A; Alché, Laura E; Barquero, Andrea A

    2010-02-26

    The 1-cinnamoyl-3,11-dihydroxymeliacarpin (CDM), isolated from extracts of Melia azedarach L., displays antiviral and immunomodulating properties. CDM is the first reported tetranortriterpenoid responsible for the alkalinization of intracellular compartments affecting both, viral endocytic and exocytic pathways. Considering that viral glycoprotein synthesis is completely dependent upon cellular membrane trafficking, we questioned whether CDM might also interfere with the normal transport of cellular glycoproteins. This study demonstrates that CDM promoted a transient block in the transport of two cellular glycoproteins, the transferrin receptor (TfR) and TNF-alpha. Nevertheless, CDM did not affect the transferrin binding ability of TfR and did not impede the TNF-alpha secretion. On the other hand, CDM disturbed the intracellular localization of capsid, glycoprotein and tegument proteins simultaneously in the same HSV-1 infected cells. Besides, we show that concanamycin A and monensin provoke a permanent blockage of viral and cellular glycoproteins, in contrast to the delay observed after CDM treatment. Thus, the delay on glycoprotein transport caused by CDM would account for the strong inhibition on virus multiplication without interfering with the bioactivity of cellular glycoproteins. PMID:20097166

  4. Effects of Herpes Simplex Virus Type 2 Glycoprotein Vaccines and CLDC Adjuvant on Genital Herpes Infection in the Guinea Pig

    PubMed Central

    Bernstein, David I; Earwood, Julie D.; Bravo, Fernando J.; Cohen, Gary H; Eisenberg, Roselyn J; Clark, Jennifer R.; Fairman, Jeffrey; Cardin, Rhonda D.

    2011-01-01

    Genital herpes simplex virus (HSV) infections are common but results from vaccine trials with HSV-2 glycoprotein D (gD) have been disappointing. We therefore compared a similar HSV gD2 vaccine, to a further truncated gD2 vaccine, to a vaccine with gD2 plus gB2 and gH2/gL2 and to a vaccine with only gB2 and gH2/gL2 in a guinea pig model of genital herpes. All vaccines were administered with cationic liposome-DNA complexes (CLDC) as an adjuvant. All vaccines significantly decreased the severity of acute genital disease and vaginal virus replication compared to the placebo group. The majority of animals in all groups developed at least one episode of recurrent disease but the frequency of recurrent disease was significantly reduced by each vaccine compared to placebo. No vaccine was significantly more protective than gD2 alone for any of the parameters described above. No vaccine decreased recurrent virus shedding. When protection against acute infection of dorsal root ganglia and the spinal cord was evaluated all vaccines decreased the per cent of animal with detectable virus and the quantity of virus but again no vaccine was significantly more protective than another. Improvements in HSV-2 vaccines may require inclusion of more T cell targets, more potent adjuvants or live virus vaccines. PMID:21238569

  5. [Comparative studies of sera from cattle with complete leukemia virus and glycoprotein antigens].

    PubMed

    Mateva, V; Vasileva, L

    1980-01-01

    One hundred cattle serums were investigated by the AGTD-test with two antigens: an antigen produced by the whole virus and an antigen containing glycoproteins. Of all serums studied 44 showed a specific precipitation in case the glycoprotein antigen was used. In case the antigen from the whole virus was used 41 serums showed a specific precipitation line, while in 3 of the serums two precipitation lines were observed. Fifty six serums proved negative, containing no antibodies against bovine leucosis virus, after antigens were used. In 2 of the serums non specific precipitation lines were obtained when the antigen from whole virus was used. the precipitation lines produced by both antigenes did not differ in intensity and time of manifestation. PMID:6251597

  6. Cytomegalovirus-based vaccine expressing Ebola virus glycoprotein protects nonhuman primates from Ebola virus infection

    PubMed Central

    Marzi, Andrea; Murphy, Aisling A.; Feldmann, Friederike; Parkins, Christopher J.; Haddock, Elaine; Hanley, Patrick W.; Emery, Matthew J.; Engelmann, Flora; Messaoudi, Ilhem; Feldmann, Heinz; Jarvis, Michael A.

    2016-01-01

    Ebolaviruses pose significant public health problems due to their high lethality, unpredictable emergence, and localization to the poorest areas of the world. In addition to implementation of standard public health control procedures, a number of experimental human vaccines are being explored as a further means for outbreak control. Recombinant cytomegalovirus (CMV)-based vectors are a novel vaccine platform that have been shown to induce substantial levels of durable, but primarily T-cell-biased responses against the encoded heterologous target antigen. Herein, we demonstrate the ability of rhesus CMV (RhCMV) expressing Ebola virus (EBOV) glycoprotein (GP) to provide protective immunity to rhesus macaques against lethal EBOV challenge. Surprisingly, vaccination was associated with high levels of GP-specific antibodies, but with no detectable GP-directed cellular immunity. PMID:26876974

  7. In vitro and in vivo characterization of glycoprotein C-deleted infectious laryngotracheitis virus.

    PubMed

    Pavlova, Sophia P; Veits, Jutta; Blohm, Ulrike; Maresch, Christina; Mettenleiter, Thomas C; Fuchs, Walter

    2010-04-01

    Infectious laryngotracheitis is an important respiratory disease of chickens that is caused by an alphaherpesvirus [infectious laryngotracheitis virus (ILTV); Gallid herpesvirus 1]. As herpesvirus envelope glycoproteins are main targets of the humoral host immune response, they are of particular interest for development of vaccines, as well as of diagnostic tools. The conserved, N-glycosylated envelope protein gC has been identified as a major surface antigen of ILTV. To study the function of gC, we now isolated a gC-deleted ILTV recombinant as well as a gC rescuant after co-transfection of permissive chicken cells with virion DNA and transfer plasmids containing engineered subgenomic fragments. Like other alphaherpesvirus homologues, ILTV gC proved to be non-essential for replication. ILTV-DeltagC exhibited delayed penetration kinetics and slightly reduced plaque sizes in cultured chicken cells, whereas virus titres were not reduced significantly compared with wild-type or gC-rescued virus. In vivo studies revealed that ILTV-DeltagC is attenuated in chickens. However, infection with high doses of ILTV-DeltagC was still fatal for approximately 20 % of the animals, whereas wild-type or gC-rescued ILTV led to 50 % mortality. Interestingly, innate and specific immune responses against ILTV-DeltagC were not reduced but enhanced, and surviving chickens were protected completely against challenge infection. Furthermore, ILTV-DeltagC might serve as a basis for marker vaccines permitting differentiation between vaccinated and field-virus-infected animals, as gC-specific antibodies could be detected easily in sera of animals infected with wild-type ILTV. PMID:19940061

  8. A Recombinant Rift Valley Fever Virus Glycoprotein Subunit Vaccine Confers Full Protection against Rift Valley Fever Challenge in Sheep.

    PubMed

    Faburay, Bonto; Wilson, William C; Gaudreault, Natasha N; Davis, A Sally; Shivanna, Vinay; Bawa, Bhupinder; Sunwoo, Sun Young; Ma, Wenjun; Drolet, Barbara S; Morozov, Igor; McVey, D Scott; Richt, Juergen A

    2016-01-01

    Rift Valley fever virus (RVFV) is a mosquito-borne zoonotic pathogen causing disease outbreaks in Africa and the Arabian Peninsula. The virus has great potential for transboundary spread due to the presence of competent vectors in non-endemic areas. There is currently no fully licensed vaccine suitable for use in livestock or humans outside endemic areas. Here we report the evaluation of the efficacy of a recombinant subunit vaccine based on the RVFV Gn and Gc glycoproteins. In a previous study, the vaccine elicited strong virus neutralizing antibody responses in sheep and was DIVA (differentiating naturally infected from vaccinated animals) compatible. In the current efficacy study, a group of sheep (n = 5) was vaccinated subcutaneously with the glycoprotein-based subunit vaccine candidate and then subjected to heterologous challenge with the virulent Kenya-128B-15 RVFV strain. The vaccine elicited high virus neutralizing antibody titers and conferred complete protection in all vaccinated sheep, as evidenced by prevention of viremia, fever and absence of RVFV-associated histopathological lesions. We conclude that the subunit vaccine platform represents a promising strategy for the prevention and control of RVFV infections in susceptible hosts. PMID:27296136

  9. A Recombinant Rift Valley Fever Virus Glycoprotein Subunit Vaccine Confers Full Protection against Rift Valley Fever Challenge in Sheep

    PubMed Central

    Faburay, Bonto; Wilson, William C.; Gaudreault, Natasha N.; Davis, A. Sally; Shivanna, Vinay; Bawa, Bhupinder; Sunwoo, Sun Young; Ma, Wenjun; Drolet, Barbara S.; Morozov, Igor; McVey, D. Scott; Richt, Juergen A.

    2016-01-01

    Rift Valley fever virus (RVFV) is a mosquito-borne zoonotic pathogen causing disease outbreaks in Africa and the Arabian Peninsula. The virus has great potential for transboundary spread due to the presence of competent vectors in non-endemic areas. There is currently no fully licensed vaccine suitable for use in livestock or humans outside endemic areas. Here we report the evaluation of the efficacy of a recombinant subunit vaccine based on the RVFV Gn and Gc glycoproteins. In a previous study, the vaccine elicited strong virus neutralizing antibody responses in sheep and was DIVA (differentiating naturally infected from vaccinated animals) compatible. In the current efficacy study, a group of sheep (n = 5) was vaccinated subcutaneously with the glycoprotein-based subunit vaccine candidate and then subjected to heterologous challenge with the virulent Kenya-128B-15 RVFV strain. The vaccine elicited high virus neutralizing antibody titers and conferred complete protection in all vaccinated sheep, as evidenced by prevention of viremia, fever and absence of RVFV-associated histopathological lesions. We conclude that the subunit vaccine platform represents a promising strategy for the prevention and control of RVFV infections in susceptible hosts. PMID:27296136

  10. Structure of a trimeric variant of the Epstein-Barr virus glycoprotein B

    SciTech Connect

    Backovic, Marija; Longnecker, Richard; Jardetzky, Theodore S

    2009-03-16

    Epstein-Barr virus (EBV) is a herpesvirus that is associated with development of malignancies of lymphoid tissue. EBV infections are life-long and occur in >90% of the population. Herpesviruses enter host cells in a process that involves fusion of viral and cellular membranes. The fusion apparatus is comprised of envelope glycoprotein B (gB) and a heterodimeric complex made of glycoproteins H and L. Glycoprotein B is the most conserved envelope glycoprotein in human herpesviruses, and the structure of gB from Herpes simplex virus 1 (HSV-1) is available. Here, we report the crystal structure of the secreted EBV gB ectodomain, which forms 16-nm long spike-like trimers, structurally homologous to the postfusion trimers of the fusion protein G of vesicular stomatitis virus (VSV). Comparative structural analyses of EBV gB and VSV G, which has been solved in its pre and postfusion states, shed light on gB residues that may be involved in conformational changes and membrane fusion. Also, the EBV gB structure reveals that, despite the high sequence conservation of gB in herpesviruses, the relative orientations of individual domains, the surface charge distributions, and the structural details of EBV gB differ from the HSV-1 protein, indicating regions and residues that may have important roles in virus-specific entry.

  11. [Ebola virus disease].

    PubMed

    Karwowska, Kornelia

    2015-01-01

    Ebola virus disease is a zoonosis causing high mortality epidemics in both human and animal populations. The virus belongs to the Filoviride family. It is composed of a single-strand of RNA. Morbidity foci appear in sub-Saharan Africa. The most probable reservoir are fruit bats, which are local delicacy. The most common route of infection is via mucosa or damaged skin. The spread of disease is rapid due to dietary habits, funeral rites and the insufficient supply of disposable equipment in hospitals. The incubation period of the disease ranges from 2 to 21 days. The beginning is abrupt, dominated by influenza-like symptoms. The disease is staggering with the predominant multi-organ failure and shock. Present-day epidemic symptoms from digestive system in the form of vomiting and diarrhoea are dominant. Currently, the research on vaccine and experimental drug is in progress. The virus is damaged by standard disinfectants used in health care units. Epidemic, which broke out in February 2014, caused by the most dangerous type Zaire, is the greatest of the existing. Morbidity and mortality is underestimated due to numerous unreported cases. PMID:25763588

  12. Horizontal transmission dynamics of a glycoprotein G deficient candidate vaccine strain of infectious laryngotracheitis virus and the effect of vaccination on transmission of virulent virus.

    PubMed

    Devlin, Joanne M; Hartley, Carol A; Gilkerson, James R; Coppo, Mauricio J C; Vaz, Paola; Noormohammadi, Amir H; Wells, Ben; Rubite, Ambrosio; Dhand, Navneet K; Browning, Glenn F

    2011-08-01

    Infectious laryngotracheitis virus (ILTV) is an alphaherpesvirus that causes acute respiratory disease in chickens worldwide. The virus is horizontally transmitted and causes large outbreaks of disease. Recent studies have shown that a glycoprotein G deficient candidate vaccine strain of ILTV (ΔgG ILTV) is safe and protects birds from disease following challenge with virulent virus. This study examined the transmission dynamics of this candidate vaccine and of ILTV in field and experimental settings. The reproduction ratio (R₀, average number of secondary infectious cases from a typical infectious case) was calculated from the growth rate of disease epidemics in broiler flocks. Assuming a latent period of 2 days and an infectious period of 4 days R₀ was estimated to be 2.43 (95% CI 2.25-2.69). In experimental settings the transmission characteristics of ΔgG ILTV were similar to those of wildtype virus, and importantly ΔgG ILTV remained safe following one in vivo passage and subsequent infection via contact-exposure. There was minimal transmission of wildtype virus in vaccinated birds. The findings from this study further demonstrate the suitability of ΔgG ILTV for use as a live attenuated vaccine. Knowledge of the basic reproduction ratio of ILTV will be valuable for future studies that aim to improve disease control using vaccination programs. PMID:21689710

  13. Anterograde Glycoprotein-Dependent Transport of Newly Generated Rabies Virus in Dorsal Root Ganglion Neurons

    PubMed Central

    Bauer, Anja; Nolden, Tobias; Schröter, Josephine; Römer-Oberdörfer, Angela; Gluska, Shani; Perlson, Eran

    2014-01-01

    ABSTRACT Rabies virus (RABV) spread is widely accepted to occur only by retrograde axonal transport. However, examples of anterograde RABV spread in peripheral neurons such as dorsal root ganglion (DRG) neurons indicated a possible bidirectional transport by an uncharacterized mechanism. Here, we analyzed the axonal transport of fluorescence-labeled RABV in DRG neurons by live-cell microscopy. Both entry-related retrograde transport of RABV after infection at axon endings and postreplicative transport of newly formed virus were visualized in compartmentalized DRG neuron cultures. Whereas entry-related transport at 1.5 μm/s occurred only retrogradely, after 2 days of infection, multiple particles were observed in axons moving in both the anterograde and retrograde directions. The dynamics of postreplicative retrograde transport (1.6 μm/s) were similar to those of entry-related retrograde transport. In contrast, anterograde particle transport at 3.4 μm/s was faster, indicating active particle transport. Interestingly, RABV missing the glycoproteins did not move anterogradely within the axon. Thus, anterograde RABV particle transport depended on the RABV glycoprotein. Moreover, colocalization of green fluorescent protein (GFP)-labeled ribonucleoproteins (RNPs) and glycoprotein in distal axonal regions as well as cotransport of labeled RNPs with membrane-anchored mCherry reporter confirmed that either complete enveloped virus particles or vesicle associated RNPs were transported. Our data show that anterograde RABV movement in peripheral DRG neurons occurs by active motor protein-dependent transport. We propose two models for postreplicative long-distance transport in peripheral neurons: either transport of complete virus particles or cotransport of RNPs and G-containing vesicles through axons to release virus at distal sites of infected DRG neurons. IMPORTANCE Rabies virus retrograde axonal transport by dynein motors supports virus spread over long distances and

  14. Recombinant infectious bursal disease virus carrying hepatitis C virus epitopes.

    PubMed

    Upadhyay, Chitra; Ammayappan, Arun; Patel, Deendayal; Kovesdi, Imre; Vakharia, Vikram N

    2011-02-01

    The delivery of foreign epitopes by a replicating nonpathogenic avian infectious bursal disease virus (IBDV) was explored. The aim of the study was to identify regions in the IBDV genome that are amenable to the introduction of a sequence encoding a foreign peptide. By using a cDNA-based reverse genetics system, insertions or substitutions of sequences encoding epitope tags (FLAG, c-Myc, or hepatitis C virus epitopes) were engineered in the open reading frames of a nonstructural protein (VP5) and the capsid protein (VP2). Attempts were also made to generate recombinant IBDV that displayed foreign epitopes in the exposed loops (P(BC) and P(HI)) of the VP2 trimer. We successfully recovered recombinant IBDVs expressing c-Myc and two different virus-neutralizing epitopes of human hepatitis C virus (HCV) envelope glycoprotein E in the VP5 region. Western blot analyses with anti-c-Myc and anti-HCV antibodies provided positive identification of both the c-Myc and HCV epitopes that were fused to the N terminus of VP5. Genetic analysis showed that the recombinants carrying the c-Myc/HCV epitopes maintained the foreign gene sequences and were stable after several passages in Vero and 293T cells. This is the first report describing efficient expression of foreign peptides from a replication-competent IBDV and demonstrates the potential of this virus as a vector. PMID:21106739

  15. [Ebola virus disease: Update].

    PubMed

    de la Calle-Prieto, Fernando; Arsuaga-Vicente, Marta; Mora-Rillo, Marta; Arnalich-Fernandez, Francisco; Arribas, Jose Ramon

    2016-01-01

    The first known Ebola outbreak occurred in 1976. Since then, 24 limited outbreaks had been reported in Central Africa, but never affecting more than 425 persons. The current outbreak in Western Africa is the largest in history with 28,220 reported cases and 11,291 deaths. The magnitude of the epidemic has caused worldwide alarm. For the first time, evacuated patients were treated outside Africa, and secondary cases have occurred in Spain and the United States. Since the start of the current epidemic, our knowledge about the epidemiology, clinical picture, laboratory findings, and virology of Ebola virus disease has considerably expanded. For the first time, experimental treatment has been tried, and there have been spectacular advances in vaccine development. A review is presented of these advances in the knowledge of Ebola virus disease. PMID:26774254

  16. A Diverse Panel of Hepatitis C Virus Glycoproteins for Use in Vaccine Research Reveals Extremes of Monoclonal Antibody Neutralization Resistance

    PubMed Central

    Urbanowicz, Richard A.; McClure, C. Patrick; Brown, Richard J. P.; Tsoleridis, Theocharis; Persson, Mats A. A.; Krey, Thomas; Irving, William L.; Tarr, Alexander W.

    2015-01-01

    therapies. A safe and effective vaccine that generates both T cell responses and neutralizing antibodies is required to eradicate the disease. Regions within the HCV surface glycoproteins E1 and E2 are essential for virus entry and are targets for neutralizing antibodies. Screening of vaccine candidates requires suitable panels of glycoproteins that represent the breadth of neutralization resistance. Use of a standard reference panel for vaccine studies will ensure comparability of data sets, as has become routine for HIV-1. Here, we describe a large panel of patient-derived HCV glycoproteins with an assessment of their neutralization sensitivity to defined monoclonal antibodies, which has enabled us to predict their likely efficacy in the wider HCV-infected population. The panel could also be important for future selection of additional therapeutic antibodies and for vaccine design. PMID:26699643

  17. Structure of Respiratory Syncytial Virus Fusion Glycoprotein in the Postfusion Conformation Reveals Preservation of Neutralizing Epitopes

    SciTech Connect

    McLellan, Jason S.; Yang, Yongping; Graham, Barney S.; Kwong, Peter D.

    2011-09-16

    Respiratory syncytial virus (RSV) invades host cells via a type I fusion (F) glycoprotein that undergoes dramatic structural rearrangements during the fusion process. Neutralizing monoclonal antibodies, such as 101F, palivizumab, and motavizumab, target two major antigenic sites on the RSV F glycoprotein. The structures of these sites as peptide complexes with motavizumab and 101F have been previously determined, but a structure for the trimeric RSV F glycoprotein ectodomain has remained elusive. To address this issue, we undertook structural and biophysical studies on stable ectodomain constructs. Here, we present the 2.8-{angstrom} crystal structure of the trimeric RSV F ectodomain in its postfusion conformation. The structure revealed that the 101F and motavizumab epitopes are present in the postfusion state and that their conformations are similar to those observed in the antibody-bound peptide structures. Both antibodies bound the postfusion F glycoprotein with high affinity in surface plasmon resonance experiments. Modeling of the antibodies bound to the F glycoprotein predicts that the 101F epitope is larger than the linear peptide and restricted to a single protomer in the trimer, whereas motavizumab likely contacts residues on two protomers, indicating a quaternary epitope. Mechanistically, these results suggest that 101F and motavizumab can bind to multiple conformations of the fusion glycoprotein and can neutralize late in the entry process. The structural preservation of neutralizing epitopes in the postfusion state suggests that this conformation can elicit neutralizing antibodies and serve as a useful vaccine antigen.

  18. Requirements for Cell Rounding and Surface Protein Down-Regulation by Ebola Virus Glycoprotein

    PubMed Central

    Francica, Joseph R.; Matukonis, Meghan K.; Bates, Paul

    2009-01-01

    Ebola virus causes an acute hemorrhagic fever that is associated with high morbidity and mortality. The viral glycoprotein is thought to contribute to pathogenesis, though precise mechanisms are unknown. Cellular pathogenesis can be modeled in vitro by expression of the Ebola viral glycoprotein (GP) in cells, which causes dramatic morphological changes, including cell rounding and surface protein down-regulation. These effects are known to be dependent on the presence of a highly glycosylated region of the glycoprotein, the mucin domain. Here we show that the mucin domain from the highly pathogenic Zaire subtype of Ebola virus is sufficient to cause characteristic cytopathology when expressed in the context of a foreign glycoprotein. Similarly to full length Ebola GP, expression of the mucin domain causes rounding, detachment from the extracellular matrix, and the down-regulation of cell surface levels of β1 integrin and major histocompatibility complex class 1. These effects were not seen when the mucin domain was expressed in the context of a glycophosphatidylinositol-anchored isoform of the foreign glycoprotein. In contrast to earlier analysis of full length Ebola glycoproteins, chimeras carrying the mucin domains from the Zaire and Reston strains appear to cause similar levels of down-modulation and cell detachment. Cytopathology associated with Ebola glycoprotein expression does not occur when GP expression is restricted to the endoplasmic reticulum. In contrast to a previously published report, our results demonstrate that GP-induced surface protein down-regulation is not mediated through a dynamin-dependent pathway. Overall, these results support a model in which the mucin domain of Ebola GP acts at the cell surface to induce protein down modulation and cytopathic effects. PMID:19013626

  19. Glycosylation of dengue virus glycoproteins and their interactions with carbohydrate receptors: possible targets for antiviral therapy.

    PubMed

    Idris, Fakhriedzwan; Muharram, Siti Hanna; Diah, Suwarni

    2016-07-01

    Dengue virus, an RNA virus belonging to the genus Flavivirus, affects 50 million individuals annually, and approximately 500,000-1,000,000 of these infections lead to dengue hemorrhagic fever or dengue shock syndrome. With no licensed vaccine or specific antiviral treatments available to prevent dengue infection, dengue is considered a major public health problem in subtropical and tropical regions. The virus, like other enveloped viruses, uses the host's cellular enzymes to synthesize its structural (C, E, and prM/M) and nonstructural proteins (NS1-5) and, subsequently, to glycosylate these proteins to produce complete and functional glycoproteins. The structural glycoproteins, specifically the E protein, are known to interact with the host's carbohydrate receptors through the viral proteins' N-glycosylation sites and thus mediate the viral invasion of cells. This review focuses on the involvement of dengue glycoproteins in the course of infection and the virus' exploitation of the host's glycans, especially the interactions between host receptors and carbohydrate moieties. We also discuss the recent developments in antiviral therapies that target these processes and interactions, focusing specifically on the use of carbohydrate-binding agents derived from plants, commonly known as lectins, to inhibit the progression of infection. PMID:27068162

  20. Delayed Infection after Immunization with a Peptide from the Transmembrane Glycoprotein of the Feline Immunodeficiency Virus

    PubMed Central

    Richardson, J.; Moraillon, A.; Crespeau, F.; Baud, S.; Sonigo, P.; Pancino, G.

    1998-01-01

    Recent advances in the quantitative assessment of viral burden, by permitting the extension of criteria applied to assess the efficacy of vaccines from all-or-none protection to diminution of the viral burden, may allow the identification of original immunogens of value in combined vaccines. Peptides corresponding to three domains of the envelope glycoproteins of feline immunodeficiency virus that are recognized during natural infection were used to immunize cats. After challenge with a primary isolate of feline immunodeficiency virus, the development of acute infection was monitored by quantitative assessment of the viral burden in plasma and tissues by competitive reverse transcription-PCR, by measurement of the humoral response developed to viral components, and by lymphocyte subset analysis. Whereas immunization with two peptides derived from the surface glycoprotein had no effect on the early course of infection, immunization with a peptide derived from the transmembrane glycoprotein delayed infection, as reflected by a diminished viral burden in the early phase of primary infection and delayed seroconversion. This peptide, located in the membrane-proximal region of the extracellular domain, has homology to an epitope of human immunodeficiency virus type 1 recognized by a broadly neutralizing monoclonal antibody. These results suggest that lentivirus transmembrane glycoproteins share a determinant in the juxtamembrane ectodomain which could be of importance in the design of vaccines against AIDS. PMID:9499101

  1. A simple, inexpensive, robust and sensitive dot-blot assay for equal detection of the nonstructural-1 glycoprotein of all dengue virus serotypes

    PubMed Central

    2013-01-01

    Background Detection of dengue virus (DENV) soluble/excreted (s/e) form of the nonstructural-1 (NS1) glycoprotein in patient acute-phase sera is ideal for diagnosis. The commercially-available detection assays are, however, too expensive for routine use and have low specificity, particularly for the s/e NS1 glycoprotein of DENV-2 and DENV-4, which are important causes of lethal human disease worldwide. Methods Mouse monoclonal antibodies (MAbs) were generated and screened against s/e NS1 glycoprotein purified from each DENV serotype to obtain those that reacted equally with each serotype, but not with yellow fever virus (YFV) s/e NS1 glycoprotein or human serum proteins. One MAb, MAb 2C4.6, was further tested against these DENV glycoproteins in human sera using simple, peroxidase-labelled secondary antibody/substrate-developed dot-blot assays. Results Optimal quenching of endogenous human serum peroxidases was attained using 3% H2O2 in H20 for 5 min. MAb 2C4.6 showed an acceptable detection sensitivity of < 32 ng/ml for the s/e NS1 glycoprotein of each DENV serotype but did not cross-react with the YFV s/e NS1 glycoprotein or human serum proteins. By contrast, the LX1 epitope-specific MAb, 3D1.4, showed similar detection sensitivity against only the DENV-1 NS1 glycoprotein, consistent with results from commercial DENV s/e NS1 glycoprotein detection assays. DENV s/e NS1 glycoproteins were stable in human sera after drying on the nitrocellulose membranes and storage for one month at ambient temperature (28°C) before being processed. The total assay time was reduced to 3 h without any loss of detection sensitivity. This dot-blot format was ideal for the circulating immune complex disruption step, which is required for increased DENV s/e NS1 glycoprotein detection. Conclusions This is the first study to determine the detection sensitivity of MAbs against known concentrations of s/e NS1 glycoprotein from each DENV serotype. The preparation of patient serum samples for

  2. Directed Selection of Recombinant Human Monoclonal Antibodies to Herpes Simplex Virus Glycoproteins from Phage Display Libraries

    NASA Astrophysics Data System (ADS)

    Sanna, Pietro Paolo; Williamson, R. Anthony; de Logu, Alessandro; Bloom, Floyd E.; Burton, Dennis R.

    1995-07-01

    Human monoclonal antibodies have considerable potential in the prophylaxis and treatment of viral disease. However, only a few such antibodies suitable for clinical use have been produced to date. We have previously shown that large panels of human recombinant monoclonal antibodies against a plethora of infectious agents, including herpes simplex virus types 1 and 2, can be established from phage display libraries. Here we demonstrate that facile cloning of recombinant Fab fragments against specific viral proteins in their native conformation can be accomplished by panning phage display libraries against viral glycoproteins "captured" from infected cell extracts by specific monoclonal antibodies immobilized on ELISA plates. We have tested this strategy by isolating six neutralizing recombinant antibodies specific for herpes simplex glycoprotein gD or gB, some of which are against conformationally sensitive epitopes. By using defined monoclonal antibodies for the antigen-capture step, this method can be used for the isolation of antibodies to specific regions and epitopes within the target viral protein. For instance, monoclonal antibodies to a nonneutralizing epitope can be used in the capture step to clone antibodies to neutralizing epitopes, or antibodies to a neutralizing epitope can be used to clone antibodies to a different neutralizing epitope. Furthermore, by using capturing antibodies to more immunodominant epitopes, one can direct the cloning to less immunogenic ones. This method should be of value in generating antibodies to be used both in the prophylaxis and treatment of viral infections and in the characterization of the mechanisms of antibody protective actions at the molecular level.

  3. Binding of a neutralizing antibody to dengue virus alters the arrangement of surface glycoproteins

    SciTech Connect

    Lok, Shee-Mei; Kostyuchenko, Victor; Nybakken, Grant E.; Holdaway, Heather A.; Battisti, Anthony J.; Sukupolvi-Petty, Soila; Sedlak, Dagmar; Fremont, Daved H.; Chipman, Paul R.; Roehrig, John T.; Diamond, Michael S.; Kuhn, Richard J.; Rossmann, Michael G.

    2008-04-02

    The monoclonal antibody 1A1D-2 has been shown to strongly neutralize dengue virus serotypes 1, 2 and 3, primarily by inhibiting attachment to host cells. A crystal structure of its antigen binding fragment (Fab) complexed with domain III of the viral envelope glycoprotein, E, showed that the epitope would be partially occluded in the known structure of the mature dengue virus. Nevertheless, antibody could bind to the virus at 37 degrees C, suggesting that the virus is in dynamic motion making hidden epitopes briefly available. A cryo-electron microscope image reconstruction of the virus:Fab complex showed large changes in the organization of the E protein that exposed the epitopes on two of the three E molecules in each of the 60 icosahedral asymmetric units of the virus. The changes in the structure of the viral surface are presumably responsible for inhibiting attachment to cells.

  4. Genotyping of Korean isolates of infectious hematopoietic necrosis virus (IHNV) based on the glycoprotein gene

    USGS Publications Warehouse

    Kim, W.-S.; Oh, M.-J.; Nishizawa, T.; Park, J.-W.; Kurath, G.; Yoshimizu, M.

    2007-01-01

    Glycoprotein (G) gene nucleotide sequences of four Korean isolates of infectious hematopoietic necrosis virus (IHNV) were analyzed to evaluate their genetic relatedness to worldwide isolates. All Korean isolates were closely related to Japanese isolates of genogroup JRt rather than to those of North American and European genogroups. It is believed that Korean IHNV has been most likely introduced from Japan to Korea by the movement of contaminated fish eggs. Among the Korean isolates, phylogenetically distinct virus types were obtained from sites north and south of a large mountain range, suggesting the possibility of more than one introduction of virus from Japan. ?? 2007 Springer-Verlag.

  5. A novel rabies vaccine based on a recombinant parainfluenza virus 5 expressing rabies virus glycoprotein.

    PubMed

    Chen, Zhenhai; Zhou, Ming; Gao, Xiudan; Zhang, Guoqing; Ren, Guiping; Gnanadurai, Clement W; Fu, Zhen F; He, Biao

    2013-03-01

    Untreated rabies virus (RABV) infection leads to death. Vaccine and postexposure treatment have been effective in preventing RABV infection. However, due to cost, rabies vaccination and treatment have not been widely used in developing countries. There are 55,000 human death caused by rabies annually. An efficacious and cost-effective rabies vaccine is needed. Parainfluenza virus 5 (PIV5) is thought to contribute to kennel cough, and kennel cough vaccines containing live PIV5 have been used in dogs for many years. In this work, a PIV5-vectored rabies vaccine was tested in mice. A recombinant PIV5 encoding RABV glycoprotein (G) (rPIV5-RV-G) was administered to mice via intranasal (i.n.), intramuscular (i.m.), and oral inoculation. The vaccinated mice were challenged with a 50% lethal challenge dose (LD(50)) of RABV challenge virus standard 24 (CVS-24) intracerebrally. A single dose of 10(6) PFU of rPIV5-RV-G was sufficient for 100% protection when administered via the i.n. route. The mice vaccinated with a single dose of 10(8) PFU of rPIV5-RV-G via the i.m. route showed very robust protection (90% to 100%). Intriguingly, the mice vaccinated orally with a single dose of 10(8) PFU of rPIV5-RV-G showed a 50% survival rate, which is comparable to the 60% survival rate among mice inoculated with an attenuated rabies vaccine strain, recombinant LBNSE. This is first report of an orally effective rabies vaccine candidate in animals based on PIV5 as a vector. These results indicate that rPIV5-RV-G is an excellent candidate for a new generation of recombinant rabies vaccine for humans and animals and PIV5 is a potential vector for oral vaccines. PMID:23269806

  6. Utilization of C-C chemokine receptor 5 by the envelope glycoproteins of a pathogenic simian immunodeficiency virus, SIVmac239.

    PubMed Central

    Marcon, L; Choe, H; Martin, K A; Farzan, M; Ponath, P D; Wu, L; Newman, W; Gerard, N; Gerard, C; Sodroski, J

    1997-01-01

    We examined chemokine receptors for the ability to facilitate the infection of CD4-expressing cells by viruses containing the envelope glycoproteins of a pathogenic simian immunodeficiency virus, SIVmac239. Expression of either human or simian C-C chemokine receptor CCR5 allowed the SIVmac239 envelope glycoproteins to mediate virus entry and cell-to-cell fusion. Thus, distantly related immunodeficiency viruses such as SIV and the primary human immunodeficiency virus type 1 isolates can utilize CCR5 as an entry cofactor. PMID:9032394

  7. Blueberry (Vaccinium corymbosum)-Virus Diseases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    At least six viruses have been found in highbush blueberry plantings in the Pacific Northwest: Blueberry mosaic virus, Blueberry red ringspot virus, Blueberry scorch virus, Blueberry shock virus, Tobacco ringspot virus, and Tomato ringspot virus. Six other virus and virus-like diseases of highbush b...

  8. Nucleotide sequence of the bovine parainfluenza 3 virus genome: the genes of the F and HN glycoproteins.

    PubMed Central

    Suzu, S; Sakai, Y; Shioda, T; Shibuta, H

    1987-01-01

    By analysing complementary DNA clones constructed from genomic RNA of bovine parainfluenza 3 virus (BPIV3), we determined the nucleotide sequence of the region containing the entire F and HN genes. Their deduced amino acid sequences showed about 80% homologies with those of human parainfluenza 3 virus (HPIV3), about 45% with those of Sendai virus, and about 20% with those of SV5 and Newcastle disease virus (NDV), indicating, together with the results described in the preceding paper on the NP, P, C and M proteins of BPIV3, that BPIV3, HPIV3 and Sendai virus constitute a paramyxovirus subgroup, and that BPIV3 and HPIV3 are very closely related. The F and HN proteins of all these viruses, including SV5 and NDV, however, were shown to have protein-specific structures as well as short but well-conserved amino acid sequences, suggesting that these structures and sequences are related to the activities of these glycoproteins. Images PMID:3031615

  9. Mutations in the putative HR-C region of the measles virus F2 glycoprotein modulate syncytium formation.

    PubMed

    Plemper, Richard K; Compans, Richard W

    2003-04-01

    The fusion (F) glycoproteins of measles virus strains Edmonston (MV-Edm) and wtF (MV-wtF) confer distinct cytopathic effects and strengths of hemagglutinin (H) interaction on a recombinant MV-Edm virus. They differ in just two amino acids, V94 and V101 in F-Edm versus M94 and F101 in F-wtF, both of which lie in the relatively uncharacterized F(2) domain. By comparing the sequence of MV F with those of the parainfluenza virus SV5 and Newcastle disease virus (NDV) F proteins, the structures of which are known, we show that MV F(2) also possesses a potential heptad repeat (HR) C domain. In NDV, the N-terminal half of HR-C interacts with HR-A in F(1) while the C-terminal half is induced to kink outward by a central proline residue. We found that this proline is part of an LXP motif conserved in all three viruses. Folding and transport of MV F require this motif to be intact and also require covalent interaction of cysteine residues that probably support the potential HR-A-HR-C interaction. Amino acids 94 and 101, both located in "d" positions of the HR-C helical wheel, lie in the potentially outwardly kinked region. We demonstrate that their effect on MV fusogenicity and glycoprotein interaction is mediated solely by amino acid 94. Substitutions at position 94 with polar or charged amino acids are tolerated poorly or not at all, while changes to smaller and more hydrophilic amino acids are tolerated in both transiently expressed F protein and recombinant virus. MV F V94A and MV F V94G viruses induce extensive syncytium formation and are relatively, or almost completely, resistant to a known inhibitor of MV glycoprotein-induced fusion. We propose that the conformational changes in MV F protein required to expose the fusion peptide involve the C-terminal half of the HR-C helix, specifically amino acid 94. PMID:12634376

  10. Disulfide bond structure of glycoprotein D of herpes simplex virus types 1 and 2.

    PubMed Central

    Long, D; Wilcox, W C; Abrams, W R; Cohen, G H; Eisenberg, R J

    1992-01-01

    Glycoprotein D (gD) is a structural component of the herpes simplex virus envelope which is essential for virus penetration. The function of this protein is highly dependent on its structure, and its structure is dependent on maintenance of three intact disulfide bonds. gD contains six cysteines in its ectodomain whose spacing is conserved among all its homologs in other alphaherpesviruses as well as Marek's disease virus. For other proteins, conservation of cysteine spacing correlates with conservation of disulfide bond structure. We have now solved the disulfide bond structure of gD-1 and gD-2 of herpes simplex virus types 1 and 2, respectively. Two approaches were used. First, we constructed 15 double-Cys mutants of gD-1, representing all possible disulfide pairs. In each case, codons for cysteines were changed to serine. We reasoned that if two cysteines normally form a disulfide bond, double mutations which eliminate one proper bond should be less harmful to gD structure than double mutations which eliminate two disulfide bonds. The mutated genes were cloned into a eucaryotic expression vector, and the proteins were expressed in transiently transfected cells. Three double mutations, Cys-1,5, Cys-2,6, and Cys-3,4 permitted gD-1 folding, processing, transport to the cell surface, and function in virus infection, whereas 12 other double mutations each produced a malfolded and nonfunctional protein. Thus, the three functional double-Cys mutants may represent the actual partners in disulfide bond linkages. The second approach was to define the actual disulfide bond structure of gD by biochemical means. Purified native gD-2 was cleaved by CNBr and proteases, and the peptides were separated by high-performance liquid chromatography. Disulfide-linked peptides were subjected to N-terminal amino acid sequencing. The results show that cysteine 1 (amino acid [aa] 66) is bonded to cysteine 5 (aa 189), cysteine 2 (aa 106) is bonded to cysteine 6 (aa 202), and cysteine 3 (aa

  11. Measles virus-induced immune suppression in the cotton rat (Sigmodon hispidus) model depends on viral glycoproteins.

    PubMed Central

    Niewiesk, S; Eisenhuth, I; Fooks, A; Clegg, J C; Schnorr, J J; Schneider-Schaulies, S; ter Meulen, V

    1997-01-01

    Immune suppression during measles accounts for most of the morbidity and mortality associated with the virus infection. Experimental study of this phenomenon has been hampered by the lack of a suitable animal model. We have used the cotton rat to demonstrate that mitogen-induced proliferation of spleen cells from measles virus-infected animals is impaired. Proliferation inhibition is seen in all lymphocyte subsets and is not dependent on viral replication. Cells which express the viral glycoproteins (hemagglutinin and fusion protein) transiently by transfection induce proliferation inhibition after intraperitoneal inoculation, whereas application of a recombinant measles virus in which measles virus glycoproteins are replaced with the vesicular stomatitis virus G protein does not have an antiproliferative effect. Therefore, in vivo expression of measles virus glycoproteins is sufficient and necessary to induce inhibition of lymphocyte proliferation. PMID:9311794

  12. Marek's disease virus morphogenesis.

    PubMed

    Denesvre, Caroline

    2013-06-01

    Marek's disease virus (MDV) is a highly contagious virus that induces T-lymphoma in chicken. This viral infection still circulates in poultry flocks despite the use of vaccines. With the emergence of new virulent strains in the field over time, MDV remains a serious threat to the poultry industry. More than 40 yr after MDV identification as a herpesvirus, the visualization and purification of fully enveloped infectious particles remain a challenge for biologists. The various strategies used to detect such hidden particles by electron microscopy are reviewed herein. It is now generally accepted that the production of cell-free virions only occurs in the feather follicle epithelium and is associated with viral, cellular, or both molecular determinants expressed in this tissue. This tissue is considered the only source of efficient virus shedding into the environment and therefore the origin of successful transmission in birds. In other avian tissues or permissive cell cultures, MDV replication only leads to a very low number of intracellular enveloped virions. In the absence of detectable extracellular enveloped virions in cell culture, the nature of the transmitted infectious material and its mechanisms of spread from cell to cell remain to be deciphered. An attempt is made to bring together the current knowledge on MDV morphogenesis and spread, and new approaches that could help understand MDV morphogenesis are discussed. PMID:23901745

  13. Rabies-virus-glycoprotein-pseudotyped recombinant baculovirus vaccine confers complete protection against lethal rabies virus challenge in a mouse model.

    PubMed

    Wu, Qunfeng; Yu, Fulai; Xu, Jinfang; Li, Yang; Chen, Huanchun; Xiao, Shaobo; Fu, Zhen F; Fang, Liurong

    2014-06-25

    Rabies virus has been an ongoing threat to humans and animals. Here, we developed a new strategy to generate a rabies virus vaccine based on a pseudotyped baculovirus. The recombinant baculovirus (BV-RVG/RVG) was pseudotyped with the rabies virus glycoprotein (RVG) and also simultaneously expressed another RVG under the control of the immediate early CMV promoter. In vitro, this RVG-pseudotyped baculovirus vector induced syncytium formation in insect cells and displayed more efficient gene delivery into mammalian cells. Mice immunized with BV-RVG/RVG developed higher levels of virus-neutralizing antibodies, and conferred 100% protection against rabies viral challenge. These data indicate that the RVG-pseudotyped baculovirus BV-RVG/RVG can be used as an alternative strategy to develop a safe and efficacious vaccine against the rabies virus. PMID:24793501

  14. Chimeric Bovine Respiratory Syncytial Virus with Attachment and Fusion Glycoproteins Replaced by Bovine Parainfluenza Virus Type 3 Hemagglutinin-Neuraminidase and Fusion Proteins

    PubMed Central

    Stope, Matthias B.; Karger, Axel; Schmidt, Ulrike; Buchholz, Ursula J.

    2001-01-01

    Chimeric bovine respiratory syncytial viruses (BRSV) expressing glycoproteins of bovine parainfluenza virus type 3 (BPIV-3) instead of BRSV glycoproteins were generated from cDNA. In the BRSV antigenome cDNA, the open reading frames of the major BRSV glycoproteins, attachment protein G and fusion protein F, were replaced individually or together by those of the BPIV-3 hemagglutinin-neuraminidase (HN) and/or fusion (F) glycoproteins. Recombinant virus could not be recovered from cDNA when the BRSV F open reading frame was replaced by the BPIV-3 F open reading frame. However, cDNA recovery of the chimeric virus rBRSV-HNF, with both glycoproteins replaced simultaneously, and of the chimeric virus rBRSV-HN, with the BRSV G protein replaced by BPIV-3 HN, was successful. The replication rates of both chimeras were similar to that of standard rBRSV. Moreover, rBRSV-HNF was neutralized by antibodies specific for BPIV-3, but not by antibodies specific to BRSV, demonstrating that the BRSV glycoproteins can be functionally replaced by BPIV-3 glycoproteins. In contrast, rBRSV-HN was neutralized by BRSV-specific antisera, but not by BPIV-3 specific sera, showing that infection of rBRSV-HN is mediated by BRSV F. Hemadsorption of cells infected with rBRSV-HNF and rBRSV-HN proved that BPIV-3 HN protein expressed by rBRSV is functional. Colocalization of the BPIV-3 glycoproteins with BRSV M protein was demonstrated by confocal laser scan microscopy. Moreover, protein analysis revealed that the BPIV-3 glycoproteins were present in chimeric virions. Taken together, these data indicate that the heterologous glycoproteins were not only expressed but were incorporated into the envelope of recombinant BRSV. Thus, the envelope glycoproteins derived from a member of the Respirovirus genus can together functionally replace their homologs in a Pneumovirus background. PMID:11533200

  15. Protective effects of recombinant glycoprotein D based prime boost approach against duck enteritis virus in mice model.

    PubMed

    Aravind, S; Kamble, Nitin Machindra; Gaikwad, Satish S; Shukla, Sanjeev Kumar; Saravanan, R; Dey, Sohini; Madhan Mohan, C

    2015-11-01

    Duck virus enteritis, also known as duck plague, is an acute herpes viral infection of ducks caused by duck enteritis virus (DEV). The method of repeated immunization with a live attenuated vaccine has been used for the prevention and control of duck enteritis virus (DEV). However, the incidence of the disease in vaccinated flocks and latency reactivation are the major constraints in the present vaccination programme. The immunogenicity and protective efficacy afforded by intramuscular inoculation of plasmid DNA encoding DEV glycoprotein D (pCDNA-gD) followed by DEV gD expressed in Saccharomyces cerevisia (rgD) was assessed in a murine model. Compared with mice inoculated with DNA (pCDNA-gD) or protein (rgD) only, mice inoculated with the combination of gD DNA and protein had enhanced ELISA antibody titers to DEV and had accelerated clearance of virus following challenge infection. Furthermore, the highest levels of lymphocyte proliferation response, IL-4, IL-12 and IFN-γ production were induced following priming with the DNA vaccine and boosting with the rgD protein. For instance, the specially designed recombinant DEV vector vaccine would be the best choice to use in ducks. It offers an excellent solution to the low vaccination coverage rate in ducks. We expect that the application of this novel vaccine in the near future will greatly decrease the virus load in the environment and reduce outbreaks of DEV in ducks. PMID:26188265

  16. Epitope dampening monotypic measles virus hemagglutinin glycoprotein results in resistance to cocktail of monoclonal antibodies.

    PubMed

    Lech, Patrycja J; Tobin, Gregory J; Bushnell, Ruth; Gutschenritter, Emily; Pham, Linh D; Nace, Rebecca; Verhoeyen, Els; Cosset, François-Loïc; Muller, Claude P; Russell, Stephen J; Nara, Peter L

    2013-01-01

    The measles virus (MV) is serologically monotypic. Life-long immunity is conferred by a single attack of measles or following vaccination with the MV vaccine. This is contrary to viruses such as influenza, which readily develop resistance to the immune system and recur. A better understanding of factors that restrain MV to one serotype may allow us to predict if MV will remain monotypic in the future and influence the design of novel MV vaccines and therapeutics. MV hemagglutinin (H) glycoprotein, binds to cellular receptors and subsequently triggers the fusion (F) glycoprotein to fuse the virus into the cell. H is also the major target for neutralizing antibodies. To explore if MV remains monotypic due to a lack of plasticity of the H glycoprotein, we used the technology of Immune Dampening to generate viruses with rationally designed N-linked glycosylation sites and mutations in different epitopes and screened for viruses that escaped monoclonal antibodies (mAbs). We then combined rationally designed mutations with naturally selected mutations to generate a virus resistant to a cocktail of neutralizing mAbs targeting four different epitopes simultaneously. Two epitopes were protected by engineered N-linked glycosylations and two epitopes acquired escape mutations via two consecutive rounds of artificial selection in the presence of mAbs. Three of these epitopes were targeted by mAbs known to interfere with receptor binding. Results demonstrate that, within the epitopes analyzed, H can tolerate mutations in different residues and additional N-linked glycosylations to escape mAbs. Understanding the degree of change that H can tolerate is important as we follow its evolution in a host whose immunity is vaccine induced by genotype A strains instead of multiple genetically distinct wild-type MVs. PMID:23300970

  17. Dissection of the Antibody Response against Herpes Simplex Virus Glycoproteins in Naturally Infected Humans

    PubMed Central

    Huang, Zhen-Yu; Whitbeck, J. Charles; Ponce de Leon, Manuel; Lou, Huan; Wald, Anna; Krummenacher, Claude; Eisenberg, Roselyn J.; Cohen, Gary H.

    2014-01-01

    ABSTRACT Relatively little is known about the extent of the polyclonal antibody (PAb) repertoire elicited by herpes simplex virus (HSV) glycoproteins during natural infection and how these antibodies affect virus neutralization. Here, we examined IgGs from 10 HSV-seropositive individuals originally classified as high or low virus shedders. All PAbs neutralized virus to various extents. We determined which HSV entry glycoproteins these PAbs were directed against: glycoproteins gB, gD, and gC were recognized by all sera, but fewer sera reacted against gH/gL. We previously characterized multiple mouse monoclonal antibodies (MAbs) and mapped those with high neutralizing activity to the crystal structures of gD, gB, and gH/gL. We used a biosensor competition assay to determine whether there were corresponding human antibodies to those epitopes. All 10 samples had neutralizing IgGs to gD epitopes, but there were variations in which epitopes were seen in individual samples. Surprisingly, only three samples contained neutralizing IgGs to gB epitopes. To further dissect the nature of these IgGs, we developed a method to select out gD- and gB-specific IgGs from four representative sera via affinity chromatography, allowing us to determine the contribution of antibodies against each glycoprotein to the overall neutralization capacity of the serum. In two cases, gD and gB accounted for all of the neutralizing activity against HSV-2, with a modest amount of HSV-1 neutralization directed against gC. In the other two samples, the dominant response was to gD. IMPORTANCE Antibodies targeting functional epitopes on HSV entry glycoproteins mediate HSV neutralization. Virus-neutralizing epitopes have been defined and characterized using murine monoclonal antibodies. However, it is largely unknown whether these same epitopes are targeted by the humoral response to HSV infection in humans. We have shown that during natural infection, virus-neutralizing antibodies are principally

  18. P-Glycoprotein and Drug Resistance in Systemic Autoimmune Diseases

    PubMed Central

    Picchianti-Diamanti, Andrea; Rosado, Maria Manuela; Scarsella, Marco; Laganà, Bruno; D’Amelio, Raffaele

    2014-01-01

    Autoimmune diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and psoriatic arthritis (PsA) are chronic inflammatory disorders of unknown etiology characterized by a wide range of abnormalities of the immune system that may compromise the function of several organs, such as kidney, heart, joints, brain and skin. Corticosteroids (CCS), synthetic and biologic immunosuppressive agents have demonstrated the capacity to improve the course of autoimmune diseases. However, a significant number of patients do not respond or develop resistance to these therapies over time. P-glycoprotein (P-gp) is a transmembrane protein that pumps several drugs out of the cell, including CCS and immunosuppressants; thus, its over-expression or hyper-function has been proposed as a possible mechanism of drug resistance in patients with autoimmune disorders. Recently, different authors have demonstrated that P-gp inhibitors, such as cyclosporine A (CsA) and its analogue Tacrolimus, are able to reduce P-gp expression and or function in SLE, RA and PsA patients. These observations suggest that P-gp antagonists could be adopted to revert drug resistance and improve disease outcome. The complex inter-relationship among drug resistance, P-gp expression and autoimmunity still remains elusive. PMID:24658440

  19. Mechanism of Binding to Ebola Virus Glycoprotein by the ZMapp, ZMAb, and MB-003 Cocktail Antibodies

    PubMed Central

    Davidson, Edgar; Bryan, Christopher; Fong, Rachel H.; Barnes, Trevor; Pfaff, Jennifer M.; Mabila, Manu; Rucker, Joseph B.

    2015-01-01

    ABSTRACT Cocktails of monoclonal antibodies (MAbs) that target the surface glycoprotein (GP) of Ebola virus (EBOV) are effective in nonhuman primate models and have been used under emergency compassionate-treatment protocols in human patients. However, the amino acids that form the detailed binding epitopes for the MAbs in the ZMapp, ZMAb, and the related MB-003 cocktails have yet to be identified. Other binding properties that define how each MAb functionally interacts with GP—such as affinity, epitope conservation, and epitope accessibility—also remain largely unknown. To help define how each MAb interacts with GP, here we used comprehensive alanine-scanning mutagenesis (shotgun mutagenesis), neutralization escape, and whole virion binding to define each MAb's specific epitope, epitope accessibility, epitope conservation, and apparent affinity. Each of the six therapeutic MAbs binds nonidentical epitopes in the GP base, glycan cap, or mucin-like domain. Their apparent affinity, epitope complementarity, and epitope accessibility helps explain why MAbs 4G7 and 13C6 are more protective than 2G4 and 1H3. The mucin-like domain MAbs 6D8 and 13F6 bind with the strongest apparent affinity, helping to explain their effectiveness in vivo despite their inability to neutralize virus. IMPORTANCE Ebola virus disease (EVD) can be caused by four different filovirus family members, including Ebola virus (EBOV), which infected 10 times more people in western Africa over the last year than all previous EVD outbreaks combined, with a number of cases distributed across the globe by travelers. Cocktails of inhibitory monoclonal antibodies (MAbs), such as ZMAb, MB-003, and in particular ZMapp, have demonstrated in animal models some of the most significant therapeutic potential for treating EVD, and in 2014, 15 patients were treated with ZMapp or ZMAb under compassionate-use protocols. Here, we have defined the epitope features for the most important therapeutic MAbs against EBOV

  20. A Single-Amino-Acid Polymorphism in Chikungunya Virus E2 Glycoprotein Influences Glycosaminoglycan Utilization

    PubMed Central

    Silva, Laurie A.; Khomandiak, Solomiia; Ashbrook, Alison W.; Weller, Romy; Heise, Mark T.; Morrison, Thomas E.

    2014-01-01

    ABSTRACT Chikungunya virus (CHIKV) is a reemerging arbovirus responsible for outbreaks of infection throughout Asia and Africa, causing an acute illness characterized by fever, rash, and polyarthralgia. Although CHIKV infects a broad range of host cells, little is known about how CHIKV binds and gains access to the target cell interior. In this study, we tested whether glycosaminoglycan (GAG) binding is required for efficient CHIKV replication using CHIKV vaccine strain 181/25 and clinical isolate SL15649. Preincubation of strain 181/25, but not SL15649, with soluble GAGs resulted in dose-dependent inhibition of infection. While parental Chinese hamster ovary (CHO) cells are permissive for both strains, neither strain efficiently bound to or infected mutant CHO cells devoid of GAG expression. Although GAGs appear to be required for efficient binding of both strains, they exhibit differential requirements for GAGs, as SL15649 readily infected cells that express excess chondroitin sulfate but that are devoid of heparan sulfate, whereas 181/25 did not. We generated a panel of 181/25 and SL15649 variants containing reciprocal amino acid substitutions at positions 82 and 318 in the E2 glycoprotein. Reciprocal exchange at residue 82 resulted in a phenotype switch; Gly82 results in efficient infection of mutant CHO cells but a decrease in heparin binding, whereas Arg82 results in reduced infectivity of mutant cells and an increase in heparin binding. These results suggest that E2 residue 82 is a primary determinant of GAG utilization, which likely mediates attenuation of vaccine strain 181/25. IMPORTANCE Chikungunya virus (CHIKV) infection causes a debilitating rheumatic disease that can persist for months to years, and yet there are no licensed vaccines or antiviral therapies. Like other alphaviruses, CHIKV displays broad tissue tropism, which is thought to be influenced by virus-receptor interactions. In this study, we determined that cell-surface glycosaminoglycans are

  1. Characterization of soluble glycoprotein D-mediated herpes simplex virus type 1 infection

    SciTech Connect

    Tsvitov, Marianna; Frampton, Arthur R.; Shah, Waris A.; Wendell, Steven K.; Ozuer, Ali; Kapacee, Zoher; Goins, William F.; Cohen, Justus B.; Glorioso, Joseph C. . E-mail: glorioso@pitt.edu

    2007-04-10

    Herpes simplex virus type 1 (HSV-1) entry into permissive cells involves attachment to cell-surface glycosaminoglycans (GAGs) and fusion of the virus envelope with the cell membrane triggered by the binding of glycoprotein D (gD) to cognate receptors. In this study, we characterized the observation that soluble forms of the gD ectodomain (sgD) can mediate entry of gD-deficient HSV-1. We examined the efficiency and receptor specificity of this activity and used sequential incubation protocols to determine the order and stability of the initial interactions required for entry. Surprisingly, virus binding to GAGs did not increase the efficiency of sgD-mediated entry and gD-deficient virus was capable of attaching to GAG-deficient cells in the absence of sgD. These observations suggested a novel binding interaction that may play a role in normal HSV infection.

  2. A recombinant Yellow Fever 17D vaccine expressing Lassa virus glycoproteins

    PubMed Central

    Bredenbeek, Peter J.; Molenkamp, Richard; Spaan, Willy J.M.; Deubel, Vincent; Marianneau, Phillippe; Salvato, Maria S.; Moshkoff, Dmitry; Zapata, Juan; Tikhonov, Ilia; Patterson, Jean; Carrion, Ricardo; Ticer, Anysha; Brasky, Kathleen; Lukashevich, Igor S.

    2006-01-01

    The Yellow Fever Vaccine 17D (YFV17D) has been used as a vector for the Lassa virus glycoprotein precursor (LASV-GPC) resulting in construction of YFV17D/LASV-GPC recombinant virus. The virus was replication-competent and processed the LASV-GPC in cell cultures. The recombinant replicated poorly in guinea pigs but still elicited specific antibodies against LASV and YFV17D antigens. A single subcutaneous injection of the recombinant vaccine protected strain 13 guinea pigs against fatal Lassa Fever. This study demonstrates the potential to develop an YFV17D-based bivalent vaccine against two viruses that are endemic in the same area of Africa. PMID:16412488

  3. A recombinant Yellow Fever 17D vaccine expressing Lassa virus glycoproteins.

    PubMed

    Bredenbeek, Peter J; Molenkamp, Richard; Spaan, Willy J M; Deubel, Vincent; Marianneau, Phillippe; Salvato, Maria S; Moshkoff, Dmitry; Zapata, Juan; Tikhonov, Ilia; Patterson, Jean; Carrion, Ricardo; Ticer, Anysha; Brasky, Kathleen; Lukashevich, Igor S

    2006-02-20

    The Yellow Fever Vaccine 17D (YFV17D) has been used as a vector for the Lassa virus glycoprotein precursor (LASV-GPC) resulting in construction of YFV17D/LASV-GPC recombinant virus. The virus was replication-competent and processed the LASV-GPC in cell cultures. The recombinant replicated poorly in guinea pigs but still elicited specific antibodies against LASV and YFV17D antigens. A single subcutaneous injection of the recombinant vaccine protected strain 13 guinea pigs against fatal Lassa Fever. This study demonstrates the potential to develop an YFV17D-based bivalent vaccine against two viruses that are endemic in the same area of Africa. PMID:16412488

  4. Fluorescent Protein-Tagged Sindbis Virus E2 Glycoprotein Allows Single Particle Analysis of Virus Budding from Live Cells

    PubMed Central

    Jose, Joyce; Tang, Jinghua; Taylor, Aaron B.; Baker, Timothy S.; Kuhn, Richard J.

    2015-01-01

    Sindbis virus (SINV) is an enveloped, mosquito-borne alphavirus. Here we generated and characterized a fluorescent protein-tagged (FP-tagged) SINV and found that the presence of the FP-tag (mCherry) affected glycoprotein transport to the plasma membrane whereas the specific infectivity of the virus was not affected. We examined the virions by transmission electron cryo-microscopy and determined the arrangement of the FP-tag on the surface of the virion. The fluorescent proteins are arranged icosahedrally on the virus surface in a stable manner that did not adversely affect receptor binding or fusion functions of E2 and E1, respectively. The delay in surface expression of the viral glycoproteins, as demonstrated by flow cytometry analysis, contributed to a 10-fold reduction in mCherry-E2 virus titer. There is a 1:1 ratio of mCherry to E2 incorporated into the virion, which leads to a strong fluorescence signal and thus facilitates single-particle tracking experiments. We used the FP-tagged virus for high-resolution live-cell imaging to study the spatial and temporal aspects of alphavirus assembly and budding from mammalian cells. These processes were further analyzed by thin section microscopy. The results demonstrate that SINV buds from the plasma membrane of infected cells and is dispersed into the surrounding media or spread to neighboring cells facilitated by its close association with filopodial extensions. PMID:26633461

  5. Ebola Virus Infections in Nonhuman Primates Are Temporally Influenced by Glycoprotein Poly-U Editing Site Populations in the Exposure Material.

    PubMed

    Trefry, John C; Wollen, Suzanne E; Nasar, Farooq; Shamblin, Joshua D; Kern, Steven J; Bearss, Jeremy J; Jefferson, Michelle A; Chance, Taylor B; Kugelman, Jeffery R; Ladner, Jason T; Honko, Anna N; Kobs, Dean J; Wending, Morgan Q S; Sabourin, Carol L; Pratt, William D; Palacios, Gustavo F; Pitt, M Louise M

    2015-12-01

    Recent experimentation with the variants of the Ebola virus that differ in the glycoprotein's poly-uridine site, which dictates the form of glycoprotein produced through a transcriptional stutter, has resulted in questions regarding the pathogenicity and lethality of the stocks used to develop products currently undergoing human clinical trials to combat the disease. In order to address these concerns and prevent the delay of these critical research programs, we designed an experiment that permitted us to intramuscularly challenge statistically significant numbers of naïve and vaccinated cynomolgus macaques with either a 7U or 8U variant of the Ebola virus, Kikwit isolate. In naïve animals, no difference in survivorship was observed; however, there was a significant delay in the disease course between the two groups. Significant differences were also observed in time-of-fever, serum chemistry, and hematology. In vaccinated animals, there was no statistical difference in survivorship between either challenge groups, with two succumbing in the 7U group compared to 1 in the 8U challenge group. In summary, survivorship was not affected, but the Ebola virus disease course in nonhuman primates is temporally influenced by glycoprotein poly-U editing site populations. PMID:26703716

  6. Nonreplicating viral vectors as potential vaccines: recombinant canarypox virus expressing measles virus fusion (F) and hemagglutinin (HA) glycoproteins.

    PubMed

    Taylor, J; Weinberg, R; Tartaglia, J; Richardson, C; Alkhatib, G; Briedis, D; Appel, M; Norton, E; Paoletti, E

    1992-03-01

    The development of canarypox virus (CPV) recombinants expressing the hemagglutinin (HA) and fusion (F) glycoproteins of measles virus (MV) is described. Inoculation of the CPV-MV recombinants into avian or nonavian tissue culture substrates led to the expression of authentic MVF and MVHA as determined by radioimmunoprecipitation and surface immunofluorescence. In contrast to avian-derived tissue culture, no productive replication of the CPV recombinant was evident in tissue culture cells derived from nonavian origin. On inoculation of dogs, a species restricted for avipoxvirus replication, the recombinants elicited a protective immune response against a lethal canine distemper virus (CDV) challenge. The level of MV neutralizing antibodies and the level of protection induced against CDV challenge achieved by the host-restricted CPV vector were equivalent to that obtained by vaccinia virus vectors expressing the same MV antigens. PMID:1736535

  7. Cleavage of a Neuroinvasive Human Respiratory Virus Spike Glycoprotein by Proprotein Convertases Modulates Neurovirulence and Virus Spread within the Central Nervous System

    PubMed Central

    Meessen-Pinard, Mathieu; Dubé, Mathieu; Day, Robert; Seidah, Nabil G.; Talbot, Pierre J.

    2015-01-01

    Human coronaviruses (HCoV) are respiratory pathogens that may be associated with the development of neurological diseases, in view of their neuroinvasive and neurotropic properties. The viral spike (S) glycoprotein is a major virulence factor for several coronavirus species, including the OC43 strain of HCoV (HCoV-OC43). In an attempt to study the role of this protein in virus spread within the central nervous system (CNS) and neurovirulence, as well as to identify amino acid residues important for such functions, we compared the sequence of the S gene found in the laboratory reference strain HCoV-OC43 ATCC VR-759 to S sequences of viruses detected in clinical isolates from the human respiratory tract. We identified one predominant mutation at amino acid 758 (from RRSR↓ G758 to RRSR↓R758), which introduces a putative furin-like cleavage (↓) site. Using a molecular cDNA infectious clone to generate a corresponding recombinant virus, we show for the first time that such point mutation in the HCoV-OC43 S glycoprotein creates a functional cleavage site between the S1 and S2 portions of the S protein. While the corresponding recombinant virus retained its neuroinvasive properties, this mutation led to decreased neurovirulence while potentially modifying the mode of virus spread, likely leading to a limited dissemination within the CNS. Taken together, these results are consistent with the adaptation of HCoV-OC43 to the CNS environment, resulting from the selection of quasi-species harboring mutations that lead to amino acid changes in viral genes, like the S gene in HCoV-OC43, which may contribute to a more efficient establishment of a less pathogenic but persistent CNS infection. This adaptative mechanism could potentially be associated with human encephalitis or other neurological degenerative pathologies. PMID:26545254

  8. Specialization of Hepatitis C Virus Envelope Glycoproteins for B Lymphocytes in Chronically Infected Patients

    PubMed Central

    Douam, Florian; Bobay, Louis-Marie; Maurin, Guillemette; Fresquet, Judith; Calland, Noémie; Maisse, Carine; Durand, Tony; Cosset, François-Loïc; Féray, Cyrille

    2015-01-01

    ABSTRACT Hepatitis C virus (HCV) productively infects hepatocytes. Virion surface glycoproteins E1 and E2 play a major role in this restricted cell tropism by mediating virus entry into particular cell types. However, several pieces of evidence have suggested the ability of patient-derived HCV particles to infect peripheral blood mononuclear cells. The viral determinants and mechanisms mediating such events remain poorly understood. Here, we aimed at isolating viral determinants of HCV entry into B lymphocytes. For this purpose, we constructed a library of full E1E2 sequences isolated from serum and B lymphocytes of four chronically infected patients. We observed a strong phylogenetic compartmentalization of E1E2 sequences isolated from B lymphocytes in one patient, indicating that E1E2 glycoproteins can represent important mediators of the strong segregation of two specialized populations in some patients. Most of the E1E2 envelope glycoproteins were functional and allowed transduction of hepatocyte cell lines using HCV-derived pseudoparticles. Strikingly, introduction of envelope glycoproteins isolated from B lymphocytes into the HCV JFH-1 replicating virus switched the entry tropism of this nonlymphotropic virus from hepatotropism to lymphotropism. Significant detection of viral RNA and viral proteins within B cells was restricted to infections with JFH-1 harboring E1E2 from lymphocytes and depended on an endocytic, pH-dependent entry pathway. Here, we achieved for the first time the isolation of HCV viral proteins carrying entry-related lymphotropism determinants. The identification of genetic determinants within E1E2 represents a first step for a better understanding of the complex relationship between HCV infection, viral persistence, and extrahepatic disorders. IMPORTANCE Hepatitis C virus (HCV) mainly replicates within the liver. However, it has been shown that patient-derived HCV particles can slightly infect lymphocytes in vitro and in vivo, highlighting

  9. Mapping the neutralizing epitopes on the glycoprotein of infectious haematopoietic necrosis virus, a fish rhabdovirus

    USGS Publications Warehouse

    Huang, C.; Chien, M.S.; Landolt, M.L.; Batts, W.; Winton, J.

    1996-01-01

    Twelve neutralizing monoclonal antibodies (MAbs) against the fish rhabdovirus, infectious haematopoietic necrosis virus (IHNV), were used to select 20 MAb escape mutants. The nucleotide sequence of the entire glycoprotein (G) gene was determined for six mutants representing differing cross-neutralization patterns and each had a single nucleotide change leading to a single amino acid substitution within one of three regions of the protein. These data were used to design nested PCR primers to amplify portions of the G gene of the 14 remaining mutants. When the PCR products from these mutants were sequenced, they also had single nucleotide substitutions coding for amino acid substitutions at the same, or nearby, locations. Of the 20 mutants for which all or part of the glycoprotein gene was sequenced, two MAbs selected mutants with substitutions at amino acids 230-231 (antigenic site I) and the remaining MAbs selected mutants with substitutions at amino acids 272-276 (antigenic site II). Two MAbs that selected mutants mapping to amino acids 272-276, selected other mutants that mapped to amino acids 78-81, raising the possibility that this portion of the N terminus of the protein was part of a discontinuous epitope defining antigenic site II. CLUSTAL alignment of the glycoproteins of rabies virus, vesicular stomatitis virus and IHNV revealed similarities in the location of the neutralizing epitopes and a high degree of conservation among cysteine residues, indicating that the glycoproteins of three different genera of animal rhabdoviruses may share a similar three-dimensional structure in spite of extensive sequence divergence.

  10. B epitopes and selection pressures in feline immunodeficiency virus envelope glycoproteins.

    PubMed Central

    Pancino, G; Chappey, C; Saurin, W; Sonigo, P

    1993-01-01

    In order to map linear B epitopes in feline immunodeficiency virus (FIV) envelope glycoproteins (Env), a random library of FIV Env polypeptides fused to beta-galactosidase and expressed in Escherichia coli was screened by using sera from experimentally FIV-infected cats. We mapped five antibody-binding domains in the surface envelope glycoprotein (SU1 to SU5) and four in the transmembrane envelope glycoprotein (TM1 to TM4). Immunological analysis with 48 serum samples from naturally or experimentally infected cats of diverse origins revealed a broad group reactivity for epitopes SU2, TM2, and TM3, whereas SU3 appeared as strictly type specific. To study selection pressures acting on the identified immunogenic domains, we analyzed structural constraints and distribution of synonymous and nonsynonymous mutations (amino acids unchanged or changed). Two linear B epitopes (SU3 and TM4) appeared to be submitted to positive selection for change, a pattern of evolution predicting their possible involvement in antiviral protection. These experiments provide a pertinent choice of oligopeptides for further analysis of the protective response against FIV envelope glycoproteins, as a model to understand the role of antibody escape in lentiviral persistence and to design feline AIDS vaccines. Images PMID:7678301

  11. Preliminary mapping of non-conserved epitopes on envelope glycoprotein E2 of Bovine viral diarrhea virus type 1 and 2.

    PubMed

    Jelsma, H; Loeffen, W L A; van Beuningen, A; van Rijn, P A

    2013-09-27

    Bovine viral diarrhea virus (BVDV) belongs together with Classical swine fever virus (CSFV) and Border disease virus (BDV) to the genus Pestivirus in the Flaviviridae family. BVDV has been subdivided into two different species, BVDV1 and BVDV2 based on phylogenetic analysis. Subsequent characterization of both strains revealed major antigenic differences. Because the envelope glycoprotein E2 is the most immunodominant protein for all pestiviruses, the present study focused on epitope mapping by constructing chimeric BVDV type 1 and 2 E2 genes in expression plasmids. These plasmids with chimeric E2-genes were transfected in SK6 cells and transient expression was studied by immunostaining with a panel of MAbs specific for E2 of BVDV1 or BVDV2, resulting in the localization of type-specific antigenic domains at similar regions. These results indicate that E2 glycoproteins of both BVDV types exhibit a comparable antigenic structure, but with type specific epitopes. In addition, the antigenic resemblance with envelope glycoprotein E2 of Classical swine fever virus is discussed. PMID:23838147

  12. Predicted 3D Model of the Rabies Virus Glycoprotein Trimer.

    PubMed

    Fernando, Bastida-González; Yersin, Celaya-Trejo; José, Correa-Basurto; Paola, Zárate-Segura

    2016-01-01

    The RABVG ectodomain is a homotrimer, and trimers are often called spikes. They are responsible for the attachment of the virus through the interaction with nicotinic acetylcholine receptors, neural cell adhesion molecule (NCAM), and the p75 neurotrophin receptor (p75NTR). This makes them relevant in viral pathogenesis. The antigenic structure differs significantly between the trimers and monomers. Surfaces rich in hydrophobic amino acids are important for trimer stabilization in which the C-terminal of the ectodomain plays an important role; to understand these interactions between the G proteins, a mechanistic study of their functions was performed with a molecular model of G protein in its trimeric form. This verified its 3D conformation. The molecular modeling of G protein was performed by a I-TASSER server and was evaluated via a Rachamandran plot and ERRAT program obtained 84.64% and 89.9% of the residues in the favorable regions and overall quality factor, respectively. The molecular dynamics simulations were carried out on RABVG trimer at 310 K. From these theoretical studies, we retrieved the RMSD values from Cα atoms to assess stability. Preliminary model of G protein of rabies virus stable at 12 ns with molecular dynamics was obtained. PMID:27294109

  13. Predicted 3D Model of the Rabies Virus Glycoprotein Trimer

    PubMed Central

    Fernando, Bastida-González; Yersin, Celaya-Trejo; José, Correa-Basurto; Paola, Zárate-Segura

    2016-01-01

    The RABVG ectodomain is a homotrimer, and trimers are often called spikes. They are responsible for the attachment of the virus through the interaction with nicotinic acetylcholine receptors, neural cell adhesion molecule (NCAM), and the p75 neurotrophin receptor (p75NTR). This makes them relevant in viral pathogenesis. The antigenic structure differs significantly between the trimers and monomers. Surfaces rich in hydrophobic amino acids are important for trimer stabilization in which the C-terminal of the ectodomain plays an important role; to understand these interactions between the G proteins, a mechanistic study of their functions was performed with a molecular model of G protein in its trimeric form. This verified its 3D conformation. The molecular modeling of G protein was performed by a I-TASSER server and was evaluated via a Rachamandran plot and ERRAT program obtained 84.64% and 89.9% of the residues in the favorable regions and overall quality factor, respectively. The molecular dynamics simulations were carried out on RABVG trimer at 310 K. From these theoretical studies, we retrieved the RMSD values from Cα atoms to assess stability. Preliminary model of G protein of rabies virus stable at 12 ns with molecular dynamics was obtained. PMID:27294109

  14. Acidic pH-Induced Conformations and LAMP1 Binding of the Lassa Virus Glycoprotein Spike

    PubMed Central

    Li, Sai; Sun, Zhaoyang; Pryce, Rhys; Parsy, Marie-Laure; Fehling, Sarah K.; Schlie, Katrin; Siebert, C. Alistair; Garten, Wolfgang; Bowden, Thomas A.; Strecker, Thomas; Huiskonen, Juha T.

    2016-01-01

    Lassa virus is an enveloped, bi-segmented RNA virus and the most prevalent and fatal of all Old World arenaviruses. Virus entry into the host cell is mediated by a tripartite surface spike complex, which is composed of two viral glycoprotein subunits, GP1 and GP2, and the stable signal peptide. Of these, GP1 binds to cellular receptors and GP2 catalyzes fusion between the viral envelope and the host cell membrane during endocytosis. The molecular structure of the spike and conformational rearrangements induced by low pH, prior to fusion, remain poorly understood. Here, we analyzed the three-dimensional ultrastructure of Lassa virus using electron cryotomography. Sub-tomogram averaging yielded a structure of the glycoprotein spike at 14-Å resolution. The spikes are trimeric, cover the virion envelope, and connect to the underlying matrix. Structural changes to the spike, following acidification, support a viral entry mechanism dependent on binding to the lysosome-resident receptor LAMP1 and further dissociation of the membrane-distal GP1 subunits. PMID:26849049

  15. Enhancement of feline immunodeficiency virus infection after immunization with envelope glycoprotein subunit vaccines.

    PubMed Central

    Siebelink, K H; Tijhaar, E; Huisman, R C; Huisman, W; de Ronde, A; Darby, I H; Francis, M J; Rimmelzwaan, G F; Osterhaus, A D

    1995-01-01

    Cats were immunized three times with different recombinant feline immunodeficiency virus (FIV) candidate vaccines. Recombinant vaccinia virus (rVV)-expressed envelope glycoprotein with (vGR657) or without (vGR657 x 15) the cleavage site and an FIV envelope bacterial fusion protein (beta-Galactosidase-Env) were incorporated into immune-stimulating complexes or adjuvanted with Quil A. Although all immunized cats developed antibodies against the envelope protein, only the cats vaccinated with the rVV-expressed envelope glycoproteins developed antibodies which neutralized FIV infection of Crandell feline kidney cells. These antibodies failed to neutralize infection of thymocytes with a molecularly cloned homologous FIV. After the third immunization the cats were challenged with homologous FIV. Two weeks after challenge the cell-associated viral load proved to be significantly higher in the cats immunized with vGR657 and vGR657 x 15 than in the other cats. The cats immunized with vGR657 and vGR657 x 15 also developed antibodies against the Gag proteins more rapidly than the cats immunized with beta-Galactosidase-Env or the control cats. This suggested that immunization with rVV-expressed glycoprotein of FIV results in enhanced infectivity of FIV. It was shown that the observed enhancement could be transferred to naive cats with plasma collected at the day of challenge. PMID:7745719

  16. Rabies virus glycoprotein pseudotyping of lentiviral vectors enables retrograde axonal transport and access to the nervous system after peripheral delivery.

    PubMed

    Mazarakis, N D; Azzouz, M; Rohll, J B; Ellard, F M; Wilkes, F J; Olsen, A L; Carter, E E; Barber, R D; Baban, D F; Kingsman, S M; Kingsman, A J; O'Malley, K; Mitrophanous, K A

    2001-09-15

    In this report it is demonstrated for the first time that rabies-G envelope of the rabies virus is sufficient to confer retrograde axonal transport to a heterologous virus/vector. After delivery of rabies-G pseudotyped equine infectious anaemia virus (EIAV) based vectors encoding a marker gene to the rat striatum, neurons in regions distal from but projecting to the injection site, such as the dopaminergic neurons of the substantia nigra pars compacta, become transduced. This retrograde transport to appropriate distal neurons was also demonstrated after delivery to substantia nigra, hippocampus and spinal cord and did not occur when vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped vectors were delivered to these sites. In addition, peripheral administration of rabies-G pseudotyped vectors to the rat gastrocnemius muscle leads to gene transfer in motoneurons of lumbar spinal cord. In contrast the same vector pseudotyped with VSV-G transduced muscle cells surrounding the injection site, but did not result in expression in any cells in the spinal cord. Long-term expression was observed after gene transfer in the nervous system and a minimal immune response which, together with the possibility of non-invasive administration, greatly extends the utility of lentiviral vectors for gene therapy of human neurological disease. PMID:11590128

  17. Growth properties and vaccine efficacy of recombinant pseudorabies virus defective in glycoprotein E and thymidine kinase genes.

    PubMed

    Wu, Ching-Ying; Liao, Chih-Ming; Chi, Jiun-Ni; Chien, Maw-Sheng; Huang, Chienjin

    2016-07-10

    Pseudorabies virus (PRV) is an alphaherpesvirus that causes pseudorabies (PR), an economically important viral disease of pigs. Marker vaccines were widely used in PR prevention and eradication programs. The purpose of this study was to construct a novel recombinant virus with deletions at defined regions in the glycoprotein E (gE) and thymine kinase (TK) genes by homologous recombination. This study also evaluated the safety and efficacy of the virus for a live attenuated marker vaccine. No significant difference was observed in virus replication between gE gene-deleted (gE(-)), gE/TK double gene-deleted (gE(-)TK(-)), and wild-type PRV by growth curve analysis. However, gE(-)TK(-) PRV was completely attenuated in mice. To evaluate the immunogenicity of gE(-)TK(-) PRV, four 12-week-old specific-pathogen-free pigs per group were immunized intramuscularly with viral titers of 1×10(4), 1×10(5), or 1×10(6) TCID50, followed by intranasal challenge infection with virulent PRV (1×10(8) TCID50) at 3 weeks post vaccination. The gE(-)TK(-) PRV-vaccinated pigs displayed no general adverse effects after immunization and had protective immune responses after PRV challenge. Thus, gE(-)TK(-) PRV was safe and efficacious and might be a potential candidate for a live attenuated marker vaccine against PRV. PMID:27164258

  18. N-Linked Glycosylation Status Of Classical Swine Fever Virus Strain Brescia E2 Glycoprotein Influences Virulence In Swine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    E2 is one of the three envelope glycoproteins of Classical Swine Fever Virus (CSFV). Previous studies indicate that E2 is involved in several functions including virus attachment and entry to target cells, production of antibodies, induction of protective immune response in swine, and virulence. Her...

  19. N-LINKED GLYCOSYLATION STATUS OF CLASSICAL SWINE FEVER VIRUS STRAIN BRECIA E2 GLYCOPROTEIN INFLUENCES VIRULENCE IN SWINE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    E2 is one of the three envelope glycoproteins of Classical Swine Fever Virus (CSFV). Although E2 have been involved in virus attachment to target cells, the induction of a protective immune response as well in the process of viral pathogenesis, the role of glycosylation in the functionality of the p...

  20. Resistance to Human Respiratory Syncytial Virus (RSV) Infection Induced by Immunization of Cotton Rats with a Recombinant Vaccinia Virus Expressing the RSV G Glycoprotein

    NASA Astrophysics Data System (ADS)

    Elango, Narayanasamy; Prince, Gregory A.; Murphy, Brian R.; Venkatesan, Sundararajan; Chanock, Robert M.; Moss, Bernard

    1986-03-01

    A cDNA copy of the G glycoprotein gene of human respiratory syncytial virus (RSV) was placed under control of a vaccinia virus promoter and inserted into the thymidine kinase locus of the vaccinia virus genome. The recombinant vaccinia virus retained infectivity and expressed a 93-kDa protein that migrated with the authentic RSV G glycoprotein upon polyacrylamide gel electrophoresis. Glycosylation of the expressed protein and transport to the cell surface were demonstrated in the absence of other RSV proteins. Cotton rats that were inoculated intradermally with the infectious recombinant virus produced serum antibody to the G glycoprotein that neutralized RSV in vitro. Furthermore, the vaccinated animals were resistant to lower respiratory tract infection upon intranasal inoculation with RSV and had reduced titers of RSV in the nose.

  1. Ebola virus glycoprotein Fc fusion protein confers protection against lethal challenge in vaccinated mice

    PubMed Central

    Konduru, Krishnamurthy; Bradfute, Steven B.; Jacques, Jerome; Manangeeswaran, Mohanraj; Nakamura, Siham; Morshed, Sufi; Wood, Steven C.; Bavari, Sina

    2011-01-01

    Ebola virus is a Filoviridae that causes hemorrhagic fever in humans and induces high morbidity and mortality rates. Filoviruses are classified as "Category A bioterrorism agents", and currently there are no licensed therapeutics or vaccines to treat and prevent infection. The Filovirus glycoprotein (GP) is sufficient to protect individuals against infection, and several vaccines based on GP are under development including recombinant adenovirus, parainfluenza virus, Venezuelan equine encephalitis virus, vesicular stomatitis virus (VSV) and virus-like particles. Here we describe the development of a GP Fc fusion protein as a vaccine candidate. We expressed the extracellular domain of the Zaire Ebola virus (ZEBOV) GP fused to the Fc fragment of human IgG1 (ZEBOVGP-Fc) in mammalian cells and showed that GP undergoes the complex furin cleavage and processing observed in the native membrane-bound GP. Mice immunized with ZEBOVGP-Fc developed T-cell immunity against ZEBOV GP and neutralizing antibodies against replication-competent VSV-G deleted recombinant VSV containing ZEBOV GP. The ZEBOVGP-Fc vaccinated mice were protected against challenge with a lethal dose of ZEBOV. These results show that vaccination with the ZEBOVGP-Fc fusion protein alone without the need of a viral vector or assembly into virus-like particles is sufficient to induce protective immunity against ZEBOV in mice. Our data suggested that Filovirus GP Fc fusion proteins could be developed as a simple, safe, efficacious, and cost effective vaccine against Filovirus infection for human use. PMID:21329775

  2. [Generation and preliminary immunological efficacy of a recombinant human adenovirus-rabies virus glycoprotein].

    PubMed

    Wang, Ying; Zhang, Shou-Feng; Liu, Ye; Zhang, Fei; Zhang, Jin-Xia; Hu, Rong-Liang

    2011-09-01

    To construct a recombinant human adenovirus type 5 expressing glycoprotein (GP) of attenuated rabies virus SRV9 and testing immunological efficacy on the immunized mice. Open reading frame of rabies virus GP gene of SRV9 strain was cloned into the shuttle vector of adenovirus expression system in multiple cloning sites to construct the recombinant shuttle plasmid pacAd5 CMV-Gs9, cotransfection was performed into 293AD cells mediated by FuGENE Transfection Reagent with linearized backbone plasmid and recombinant shuttle plasmid, cell cultures were collected after CPE appearance and were identified by PCR and electronmicroscopy, virus titer was measured in 293AD cells. Kunming mice were intraperitoneally injected with 10(6) TCID50 adenovirus, blood for serum preparation was collected through caudal vein pre-immune and post-immune and tested for VNA appearance by fluorescent antibody virus neutralization test (FAVN) detection. Recombinant shuttle plasmid pacAd5 CMV-Gs9 was constructed correctly. A recombinant human adenovirus type 5 was obtained expressing GP protein of rabies virus SRV9. The virus titer reached 10(6) CFU/mL at the least. All mice developed a certain amount of the anti-rabies neutralizing antibody 14 days after intraperitoneal inoculation, while the effective protection rates were 90%. In conclusion, Recombinant adenovirus expressing the rabies virus GP was constructed successfully and a certain amount of neutralizing antibodies were induced in mice, which laid the material foundation for further development of new rabies vaccine. PMID:21998956

  3. Crystal Structure of West Nile Virus Envelope Glycoprotein Reveals Viral Surface Epitopes

    SciTech Connect

    Kanai,R.; Kar, K.; Anthony, K.; Gould, L.; Ledizet, M.; Fikrig, E.; Marasco, W.; Koski, R.; Modis, Y.

    2006-01-01

    West Nile virus, a member of the Flavivirus genus, causes fever that can progress to life-threatening encephalitis. The major envelope glycoprotein, E, of these viruses mediates viral attachment and entry by membrane fusion. We have determined the crystal structure of a soluble fragment of West Nile virus E. The structure adopts the same overall fold as that of the E proteins from dengue and tick-borne encephalitis viruses. The conformation of domain II is different from that in other prefusion E structures, however, and resembles the conformation of domain II in postfusion E structures. The epitopes of neutralizing West Nile virus-specific antibodies map to a region of domain III that is exposed on the viral surface and has been implicated in receptor binding. In contrast, we show that certain recombinant therapeutic antibodies, which cross-neutralize West Nile and dengue viruses, bind a peptide from domain I that is exposed only during the membrane fusion transition. By revealing the details of the molecular landscape of the West Nile virus surface, our structure will assist the design of antiviral vaccines and therapeutics.

  4. Autophagy and the Effects of Its Inhibition on Varicella-Zoster Virus Glycoprotein Biosynthesis and Infectivity

    PubMed Central

    Buckingham, Erin M.; Carpenter, John E.; Jackson, Wallen

    2014-01-01

    Autophagy and the effects of its inhibition or induction were investigated during the entire infectious cycle of varicella-zoster virus (VZV), a human herpesvirus. As a baseline, we first enumerated the number of autophagosomes per cell after VZV infection compared with the number after induction of autophagy following serum starvation or treatment with tunicamycin or trehalose. Punctum induction by VZV was similar in degree to punctum induction by trehalose in uninfected cells. Treatment of infected cells with the autophagy inhibitor 3-methyladenine (3-MA) markedly reduced the viral titer, as determined by assays measuring both cell-free virus and infectious foci (P < 0.0001). We next examined a virion-enriched band purified by density gradient sedimentation and observed that treatment with 3-MA decreased the amount of VZV gE, while treatment with trehalose increased the amount of gE in the same band. Because VZV gE is the most abundant glycoprotein, we selected gE as a representative viral glycoprotein. To further investigate the role of autophagy in VZV glycoprotein biosynthesis as well as confirm the results obtained with 3-MA inhibition, we transfected cells with ATG5 small interfering RNA to block autophagosome formation. VZV-induced syncytium formation was markedly reduced by ATG5 knockdown (P < 0.0001). Further, we found that both expression and glycan processing of VZV gE were decreased after ATG5 knockdown, while expression of the nonglycosylated IE62 tegument protein was unchanged. Taken together, our cumulative results not only documented abundant autophagy within VZV-infected cells throughout the infectious cycle but also demonstrated that VZV-induced autophagy facilitated VZV glycoprotein biosynthesis and processing. PMID:24198400

  5. The effect of ammonium chloride and tunicamycin on the glycoprotein content and infectivity of herpes simplex virus type 1.

    PubMed

    Kousoulas, K G; Bzik, D J; DeLuca, N; Person, S

    1983-03-01

    Infectious virions of MP, a syncytial strain of herpes simplex virus type 1, are formed in the presence of 50 mM NH4Cl. Underglycosylated virion glycoproteins are synthesized in infected cells and are incorporated into virions in the presence of the same concentration of NH4Cl. We conclude that fully glycosylated glycoproteins are not required for viral infectivity. Virus particles, deficient in glycosylated glycoproteins, are assembled in the presence of tunicamycin but they are not infectious. The decrease in infectivity could be due to the decreased amount of the gB or possibly other peptides and/or to the lack of the high-mannose saccharides of precursor glycoproteins. PMID:6301148

  6. Effect of ammonium chloride and tunicamycin on the glycoprotein content and infectivity of herpes simplex virus type 1

    SciTech Connect

    Kousoulas, K.G.; Bzik, D.J.; DeLuca, N.; Person, S.

    1983-01-01

    Infectious virions of MP, a syncytial strain of herpes simplex virus type 1, are formed in the presence of 50 mM NH/sub 4/Cl. Underglycosylated virion glycoproteins are synthesized in infected cells and are incorporated into virions in the presence of the same concentration of NH/sub 4/Cl. We conclude that fully glycosylated glycoproteins are not required for viral infectivity. Virus particles, deficient in glycosylated glycoproteins, are assembled in the presence of tunicamycin but they are not infectious. The decrease in infectivity could be due to the decreased amount of the gB or possibly other peptides and/or to the lack of the high-mannose saccharides of precursor glycoproteins. 32 references, 4 figures.

  7. Rabies virus glycoprotein variants display different patterns in rabies monosynaptic tracing

    PubMed Central

    Mori, Takuma; Morimoto, Kinjiro

    2014-01-01

    Rabies virus (RV) has been widely used to trace multi-synaptic neuronal circuits. The recent development of glycoprotein-deficient rabies virus (RV-ΔG) expressing various proteins has enabled analyzes of both the structure and function of neuronal circuits. The main advantage of RV-ΔG is its ability to trace monosynaptic circuits by the complementation of rabies virus glycoprotein (RVG), but it has the disadvantage of cytotoxicity. Several strain variants of RV have different biological characteristics, such as synaptic spreading and cytotoxicity, mainly due to amino acid mutations in RVG. We developed an improved protocol for the production of a highly attenuated strain of RV-ΔG and assessed whether RVG variants affect rabies monosynaptic tracing and the health of infected neurons. We demonstrated that (1) rabies monosynaptic tracing with RVG variants traced different subsets of presynaptic partners, (2) RVG of the attenuated strain also labeled astrocytes, and (3) the cytotoxicity of RV-ΔG did not depend on RVG but on RV-ΔG. These findings indicate that RVG variants are an important determinant of rabies monosynaptic tracing. PMID:24427117

  8. N-Glycosylation Profiling of Porcine Reproductive and Respiratory Syndrome Virus Envelope Glycoprotein 5

    PubMed Central

    Li, Juan; Tao, Shujuan; Orlando, Ron; Murtaugh, Michael P.

    2015-01-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) is a positive-sense ssRNA virus whose envelope contains four glycoproteins and three nonglycosylated proteins. Glycans of major envelope glycoprotein 5 (GP5) are proposed as important for virus assembly and entry into permissive cells. Structural characterization of GP5 glycans would facilitate the mechanistic understanding of these processes. Thus, we purified the PRRSV type 2 prototype strain, VR2332, and analyzed the virion-associated glycans by both biochemical and mass spectrometric methods. Endoglycosidase digestion showed that GP5 was the primary protein substrate, and that the carbohydrate moieties were primarily complex-type N-glycans. Mass spectrometric analysis (HPLC-ESI-MS/MS) of GP5 N-glycans revealed an abundance of N-acetylglucosamine (GlcNAc) and N-acetyllactosamine (LacNAc) oligomers in addition to sialic acids. GlcNAc and LacNAc accessibility to ligands was confirmed by lectin co-precipitation. Our findings help to explain PRRSV infection of cells lacking sialoadhesin and provide a glycan database to facilitate molecular structural studies of PRRSV. PMID:25726973

  9. Functional characterization of the Sindbis virus E2 glycoprotein by transposon linker-insertion mutagenesis

    SciTech Connect

    Navaratnarajah, Chanakha K.; Kuhn, Richard J. . E-mail: kuhnr@purdue.edu

    2007-06-20

    The glycoprotein envelope of alphaviruses consists of two proteins, E1 and E2. E1 is responsible for fusion and E2 is responsible for receptor binding. An atomic structure is available for E1, but one for E2 has not been reported. In this study, transposon linker-insertion mutagenesis was used to probe the function of different domains of E2. A library of mutants, containing 19 amino acid insertions in the E2 glycoprotein sequence of the prototype alphavirus, Sindbis virus (SINV), was generated. Fifty-seven independent E2 insertions were characterized, of which more than half (67%) gave rise to viable virus. The wild-type-like mutants identify regions that accommodate insertions without perturbing virus production and can be used to insert targeting moieties to direct SINV to specific receptors. The defective and lethal mutants give insight into regions of E2 important for protein stability, transport to the cell membrane, E1-E2 contacts, and receptor binding.

  10. Phylogenetic relationships of Iranian infectious hematopoietic necrosis virus of rainbow trout (Oncorhynchus mykiss) based on the glycoprotein gene

    USGS Publications Warehouse

    Adel, Milad; Amiri, Alireza Babaalian; Dada, Maryam; Kurath, Gael; Laktarashi, Bahram; Ghajari, Amrolah; Breyta, Rachel

    2016-01-01

    Infectious hematopoietic necrosis virus (IHNV), a member of family Rhabdoviridae and genus Novirhabdoviridae, causes a highly lethal disease of salmon and trout. In Iran IHNV was first detected in 2001 on farms rearing rainbow trout (Oncorhynchus mykiss). To evaluate the genetic relationships of IHNV from northern and western Iran, the sequences of a 651-nt region of the glycoprotein gene were determined for two Iranian isolates. These sequences were analyzed to evaluate their genetic relatedness to worldwide isolates representing the five known genogroups of IHNV. Iranian isolates were most closely related to European isolates within the genogroup E rather than those of North American genogroups U, M and L, or the Asian genogroup J. It appears that Iranian IHNV was most likely introduced to Iran from a source in Europe by the movement of contaminated fish eggs.

  11. Phylogenetic relationships of Iranian infectious hematopoietic necrosis virus of rainbow trout (Oncorhynchus mykiss) based on the glycoprotein gene.

    PubMed

    Adel, Milad; Amiri, Alireza Babaalian; Dadar, Maryam; Breyta, Rachel; Kurath, Gael; Laktarashi, Bahram; Ghajari, Amrolah

    2016-03-01

    Infectious hematopoietic necrosis virus (IHNV), a member of family Rhabdoviridae and genus Novirhabdoviridae, causes a highly lethal disease of salmon and trout. In Iran IHNV was first detected in 2001 on farms rearing rainbow trout (Oncorhynchus mykiss). To evaluate the genetic relationships of IHNV from northern and western Iran, the sequences of a 651-nt region of the glycoprotein gene were determined for two Iranian isolates. These sequences were analyzed to evaluate their genetic relatedness to worldwide isolates representing the five known genogroups of IHNV. Iranian isolates were most closely related to European isolates within the genogroup E rather than those of North American genogroups U, M and L, or the Asian genogroup J. It appears that Iranian IHNV was most likely introduced to Iran from a source in Europe by the movement of contaminated fish eggs. PMID:26602428

  12. Vesicular stomatitis virus glycoprotein mutations that affect membrane fusion activity and abolish virus infectivity.

    PubMed Central

    Fredericksen, B L; Whitt, M A

    1995-01-01

    We have introduced amino acid substitutions into two regions of the extracellular domain of the vesicular stomatitis virus (VSV) glycoprotein (G protein) and examined the effect of these mutations on protein transport, low-pH-induced stability of G protein oligomers, and membrane fusion activity. We suggested previously that the region between amino acids 118 and 139 may be important for the membrane fusion activity of G protein, on the basis of the characterization of a fusion-defective G protein mutant (M. A. Whitt, P. Zagouras, B. Crise, and J. K. Rose, J. Virol. 64:4907-4913, 1990). It has also been postulated by others that this region as well as the region between amino acids 181 and 212 may constitute putative internal fusion domains of VSV G protein. In this report, we show that three different amino acids substitutions between residues 118 and 139 (G-124-->E, P-127-->D, and A-133-->K) either altered or abolished low-pH-dependent membrane fusion activity. In contrast, substitutions between residues 192 and 212 resulted either in G proteins that had wild-type fusion activity or in mutant proteins in which the mutation prevented transport of G protein to the cell surface. Two of the substitutions between residues 118 and 139 (G-124-->E and P-127-->D) resulted in G proteins that were fusion defective at pH 5.7, although syncytia were observed after cells were treated with fusion buffer at pH 5.5, albeit at levels significantly less than that induced by wild-type G protein. Interestingly, when either G-124-->E or P-127-->D was incorporated into tsO45 virions, the resulting particles were not infectious, presumably because the viral envelope was not able to fuse with the proper intracellular membrane. These results support the hypothesis that the region between amino acids 118 and 139 is important for the membrane fusion activity of VSV G protein and may constitute an internal fusion domain. PMID:7853475

  13. A molecular epidemiological study targeting the glycoprotein gene of rabies virus isolates from China.

    PubMed

    Meng, Sheng-Li; Yan, Jia-Xin; Xu, Ge-Lin; Nadin-Davis, Susan A; Ming, Ping-Gang; Liu, Sheng-Ya; Wu, Jie; Ming, He-Tian; Zhu, Feng-Cai; Zhou, Dun-Jin; Xiao, Qi-You; Dong, Guan-Mu; Yang, Xiao-Ming

    2007-03-01

    A group of 31 rabies viruses (RABVs), recovered primarily from dogs, one deer and one human case, were collected from various areas in China between 1989 and 2006. Complete G gene sequences determined for these isolates indicated identities of nucleotide and amino acid sequences of >or=87% and 93.8%, respectively. Phylogenetic analysis of these and some additional Chinese isolates clearly supported the placement of all Chinese viruses in Lyssavirus genotype 1 and divided all Chinese isolates between four distinct groups (I-IV). Several variants identified within the most commonly encountered group I were distributed according to their geographical origins. A comparison of representative Chinese viruses with other isolates retrieved world-wide indicated a close evolutionary relationship between China group I and II viruses and those of Indonesia while China group III viruses formed an outlying branch to variants from Malaysia and Thailand. China group IV viruses were closely related to several vaccine strains. The predicted glycoprotein sequences of these RABVs variants are presented and discussed with respect to the utility of the anti-rabies biologicals currently employed in China. PMID:17129631

  14. Mutations in the Carboxi Terminal Region of E2 Glycoprotein of Classical Swine Fever Virus is Responsible for Viral Attenuation in Swine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have reported that chimeric virus 319.1 virus containing the E2 glycoprotein gene from Classical Swine Fever Virus (CSFV) vaccine strain CS with the genetic background of virulent CSFV strain Brescia (BIC virus) was attenuated in pigs. To identify the amino acids mediating 319.1 virus attenuation...

  15. A Tyrosine-to-Histidine Switch at Position 18 of the Ross River Virus E2 Glycoprotein Is a Determinant of Virus Fitness in Disparate Hosts

    PubMed Central

    Jupille, Henri J.; Medina-Rivera, Melisa; Hawman, David W.; Oko, Lauren

    2013-01-01

    Arthritogenic alphaviruses are human pathogens maintained in nature through alternating replication in vertebrates and mosquitoes. Using chimeric viruses, we previously reported that replacement of the PE2 coding region of the T48 strain of Ross River virus (RRV-T48) with that from the attenuated DC5692 strain, which differ by 7 amino acids, resulted in an attenuated disease phenotype in a mouse model of RRV-induced rheumatic disease. Here, we demonstrate that introduction of one of these amino acid differences, a tyrosine (Y)-to-histidine (H) change at position 18 of the E2 glycoprotein (E2 Y18H), into the RRV-T48 genetic background was sufficient to generate a virus that caused dramatically less severe musculoskeletal disease in mice. The attenuated phenotype of RRV-T48 E2 Y18H was associated with reduced viral loads in musculoskeletal tissues, reduced viremia, and less efficient virus spread. Consistent with these findings, RRV-T48 E2 Y18H replicated less well in mammalian cells in vitro due to significantly reduced PFU released per infected cell. In contrast, RRV-T48 E2 Y18H replicated more efficiently than RRV-T48 in C6/36 mosquito cells. Competition studies confirmed that RRV-T48 E2 Y18H had a fitness advantage in mosquito cells and a fitness disadvantage in mammalian cells. Interestingly, all sequenced Ross River viruses encode either a tyrosine or a histidine at E2 position 18, and this holds true for other alphaviruses in the Semliki Forest antigenic complex. Taken together, these findings suggest that a tyrosine-to-histidine switch at E2 position 18 functions as a regulator of RRV fitness in vertebrate and invertebrate cells. PMID:23514884

  16. Hepatitis C Virus (HCV) Envelope Glycoproteins E1 and E2 Contain Reduced Cysteine Residues Essential for Virus Entry*

    PubMed Central

    Fraser, Johanna; Boo, Irene; Poumbourios, Pantelis; Drummer, Heidi E.

    2011-01-01

    The HCV envelope glycoproteins E1 and E2 contain eight and 18 highly conserved cysteine residues, respectively. Here, we examined the oxidation state of E1E2 heterodimers incorporated into retroviral pseudotyped particles (HCVpp) and investigated the significance of free sulfhydryl groups in cell culture-derived HCV (HCVcc) and HCVpp entry. Alkylation of free sulfhydryl groups on HCVcc/pp with a membrane-impermeable sulfhydryl-alkylating reagent 4-(N-maleimido)benzyl-α-trimethylammonium iodide (M135) prior to virus attachment to cells abolished infectivity in a dose-dependent manner. Labeling of HCVpp envelope proteins with EZ-Link maleimide-PEG2-biotin (maleimide-biotin) detected free thiol groups in both E1 and E2. Unlike retroviruses that employ disulfide reduction to facilitate virus entry, the infectivity of alkylated HCVcc could not be rescued by addition of exogenous reducing agents. Furthermore, the infectivity of HCVcc bound to target cells was not affected by addition of M135 indicative of a change in glycoprotein oxidation state from reduced to oxidized following virus attachment to cells. By contrast, HCVpp entry was reduced by 61% when treated with M135 immediately following attachment to cells, suggesting that the two model systems might demonstrate variations in oxidation kinetics. Glycoprotein oxidation was not altered following binding of HCVpp incorporated E1E2 to soluble heparin or recombinant CD81. These results suggest that HCV entry is dependent on the presence of free thiol groups in E1 and E2 prior to cellular attachment and reveals a new essential component of the HCV entry process. PMID:21768113

  17. Hepatitis C virus (HCV) envelope glycoproteins E1 and E2 contain reduced cysteine residues essential for virus entry.

    PubMed

    Fraser, Johanna; Boo, Irene; Poumbourios, Pantelis; Drummer, Heidi E

    2011-09-16

    The HCV envelope glycoproteins E1 and E2 contain eight and 18 highly conserved cysteine residues, respectively. Here, we examined the oxidation state of E1E2 heterodimers incorporated into retroviral pseudotyped particles (HCVpp) and investigated the significance of free sulfhydryl groups in cell culture-derived HCV (HCVcc) and HCVpp entry. Alkylation of free sulfhydryl groups on HCVcc/pp with a membrane-impermeable sulfhydryl-alkylating reagent 4-(N-maleimido)benzyl-α-trimethylammonium iodide (M135) prior to virus attachment to cells abolished infectivity in a dose-dependent manner. Labeling of HCVpp envelope proteins with EZ-Link maleimide-PEG2-biotin (maleimide-biotin) detected free thiol groups in both E1 and E2. Unlike retroviruses that employ disulfide reduction to facilitate virus entry, the infectivity of alkylated HCVcc could not be rescued by addition of exogenous reducing agents. Furthermore, the infectivity of HCVcc bound to target cells was not affected by addition of M135 indicative of a change in glycoprotein oxidation state from reduced to oxidized following virus attachment to cells. By contrast, HCVpp entry was reduced by 61% when treated with M135 immediately following attachment to cells, suggesting that the two model systems might demonstrate variations in oxidation kinetics. Glycoprotein oxidation was not altered following binding of HCVpp incorporated E1E2 to soluble heparin or recombinant CD81. These results suggest that HCV entry is dependent on the presence of free thiol groups in E1 and E2 prior to cellular attachment and reveals a new essential component of the HCV entry process. PMID:21768113

  18. Development of recombinant canine adenovirus type-2 expressing the Gn glycoprotein of Seoul virus.

    PubMed

    Yuan, Ziguo; Zhang, Xiuxiang; Zhang, Shoufeng; Liu, Ye; Gao, Shengyan; Zhang, Fei; Xu, Huijuan; Wang, Xiaohu; Hu, Rongliang

    2008-05-01

    Seoul virus glycoprotein Gn is a major structural protein and candidate antigen of hantavirus that induces a highly immunogenic response for hantavirus vaccine. In this study, a replication-competent recombinant canine adenovirus type-2 expressing Gn was constructed by the in vitro ligation method. The Gn expression cassette, including the human cytomegalovirus (hCMV) promoter/enhancer and the SV40 early mRNA polyadenylation signal, was cloned into the SspI site of the E3 region which is not essential for proliferation of CAV-2. Expression of Gn was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. PMID:18249007

  19. Resting lymphocyte transduction with measles virus glycoprotein pseudotyped lentiviral vectors relies on CD46 and SLAM

    SciTech Connect

    Zhou Qi; Schneider, Irene C.; Gallet, Manuela; Kneissl, Sabrina; Buchholz, Christian J.

    2011-05-10

    The measles virus (MV) glycoproteins hemagglutinin (H) and fusion (F) were recently shown to mediate transduction of resting lymphocytes by lentiviral vectors. MV vaccine strains use CD46 or signaling lymphocyte activation molecule (SLAM) as receptor for cell entry. A panel of H protein mutants derived from vaccine strain or wild-type MVs that lost or gained CD46 or SLAM receptor usage were investigated for their ability to mediate gene transfer into unstimulated T lymphocytes. The results demonstrate that CD46 is sufficient for efficient vector particle association with unstimulated lymphocytes. For stable gene transfer into these cells, however, both MV receptors were found to be essential.

  20. Alteration of a second putative fusion peptide of structural glycoprotein E2 of Classical Swine Fever Virus alters virus replication and virulence in swine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    E2, the major envelope glycoprotein of Classical Swine Fever Virus (CSFV), is involved in several critical virus functions including cell attachment, host range susceptibility, and virulence in natural hosts. Functional structural analysis of E2 based on Wimley-White interfacial hydrophobicity dis...

  1. Application of recombinant adenoviruses expressing glycoprotein or nucleoprotein of rabies virus to Korean raccoon dogs

    PubMed Central

    Choi, Jiyoung; Kim, Ha-Hyun; Jo, Hyun-Ye; Choi, Sung-Suk; Kim, Jong-Taek; Cho, In-Soo; Kim, Hee-Won

    2015-01-01

    Purpose A new rabies vaccine for animals, including raccoon dogs, in Korea is needed to eradicate rabies infection. In this study, we constructed two recombinant adenoviruses expressing the glycoprotein or nucleoprotein of the rabies virus (RABV). We then investigated the safety and immunogenicity of these strains in raccoon dogs, depending on inoculation route. Materials and Methods Recombinant adenoviruses expressing the glycoprotein (Ad-0910G) or nucleoprotein (Ad-0910N) of rabies were constructed in 293A cells using an adenoviral system. One-year-old raccoon dogs underwent intramuscular (IM) inoculation or oral administration of the recombinant Ad-0910G and Ad-0910N. Clinical symptoms were observed and virus-neutralizing antibodies (VNA) against RABV were measured at 0, 2, 4, and 6 weeks after the immunization. Raccoons were considered positive if VNA titers were ≥ 0.1 IU/mL. Results Raccoon dogs inoculated with the combined Ad-0910G and Ad-0910N virus via the IM route did not exhibit any clinical sign of rabies during the observation period. All raccoon dogs (n = 7) immunized IM had high VNA titers, ranging from 0.17 to 41.6 IU/mL at 2 weeks after inoculation, but 70% (7/10) of raccoon dogs administered viruses via the oral route responded by 6 weeks after administration against RABV. Conclusion Raccoon dogs inoculated with Ad-0910G and Ad-0910N viruses showed no adverse effects. Immunization with the combined Ad-0910G and Ad-0910N strains may play an important role in inducing VNA against RABV in raccoon dogs. PMID:26273578

  2. Evidence suggesting that HCV p7 protects E2 glycoprotein from premature degradation during virus production.

    PubMed

    Atoom, Ali M; Jones, Daniel M; Russell, Rodney S

    2013-09-01

    The hepatitis C virus (HCV) genome encodes a 63 amino acid (aa) protein, p7, which is located between the structural and non-structural proteins. p7 localizes to endoplasmic reticulum membranes and is composed of two transmembrane domains (TM1 and TM2) and a cytoplasmic loop. While its exact role is unknown, p7 is crucial for assembly and/or release of infectious virus production in cell culture, as well as infectivity in chimpanzees. The contribution of p7 to the HCV life cycle may result from at least two distinct roles. Firstly, several studies have shown that p7 acts as an ion channel, the functionality of which is critical for infection. Secondly, p7 interacts with NS2 in a manner that may regulate the targeting of other structural proteins during the assembly process. In this study, we observed that mutations in TM1 and the cytoplasmic loop of p7 decreased infectious virus production in a single-cycle virus production assay. Analysis of intra- and extracellular virus titers indicated that p7 functions at a stage prior to generation of infectious particles. These effects were not due to altered RNA replication since no effects on levels of NS3 or NS5A protein were observed, and were not a consequence of altered recruitment of core protein to lipid droplets. Similarly, these mutations seemingly did not prevent nucleocapsid oligomerization. Importantly, we found that an alanine triplet substitution including the two basic residues of the cytoplasmic loop, which is integral to p7 ion channel function, significantly reduced E2 glycoprotein levels. A time course experiment tracking E2 levels indicated that E2 was degraded over time, as opposed to being synthesized in reduced quantities. The results of this study provide strong evidence that one of the functions of p7 is to protect HCV glycoproteins from premature degradation during virion morphogenesis. PMID:23816605

  3. Vaccinia virus recombinants expressing either the measles virus fusion or hemagglutinin glycoprotein protect dogs against canine distemper virus challenge.

    PubMed

    Taylor, J; Pincus, S; Tartaglia, J; Richardson, C; Alkhatib, G; Briedis, D; Appel, M; Norton, E; Paoletti, E

    1991-08-01

    cDNA clones of the genes encoding either the hemagglutinin (HA) or fusion (F) proteins of the Edmonston strain of measles virus (MV) were expressed in vaccinia virus recombinants. Immunofluorescence analysis detected both proteins on the plasma membranes of unfixed cells as well as internally in fixed cells. Immunoprecipitation of metabolically radiolabeled infected-cell extracts by using specific sera demonstrated a 76-kDa HA polypeptide and gene products of 60, 44, and 23 kDa which correspond to a MV F precursor and cleavage products F0, F1, and F2, respectively. Neither recombinant induced cell fusion of Vero cells when inoculated individually, but efficient cell fusion was readily observed upon coinfection of cells with both recombinants. Inoculation of dogs with the vaccinia virus-MV F recombinant (VV-MVF) did not give rise to detectable MV-neutralizing antibody. Inoculation of dogs with the vaccinia virus-MV HA recombinant (VV-MVHA) or coinoculation with both recombinants (VV-MVF and VV-MVHA) induced significant MV-neutralizing titers that were increased following a booster inoculation. Inoculation of dogs with the vaccinia virus recombinants or with MV failed to induce canine distemper virus (CDV)-neutralizing antibodies. Upon challenge with a lethal dose of virulent CDV, signs of infection were observed in dogs inoculated with (VV-MVF). No symptoms of disease were observed in dogs that had been vaccinated with VV-MVHA or with VV-MVHA and VV-MVF and then challenged with CDV. All dogs vaccinated with the recombinant viruses as well as those inoculated with MV or a vaccine strain of CDV survived CDV challenge. PMID:1830113

  4. Vaccinia virus recombinants expressing either the measles virus fusion or hemagglutinin glycoprotein protect dogs against canine distemper virus challenge.

    PubMed Central

    Taylor, J; Pincus, S; Tartaglia, J; Richardson, C; Alkhatib, G; Briedis, D; Appel, M; Norton, E; Paoletti, E

    1991-01-01

    cDNA clones of the genes encoding either the hemagglutinin (HA) or fusion (F) proteins of the Edmonston strain of measles virus (MV) were expressed in vaccinia virus recombinants. Immunofluorescence analysis detected both proteins on the plasma membranes of unfixed cells as well as internally in fixed cells. Immunoprecipitation of metabolically radiolabeled infected-cell extracts by using specific sera demonstrated a 76-kDa HA polypeptide and gene products of 60, 44, and 23 kDa which correspond to a MV F precursor and cleavage products F0, F1, and F2, respectively. Neither recombinant induced cell fusion of Vero cells when inoculated individually, but efficient cell fusion was readily observed upon coinfection of cells with both recombinants. Inoculation of dogs with the vaccinia virus-MV F recombinant (VV-MVF) did not give rise to detectable MV-neutralizing antibody. Inoculation of dogs with the vaccinia virus-MV HA recombinant (VV-MVHA) or coinoculation with both recombinants (VV-MVF and VV-MVHA) induced significant MV-neutralizing titers that were increased following a booster inoculation. Inoculation of dogs with the vaccinia virus recombinants or with MV failed to induce canine distemper virus (CDV)-neutralizing antibodies. Upon challenge with a lethal dose of virulent CDV, signs of infection were observed in dogs inoculated with (VV-MVF). No symptoms of disease were observed in dogs that had been vaccinated with VV-MVHA or with VV-MVHA and VV-MVF and then challenged with CDV. All dogs vaccinated with the recombinant viruses as well as those inoculated with MV or a vaccine strain of CDV survived CDV challenge. Images PMID:1830113

  5. Molecular Evolution and Circulation Patterns of Human Respiratory Syncytial Virus Subgroup A: Positively Selected Sites in the Attachment G Glycoprotein

    PubMed Central

    Zlateva, Kalina T.; Lemey, Philippe; Vandamme, Anne-Mieke; Van Ranst, Marc

    2004-01-01

    Human respiratory syncytial virus (HRSV) is the most common etiological agent of acute lower respiratory tract disease in infants and can cause repeated infections throughout life. In this study, we have analyzed nucleotide sequences encompassing 629 bp at the carboxy terminus of the G glycoprotein gene for HRSV subgroup A strains isolated over 47 years, including 112 Belgian strains isolated over 19 consecutive years (1984 to 2002). By using a maximum likelihood method, we have tested the presence of diversifying selection and identified 13 positively selected sites with a posterior probability above 0.5. The sites under positive selection correspond to sites of O glycosylation or to amino acids that were previously described as monoclonal antibody-induced in vitro escape mutants. Our findings suggest that the evolution of subgroup A HRSV G glycoprotein is driven by immune pressure operating in certain codon positions located mainly in the second hypervariable region of the ectodomain. Phylogenetic analysis revealed the prolonged cocirculation of two subgroup A lineages among the Belgian population and the possible extinction of three other lineages. The evolutionary rate of HRSV subgroup A isolates was estimated to be 1.83 × 10−3 nucleotide substitutions/site/year, projecting the most recent common ancestor back to the early 1940s. PMID:15078950

  6. Genetic Diversity Underlying the Envelope Glycoproteins of Hepatitis C Virus: Structural and Functional Consequences and the Implications for Vaccine Design

    PubMed Central

    Tarr, Alexander W.; Khera, Tanvi; Hueging, Kathrin; Sheldon, Julie; Steinmann, Eike; Pietschmann, Thomas; Brown, Richard J. P.

    2015-01-01

    In the 26 years since the discovery of Hepatitis C virus (HCV) a major global research effort has illuminated many aspects of the viral life cycle, facilitating the development of targeted antivirals. Recently, effective direct-acting antiviral (DAA) regimens with >90% cure rates have become available for treatment of chronic HCV infection in developed nations, representing a significant advance towards global eradication. However, the high cost of these treatments results in highly restricted access in developing nations, where the disease burden is greatest. Additionally, the largely asymptomatic nature of infection facilitates continued transmission in at risk groups and resource constrained settings due to limited surveillance. Consequently a prophylactic vaccine is much needed. The HCV envelope glycoproteins E1 and E2 are located on the surface of viral lipid envelope, facilitate viral entry and are the targets for host immunity, in addition to other functions. Unfortunately, the extreme global genetic and antigenic diversity exhibited by the HCV glycoproteins represents a significant obstacle to vaccine development. Here we review current knowledge of HCV envelope protein structure, integrating knowledge of genetic, antigenic and functional diversity to inform rational immunogen design. PMID:26193307

  7. Genetic Diversity Underlying the Envelope Glycoproteins of Hepatitis C Virus: Structural and Functional Consequences and the Implications for Vaccine Design.

    PubMed

    Tarr, Alexander W; Khera, Tanvi; Hueging, Kathrin; Sheldon, Julie; Steinmann, Eike; Pietschmann, Thomas; Brown, Richard J P

    2015-07-01

    In the 26 years since the discovery of Hepatitis C virus (HCV) a major global research effort has illuminated many aspects of the viral life cycle, facilitating the development of targeted antivirals. Recently, effective direct-acting antiviral (DAA) regimens with >90% cure rates have become available for treatment of chronic HCV infection in developed nations, representing a significant advance towards global eradication. However, the high cost of these treatments results in highly restricted access in developing nations, where the disease burden is greatest. Additionally, the largely asymptomatic nature of infection facilitates continued transmission in at risk groups and resource constrained settings due to limited surveillance. Consequently a prophylactic vaccine is much needed. The HCV envelope glycoproteins E1 and E2 are located on the surface of viral lipid envelope, facilitate viral entry and are the targets for host immunity, in addition to other functions. Unfortunately, the extreme global genetic and antigenic diversity exhibited by the HCV glycoproteins represents a significant obstacle to vaccine development. Here we review current knowledge of HCV envelope protein structure, integrating knowledge of genetic, antigenic and functional diversity to inform rational immunogen design. PMID:26193307

  8. In ovo vaccination of commercial broilers with a glycoprotein J gene-deleted strain of infectious laryngotracheitis virus.

    PubMed

    Mashchenko, Anna; Riblet, Sylva M; Zavala, Guillermo; García, Maricarmen

    2013-06-01

    Conventional live attenuated vaccines have been used as the main tool worldwide for the control of infectious laryngotracheitis. However, their suboptimal attenuation combined with poor mass administration practices allowed chicken embryo origin vaccine-derived isolates to circulate in the field, regain virulence, and be the cause of continuous outbreaks of the disease. Previous studies indicated that stable attenuation of infectious laryngotracheitis virus (ILTV) can be achieved by the deletion of individual viral genes that are not essential for viral replication in vitro. One of these genes is the glycoprotein J (gJ) gene. Its deletion provided significant attenuation to virulent ILTV strains from Europe and the United States. The objective of this study was to construct an attenuated gJ-deleted ILTV strain and evaluate its safety and efficacy for in ovo (IO) administration of commercial broilers. A novel gJ-deleted virus (N(delta)gJ) was constructed, and a 10(3) median tissue culture infective dose administered at 18 days of embryo age was considered safe because it did not affect hatchability or survivability of chickens during the first week posthatch. Broilers vaccinated IO and IO + eye drop at 14 days of age presented a significant reduction in clinical signs and reduction of virus loads after challenge, as compared with the nonvaccinated challenged group of chickens. Therefore, this study presents initial proof that the N(delta)gJ strain is a potential ILTV live-attenuated vaccine candidate suitable for IO vaccination of commercial broilers. PMID:23901771

  9. Importance of the short cytoplasmic domain of the feline immunodeficiency virus transmembrane glycoprotein for fusion activity and envelope glycoprotein incorporation into virions

    SciTech Connect

    Celma, Cristina C.P.; Paladino, Monica G.; Gonzalez, Silvia A.; Affranchino, Jose L.

    2007-09-30

    The mature form of the envelope (Env) glycoprotein of lentiviruses is a heterodimer composed of the surface (SU) and transmembrane (TM) subunits. Feline immunodeficiency virus (FIV) possesses a TM glycoprotein with a cytoplasmic tail of approximately 53 amino acids which is unusually short compared with that of the other lentiviral glycoproteins (more than 100 residues). To investigate the relevance of the FIV TM cytoplasmic domain to Env-mediated viral functions, we characterized the biological properties of a series of Env glycoproteins progressively shortened from the carboxyl terminus. All the mutant Env proteins were efficiently expressed in feline cells and processed into the SU and TM subunits. Deletion of 5 or 11 amino acids from the TM C-terminus did not significantly affect Env surface expression, fusogenic activity or Env incorporation into virions, whereas removal of 17 or 23 residues impaired Env-mediated cell-to-cell fusion. Further truncation of the FIV TM by 29 residues resulted in an Env glycoprotein that was poorly expressed at the cell surface, exhibited only 20% of the wild-type Env fusogenic capacity and was inefficiently incorporated into virions. Remarkably, deletion of the TM C-terminal 35 or 41 amino acids restored or even enhanced Env biological functions. Indeed, these mutant Env glycoproteins bearing cytoplasmic domains of 18 or 12 amino acids were found to be significantly more fusogenic than the wild-type Env and were efficiently incorporated into virions. Interestingly, truncation of the TM cytoplasmic domain to only 6 amino acids did not affect Env incorporation into virions but abrogated Env fusogenicity. Finally, removal of the entire TM cytoplasmic tail or deletion of as many as 6 amino acids into the membrane-spanning domain led to a complete loss of Env functions. Our results demonstrate that despite its relatively short length, the FIV TM cytoplasmic domain plays an important role in modulating Env-mediated viral functions.

  10. Genetic and phylogenetic analysis of glycoprotein of rabies virus isolated from several species in Brazil.

    PubMed

    Sato, Go; Itou, Takuya; Shoji, Youko; Miura, Yasuo; Mikami, Takeshi; Ito, Mikako; Kurane, Ichiro; Samara, Samir I; Carvalho, Adolorata A B; Nociti, Darci P; Ito, Fumio H; Sakai, Takeo

    2004-07-01

    Genetic and phylogenetic analyses of the region containing the glycoprotein (G) gene, which is related to pathogenicity and antigenicity, and the G-L intergenic region were carried out in 14 Brazilian rabies virus isolates. The isolates were classified as dog-related rabies virus (DRRV) or vampire bat-related rabies virus (VRRV), by nucleoprotein (N) analysis. The nucleotide and amino acid (AA) homologies of the area containing the G protein gene and G-L intergenic region were generally lower than those of the ectodomain. In both regions, nucleotide and deduced AA homologies were lower among VRRVs than among DRRVs. There were AA differences between DRRV and VRRV at 3 antigenic sites and epitopes (IIa, WB+ and III), suggesting that DRRV and VRRV can be distinguished by differences of antigenicity. In a comparison of phylogenetic trees between the ectodomain and the area containing the G protein gene and G-L intergenic region, the branching patterns of the chiropteran and carnivoran rabies virus groups differed, whereas there were clear similarities in patterns within the DRRV and VRRV groups. Additionally, the VRRV isolates were more closely related to chiropteran strains isolated from Latin America than to Brazilian DRRV. These results indicate that Brazilian rabies virus isolates can be classified as DRRV or VRRV by analysis of the G gene and the G-L intergenic region, as well as by N gene analysis. PMID:15297743

  11. Ebola virus disease

    MedlinePlus

    ... urine, saliva, sweat, feces, vomit, breast milk, and semen. The virus can enter the body through a ... use condoms for 12 months or until their semen has twice tested negative. Long-term complications can ...

  12. Comparison of the immunogenicity of two inactivated recombinant rabies viruses overexpressing the glycoprotein.

    PubMed

    Navid, Muhammad Tariq; Li, Yingying; Zhou, Ming; Cui, Min; Fu, Zhen F; Tang, Lijun; Zhao, Ling

    2016-10-01

    Two recombinant rabies viruses overexpressing their glycoprotein (G) were compared in this study, with the overexpressed G inserted between P and M genes (named LBNSE-PM-G), and between the G and L genes (named LBNSE-GL-G), respectively. LBNSE-PM-G produced more G protein and induced stronger apoptosis than LBNSE-GL-G in infected cells, while the amount of virion-incorporated G in LBNSE-PM-G was less than in LBNSE-GL-G. Mice immunized with inactivated LBNSE-PM-G produced lower titers of virus-neutralizing antibody, and this recombinant conferred worse protection than LBNSE-GL-G. Our results suggest that over expressed G gene inserted between G and L, but not between P and M, enhanced the immunogenicity when used as an inactivated rabies vaccine. PMID:27438075

  13. The Ebola virus glycoprotein mediates entry via a non-classical dynamin-dependent macropinocytic pathway

    SciTech Connect

    Mulherkar, Nirupama; Raaben, Matthijs; Torre, Juan Carlos de la; Whelan, Sean P.; Chandran, Kartik

    2011-10-25

    Ebola virus (EBOV) has been reported to enter cultured cell lines via a dynamin-2-independent macropinocytic pathway or clathrin-mediated endocytosis. The route(s) of productive EBOV internalization into physiologically relevant cell types remain unexplored, and viral-host requirements for this process are incompletely understood. Here, we use electron microscopy and complementary chemical and genetic approaches to demonstrate that the viral glycoprotein, GP, induces macropinocytic uptake of viral particles into cells. GP's highly-glycosylated mucin domain is dispensable for virus-induced macropinocytosis, arguing that interactions between other sequences in GP and the host cell surface are responsible. Unexpectedly, we also found a requirement for the large GTPase dynamin-2, which is proposed to be dispensable for several types of macropinocytosis. Our results provide evidence that EBOV uses an atypical dynamin-dependent macropinocytosis-like entry pathway to enter Vero cells, adherent human peripheral blood-derived monocytes, and a mouse dendritic cell line.

  14. The paramyxovirus simian virus 5 hemagglutinin-neuraminidase glycoprotein, but not the fusion glycoprotein, is internalized via coated pits and enters the endocytic pathway.

    PubMed Central

    Leser, G P; Ector, K J; Lamb, R A

    1996-01-01

    The hemagglutinin-neuraminidase (HN) and fusion (F) glycoproteins of the paramyxovirus simian virus 5 (SV5) are expressed on the surface of virus-infected cells. Although the F protein was found to be expressed stably, the HN protein was internalized from the plasma membrane. HN protein lacks known internalization signals in its cytoplasmic domain that are common to many integral membrane proteins that are internalized via clathrin-coated pits. Thus, the cellular pathway of HN protein internalization was examined. Biochemical analysis indicated that HN was lost from the cell surface with a t1/2 of approximately 45-50 min and turned over with a t1/2 of approximately 2 h. Immunofluorescent analysis showed internalized SV5 HN in vesicle-like structures in a juxtanuclear pattern coincident with the localization of ovalbumin. In contrast the SV5 F glycoprotein and the HN glycoprotein of the highly related parainfluenza virus 3 (hPIV-3) were found only on the cell surface. Immunogold staining of HN on the surface of SV5-infected CV-1 cells and examination using electron microscopy, showed heavy surface labeling that gradually decreased with time. Concomitantly, gold particles were detected in the endosomal system and with increasing time, gold-labeled structures having the morphology of lysosomes were observed. On the plasma membrane approximately 5% of the gold-labeled HN was found in coated pits. The inhibition of the pinching-off of coated pits from the plasma membrane by cytosol acidification significantly reduced HN internalization. Internalized HN was co-localized with gold-conjugated transferrin, a marker for the early endosomal compartments, and with gold-conjugated bovine serum albumin, a marker for late endosomal compartments. Taken together, these data strongly suggest that the HN glycoprotein is internalized via clathrin-coated pits and delivered to the endocytic pathway. Images PMID:8741847

  15. A Glycoprotein Subunit Vaccine Elicits a Strong Rift Valley Fever Virus Neutralizing Antibody Response in Sheep

    PubMed Central

    Lebedev, Maxim; McVey, D. Scott; Wilson, William; Morozov, Igor; Young, Alan

    2014-01-01

    Abstract Rift Valley fever virus (RVFV), a member of the Bunyaviridae family, is a mosquito-borne zoonotic pathogen that causes serious morbidity and mortality in livestock and humans. The recent spread of the virus beyond its traditional endemic boundaries in Africa to the Arabian Peninsula coupled with the presence of susceptible vectors in nonendemic countries has created increased interest in RVF vaccines. Subunit vaccines composed of specific virus proteins expressed in eukaryotic or prokaryotic expression systems are shown to elicit neutralizing antibodies in susceptible hosts. RVFV structural proteins, amino-terminus glycoprotein (Gn), and carboxyl-terminus glycoprotein (Gc), were expressed using a recombinant baculovirus expression system. The recombinant proteins were reconstituted as a GnGc subunit vaccine formulation and evaluated for immunogenicity in a target species, sheep. Six sheep were each immunized with a primary dose of 50 μg of each vaccine immunogen with the adjuvant montanide ISA25; at day 21, postvaccination, each animal received a second dose of the same vaccine. The vaccine induced a strong antibody response in all animals as determined by indirect enzyme-linked immunosorbent assay (ELISA). A plaque reduction neutralization test (PRNT80) showed the primary dose of the vaccine was sufficient to elicit potentially protective virus neutralizing antibody titers ranging from 40 to 160, and the second vaccine dose boosted the titer to more than 1280. Furthermore, all animals tested positive for neutralizing antibodies at day 328 postvaccination. ELISA analysis using the recombinant nucleocapsid protein as a negative marker antigen indicated that the vaccine candidate is DIVA (differentiating infected from vaccinated animals) compatible and represents a promising vaccine platform for RVFV infection in susceptible species. PMID:25325319

  16. Receptor choice determinants in the envelope glycoproteins of amphotropic, xenotropic, and polytropic murine leukemia viruses.

    PubMed Central

    Battini, J L; Heard, J M; Danos, O

    1992-01-01

    The envelope glycoproteins (SU) of mammalian type C retroviruses possess an amino-terminal domain of about 200 residues, which is involved in binding a cell surface receptor. In this domain, highly conserved amino acid sequences are interrupted by two segments of variable length and sequence, VRA and VRB. We have studied the role of these variable regions in receptor recognition and binding by constructing chimeric molecules in which portions of the amino-terminal domains from amphotropic (4070A), xenotropic (NZB), and polytropic (MCF 247) murine leukemia virus SU proteins were permuted. These chimeras, which exchanged either one or two variable regions, were expressed at the surface of replication-defective viral particles by a pseudotyping assay. Wild-type or recombinant env genes were transfected into a cell line producing Moloney murine leukemia virus particles devoid of envelope glycoproteins in which a retrovirus vector genome carrying an Escherichia coli lacZ gene was packaged. The host range and sensitivity to interference of pseudotyped virions were assayed, and we observed which permutations resulted in receptor switch or loss of function. Our results indicate that the determinants of receptor choice are found within the just 120 amino acids of SU proteins. Downstream sequences contribute to the stabilization of the receptor-specific structure. PMID:1310758

  17. Folding of rabies virus glycoprotein: epitope acquisition and interaction with endoplasmic reticulum chaperones.

    PubMed Central

    Gaudin, Y

    1997-01-01

    Four well-characterized monoclonal antibodies (MAbs) directed against rabies virus glycoprotein (G) were used to study G folding in vivo. Two of the MAbs were able to immunoprecipitate incompletely oxidized folding intermediates. The two others recognized G only after folding was completed. By using these MAbs, the ability of G to undergo low-pH-induced conformational changes during folding was also investigated. It appeared that some domains acquire this ability before folding is completed. In addition, interactions between unfolded G and some of the molecular chaperones were analyzed. Unfolded G was associated with BiP and calnexin. Association with BiP was maximal immediately after the pulse, whereas association with calnexin was maximal after 5 to 10 min of chase. The effects of tunicamycin and castanospermine on chaperone binding and folding were also studied. In the presence of both drugs, calnexin binding was reduced, consistent with the view that calnexin specifically recognizes monoglucosylated oligosaccharides, but some residual binding was still observed, indicating that calnexin also recognizes the polypeptide chain. In the presence of both drugs, association with BiP was increased and prolonged and folding was impaired. However, the global effects of the drugs were different, since folding was much more efficient in the presence of castanospermine than in the presence of tunicamycin. Taken together, these results provide the basis to draw a schematic view of rabies virus glycoprotein folding. PMID:9094649

  18. Serological responses in chimpanzees inoculated with human immunodeficiency virus glycoprotein (gp120) subunit vaccine

    SciTech Connect

    Arthur, L.O.; Pyle, S.W.; Nara, P.L.; Bess, J.W. Jr.; Gonda, M.A.; Kelliher, J.C.; Gilden, R.V.; Robey, W.G.; Bolognesi, D.P.; Gallo, R.C.

    1987-12-01

    The major envelope glycoprotein of a human immunodeficiency virus (HIV) has been purified and was utilized as a prototype vaccine in chimpanzees. The 120,000-dalton glycoprotein (gp120) was purified from membranes of human T-lymphotropic virus (HTLV)-IIIB-infected cells and the final preparation contained low levels to no detectable HTLV-IIIB core antigen (p24) and low levels of endotoxin. Chimpanzees inoculated with gp120 responded by developing antibodies that precipitated radiolabeled gp120 and neutralized in vitro infection of HTLV-IIIB. Antibodies to HTLV-IIIB p24 were not detected in the gp120-immunized chimpanzees. Peripheral blood leukocytes from the vaccinated animals were examined for T4/sup +/ and T8/sup +/ cells, and no decrease in the T4/T8 ratio was found, indicating that immunization with a ligand (gp120) that binds to T4 has not detectable adverse effect on the population of T4/sup +/ cells. The only current animal model that can be reproducibly infected with HIV is the chimpanzee. Immunization of chimpanzees with HIV proteins will provide an experimental system for testing the effectiveness of prototype vaccines for preventing HIV infection in vivo.

  19. Membrane proteins specified by herpes simplex viruses. V. Identification of an Fc-binding glycoprotein.

    PubMed Central

    Baucke, R B; Spear, P G

    1979-01-01

    A glycoprotein with affinity for the Fc region of immunoglobulin was isolated from extracts of cultured cells infected with herpes simplex virus type 1, and experiments were done to characterize its properties and to investigate whether it could account for the Fc-binding activity previously demonstrated on the surfaces of intact herpes simplex virus-infected cells. The technique of affinity chromatography was used to identify and isolate the Fc-binding glycoprotein and to demonstrate the specificity of its interaction with immunoglobulin G-Fc. Although three electrophoretically distinguishable Fc-binding polypeptides were identified by affinity chromatography, these three species appear to be different forms of the same translation product, based on comparisons of proteolytic digestion products and on the kinetics of appearance of each form after a brief pulse with radioactive amino acids. The results suggest that one polypeptide, designated pE, is processed to yield gE1, which is in turn processed to yield gE2. Both gE1 and gE2 are glycosylated membrane proteins and both can be labeled by the lactoperoxidase-catalyzed radioiodination of intact infected cells, indicating the presence of these proteins in surface membranes of the cells. Increases in the amounts of gE1 and gE2 at the cell surface were found to parallel the increase in Fc-binding activity of intact infected cells. Images PMID:229267

  20. Bioinformatics and evolutionary insight on the spike glycoprotein gene of QX-like and Massachusetts strains of infectious bronchitis virus

    PubMed Central

    2012-01-01

    Background Infectious bronchitis virus (IBV) is a Gammacoronavirus of the family Coronaviridae and is a causative agent of an economically important disease in poultry. The spike glycoprotein of IBV is essential for host cell attachment, neutralization, and is involved in the induction of protective immunity. Previously obtained sequence data of the spike gene of IBV QX-like and Massachusetts strains were subjected to bioinformatics analysis. Findings On analysis of potential phosphorylation sites, the Ser542 and Ser563 sites were not present in Massachusetts strains, while QX-like isolates did not have the Ser534 site. Massachusetts and QX-like strains showed different cleavage site motifs. The N-glycosylation sites ASN-XAA-SER/THR-55, 147, 200 and 545 were additionally present in QX-like strains. The leucine-rich repeat regions in Massachusetts strains consisted of stretches of 63 to 69 amino acids, while in the QX-like strains they contained 59 amino acids in length. An additional palmitoylation site was observed in CK/SWE/082066/2010 a QX-like strain. Primary structure data showed difference in the physical properties and hydrophobic nature of both genotypes. The comparison of secondary structures revealed no new structural domains in the genotypic variants. The phylogenetic analyses based on avian and mammalian coronaviruses showed the analysed IBV as closely related to turkey coronaviruses and distantly related to thrush and munia coronaviruses. Conclusion The study demonstrated that spike glycoprotein of the Massachusetts and the QX-like variants of IBV are molecularly distinct and that this may reflect in differences in the behavior of these viruses in vivo. PMID:22992336

  1. Neutralizing antibodies in Borna disease virus-infected rats.

    PubMed Central

    Hatalski, C G; Kliche, S; Stitz, L; Lipkin, W I

    1995-01-01

    Borna disease is a neurologic syndrome caused by infection with a nonsegmented, negative-strand RNA virus, Borna disease virus. Infected animals have antibodies to two soluble viral proteins, p40 and p23, and a membrane-associated viral glycoprotein, gp18. We examined the time course for the development of neutralization activity and the expression of antibodies to individual viral proteins in sera of infected rats. The appearance of neutralizing activity correlated with the development of immunoreactivity to gp18, but not p40 or p23. Monospecific and monoclonal antibodies to native gp18 and recombinant nonglycosylated gp18 were also found to have neutralizing activity and to immunoprecipitate viral particles or subparticles. These findings suggest that gp18 is likely to be present on the surface of the viral particles and is likely to contain epitopes important for virus neutralization. PMID:7815538

  2. Structure of a Major Antigenic Site on the Respiratory Syncytial Virus Fusion Glycoprotein in Complex with Neutralizing Antibody 101F

    SciTech Connect

    McLellan, Jason S.; Chen, Man; Chang, Jung-San; Yang, Yongping; Kim, Albert; Graham, Barney S.; Kwong, Peter D.

    2010-11-19

    Respiratory syncytial virus (RSV) is a major cause of pneumonia and bronchiolitis in infants and elderly people. Currently there is no effective vaccine against RSV, but passive prophylaxis with neutralizing antibodies reduces hospitalizations. To investigate the mechanism of antibody-mediated RSV neutralization, we undertook structure-function studies of monoclonal antibody 101F, which binds a linear epitope in the RSV fusion glycoprotein. Crystal structures of the 101F antigen-binding fragment in complex with peptides from the fusion glycoprotein defined both the extent of the linear epitope and the interactions of residues that are mutated in antibody escape variants. The structure allowed for modeling of 101F in complex with trimers of the fusion glycoprotein, and the resulting models suggested that 101F may contact additional surfaces located outside the linear epitope. This hypothesis was supported by surface plasmon resonance experiments that demonstrated 101F bound the peptide epitope {approx}16,000-fold more weakly than the fusion glycoprotein. The modeling also showed no substantial clashes between 101F and the fusion glycoprotein in either the pre- or postfusion state, and cell-based assays indicated that 101F neutralization was not associated with blocking virus attachment. Collectively, these results provide a structural basis for RSV neutralization by antibodies that target a major antigenic site on the fusion glycoprotein.

  3. Pathogenicity of Hantaan Virus in Newborn Mice: Genetic Reassortant Study Demonstrating that a Single Amino Acid Change in Glycoprotein G1 Is Related to Virulence

    PubMed Central

    Ebihara, Hideki; Yoshimatsu, Kumiko; Ogino, Michiko; Araki, Koichi; Ami, Yasushi; Kariwa, Hiroaki; Takashima, Ikuo; Li, Dexin; Arikawa, Jiro

    2000-01-01

    Two Hantaan virus strains, clone 1 (cl-1), which is virulent in newborn mice, and its attenuated mutant (mu11E10), were used to examine the pathogenesis of Hantaan virus infection in a mouse model and identify virus factors relating to virulence. After subcutaneous inoculation of newborn BALB/c mice, cl-1 caused fatal disease with high viral multiplication in peripheral organs, but mu11E10 produced nonfatal infection with a low level of virus multiplication. Intracerebral inoculation of either strain caused fatal disease. Histopathological changes in the dead animals were prominent in the brain, indicating that the brain is the target organ and produces the fatal outcome. These results indicate that mu11E10 has a generally less virulent phenotype, and because of decreased multiplication in peripheral tissues, neuroinvasiveness is also decreased. An experiment with genetic reassortant viruses showed that in newborn mice the M segment is the most related to virulence and the L segment is partly related. Sequence comparison detected a single deduced amino acid change (cl-1 Ile to mu11E10 Thr) at amino acid number 515 in glycoprotein G1. One nucleotide change, but no amino acid substitution, was observed in the noncoding region of the L segment. In mouse brain microvascular endothelial cells in vitro, viruses possessing a cl-1-derived M segment grew more rapidly than viruses containing a mu11E10-derived M segment. These results suggest that the single amino acid change in the glycoprotein alters peripheral growth, which affects invasion of the central nervous system in mice. PMID:10982372

  4. Ebola Virus Infections in Nonhuman Primates Are Temporally Influenced by Glycoprotein Poly-U Editing Site Populations in the Exposure Material

    PubMed Central

    Trefry, John C.; Wollen, Suzanne E.; Nasar, Farooq; Shamblin, Joshua D.; Kern, Steven J.; Bearss, Jeremy J.; Jefferson, Michelle A.; Chance, Taylor B.; Kugelman, Jeffery R.; Ladner, Jason T.; Honko, Anna N.; Kobs, Dean J.; Wending, Morgan Q.S.; Sabourin, Carol L.; Pratt, William D.; Palacios, Gustavo F.; Pitt, M. Louise M.

    2015-01-01

    Recent experimentation with the variants of the Ebola virus that differ in the glycoprotein’s poly-uridine site, which dictates the form of glycoprotein produced through a transcriptional stutter, has resulted in questions regarding the pathogenicity and lethality of the stocks used to develop products currently undergoing human clinical trials to combat the disease. In order to address these concerns and prevent the delay of these critical research programs, we designed an experiment that permitted us to intramuscularly challenge statistically significant numbers of naïve and vaccinated cynomolgus macaques with either a 7U or 8U variant of the Ebola virus, Kikwit isolate. In naïve animals, no difference in survivorship was observed; however, there was a significant delay in the disease course between the two groups. Significant differences were also observed in time-of-fever, serum chemistry, and hematology. In vaccinated animals, there was no statistical difference in survivorship between either challenge groups, with two succumbing in the 7U group compared to 1 in the 8U challenge group. In summary, survivorship was not affected, but the Ebola virus disease course in nonhuman primates is temporally influenced by glycoprotein poly-U editing site populations. PMID:26703716

  5. Aberrant trafficking of hepatitis B virus glycoproteins in cells in which N-glycan processing is inhibited

    PubMed Central

    Lu, Xuanyong; Mehta, Anand; Dadmarz, Mitra; Dwek, Raymond; Blumberg, Baruch S.; Block, Timothy M.

    1997-01-01

    The role of N-glycan trimming in glycoprotein fate and function is unclear. We have recently shown that hepatitis B virus (HBV) DNA is not efficiently secreted from cells in which α-glucosidase mediated N-glycan trimming is inhibited. Here it is shown that, in cells in glucosidase-inhibited cells, viral DNA, accompanied by envelope and core proteins, most likely accumulate within lysosomal compartments. Pulse–chase experiments show that although the viral glycoproteins (L, M, and S) are dysfunctional, in the sense that they do not mediate virion egress and are not efficiently secreted from the cell, they all still leave the endoplasmic reticulum (ER). Surprisingly, however, the glycoproteins retained within the cell were not rapidly degraded, appearing as aggregates, enriched for L and M, with intracellular half-lives exceeding 20 h. Moreover, by 24 h after synthesis, a substantial fraction of the detained glycoproteins appeared to return to the ER, although a considerable amount was also found in the lysosomes. To our knowledge, this is the first report that shows, as a consequence of inhibiting glycosylation processing, certain glycoproteins (i) become dysfunctional and aggregate, yet still depart from the ER, and (ii) have extended rather than shortened half-lives. Taken together, these data suggest that proper intracellular routing of HBV glycoproteins requires ER glucosidase function. It is hypothesized that failure to process N-glycan causes HBV glycoproteins to aggregate and that impaired protein–protein interactions and trafficking are the result of misfolding. PMID:9122203

  6. The measles virus (MV) glycoproteins interact with cellular chaperones in the endoplasmic reticulum and MV infection upregulates chaperone expression.

    PubMed

    Bolt, G

    2001-01-01

    The present study examines the coprecipitation of measles virus (MV) glycoproteins with host cell endoplasmic reticulum (ER) chaperone proteins. Both the haemagglutinin (H) and fusion (F) glycoproteins interacted with calnexin and GRP78, whereas interaction with calreticulin was only demonstrated for the H glycoprotein. The alpha-glucosidase inhibitor castanospermine reduced and delayed the association of F proteins with calnexin. We have previously shown that alpha-glucosidase activity is important for the functionality and antigenicity of the MV F glycoprotein and for release of MV particles from infected cells. Thus, interaction with calnexin appears vital for processing of nascent MV F protein into its functional conformation. In contrast to many other viral glycoproteins, a substantial proportion of the pulsed MV glycoproteins remained associated with ER chaperones for more than 2(1/2) h. Thus, the slow and incomplete migration of MV glycoproteins to the cell surface may result from their retention by ER chaperones, probably due to malfolding. MV infection upregulated the cellular expression of calreticulin and GRP78 and also increased their presence at the cell surface. The chaperone proteins are involved in a wide range of cellular processes, and their induction by MV may play a role for the pathogenesis of measles and its sequelae. PMID:11765911

  7. Crystal Structure of the Pre-fusion Nipah Virus Fusion Glycoprotein Reveals a Novel Hexamer-of-Trimers Assembly

    PubMed Central

    Dutta, Somnath; Yan, Lianying; Feng, YanRu; Wang, Lin-Fa; Skiniotis, Georgios; Lee, Benhur; Zhou, Z. Hong; Broder, Christopher C.; Aguilar, Hector C.; Nikolov, Dimitar B.

    2015-01-01

    Nipah virus (NiV) is a paramyxovirus that infects host cells through the coordinated efforts of two envelope glycoproteins. The G glycoprotein attaches to cell receptors, triggering the fusion (F) glycoprotein to execute membrane fusion. Here we report the first crystal structure of the pre-fusion form of the NiV-F glycoprotein ectodomain. Interestingly this structure also revealed a hexamer-of-trimers encircling a central axis. Electron tomography of Nipah virus-like particles supported the hexameric pre-fusion model, and biochemical analyses supported the hexamer-of-trimers F assembly in solution. Importantly, structure-assisted site-directed mutagenesis of the interfaces between F trimers highlighted the functional relevance of the hexameric assembly. Shown here, in both cell-cell fusion and virus-cell fusion systems, our results suggested that this hexamer-of-trimers assembly was important during fusion pore formation. We propose that this assembly would stabilize the pre-fusion F conformation prior to cell attachment and facilitate the coordinated transition to a post-fusion conformation of all six F trimers upon triggering of a single trimer. Together, our data reveal a novel and functional pre-fusion architecture of a paramyxoviral fusion glycoprotein. PMID:26646856

  8. Crystal structure of glycoprotein E2 from bovine viral diarrhea virus

    PubMed Central

    Li, Yue; Wang, Jimin; Kanai, Ryuta; Modis, Yorgo

    2013-01-01

    Pestiviruses, including bovine viral diarrhea virus, are important animal pathogens and are closely related to hepatitis C virus, which remains a major global health threat. They have an outer lipid envelope bearing two glycoproteins, E1 and E2, required for cell entry. They deliver their genome into the host cell cytoplasm by fusion of their envelope with a cellular membrane. The crystal structure of bovine viral diarrhea virus E2 reveals a unique protein architecture consisting of two Ig-like domains followed by an elongated β-stranded domain with a new fold. E2 forms end-to-end homodimers with a conserved C-terminal motif rich in aromatic residues at the contact. A disulfide bond across the interface explains the acid resistance of pestiviruses and their requirement for a redox activation step to initiate fusion. From the structure of E2, we propose alternative possible membrane fusion mechanisms. We expect the pestivirus fusion apparatus to be conserved in hepatitis C virus. PMID:23569276

  9. Amino-terminal sequence of glycoprotein D of herpes simplex virus types 1 and 2

    SciTech Connect

    Eisenberg, R.J.; Long, D.; Hogue-Angeletti, R.; Cohen, G.H.

    1984-01-01

    Glycoprotein D (gD) of herpes simplex virus is a structural component of the virion envelope which stimulates production of high titers of herpes simplex virus type-common neutralizing antibody. The authors caried out automated N-terminal amino acid sequencing studies on radiolabeled preparations of gD-1 (gD of herpes simplex virus type 1) and gD-2 (gD of herpes simplex virus type 2). Although some differences were noted, particularly in the methionine and alanine profiles for gD-1 and gD-2, the amino acid sequence of a number of the first 30 residues of the amino terminus of gD-1 and gD-2 appears to be quite similar. For both proteins, the first residue is a lysine. When we compared out sequence data for gD-1 with those predicted by nucleic acid sequencing, the two sequences could be aligned (with one exception) starting at residue 26 (lysine) of the predicted sequence. Thus, the first 25 amino acids of the predicted sequence are absent from the polypeptides isolated from infected cells.

  10. Ability of the Encephalitic Arbovirus Semliki Forest Virus To Cross the Blood-Brain Barrier Is Determined by the Charge of the E2 Glycoprotein

    PubMed Central

    Ferguson, Mhairi C.; Saul, Sirle; Fragkoudis, Rennos; Weisheit, Sabine; Cox, Jonathan; Patabendige, Adjanie; Sherwood, Karen; Watson, Mick; Merits, Andres

    2015-01-01

    ABSTRACT Semliki Forest virus (SFV) provides a well-characterized model system to study the pathogenesis of virus encephalitis. Several studies have used virus derived from the molecular clone SFV4. SFV4 virus does not have the same phenotype as the closely related L10 or the prototype virus from which its molecular clone was derived. In mice, L10 generates a high-titer plasma viremia, is efficiently neuroinvasive, and produces a fatal panencephalitis, whereas low-dose SFV4 produces a low-titer viremia, rarely enters the brain, and generally is avirulent. To determine the genetic differences responsible, the consensus sequence of L10 was determined and compared to that of SFV4. Of the 12 nucleotide differences, six were nonsynonymous; these were engineered into a new molecular clone, termed SFV6. The derived virus, SFV6, generated a high-titer viremia and was efficiently neuroinvasive and virulent. The phenotypic difference mapped to a single amino acid residue at position 162 in the E2 envelope glycoprotein (lysine in SFV4, glutamic acid in SFV6). Analysis of the L10 virus showed it contained different plaque phenotypes which differed in virulence. A lysine at E2 247 conferred a small-plaque avirulent phenotype and glutamic acid a large-plaque virulent phenotype. Viruses with a positively charged lysine at E2 162 or 247 were more reliant on glycosaminoglycans (GAGs) to enter cells and were selected for by passage in BHK-21 cells. Interestingly, viruses with the greatest reliance on binding to GAGs replicated to higher titers in the brain and more efficiently crossed an in vitro blood-brain barrier (BBB). IMPORTANCE Virus encephalitis is a major disease, and alphaviruses, as highlighted by the recent epidemic of chikungunya virus (CHIKV), are medically important pathogens. In addition, alphaviruses provide well-studied experimental systems with extensive literature, many tools, and easy genetic modification. In this study, we elucidate the genetic basis for the

  11. Efficient generation of vesicular stomatitis virus (VSV)-pseudotypes bearing morbilliviral glycoproteins and their use in quantifying virus neutralising antibodies

    PubMed Central

    Logan, Nicola; McMonagle, Elizabeth; Drew, Angharad A.; Takahashi, Emi; McDonald, Michael; Baron, Michael D.; Gilbert, Martin; Cleaveland, Sarah; Haydon, Daniel T.; Hosie, Margaret J.; Willett, Brian J.

    2016-01-01

    Morbillivirus neutralising antibodies are traditionally measured using either plaque reduction neutralisation tests (PRNTs) or live virus microneutralisation tests (micro-NTs). While both test formats provide a reliable assessment of the strength and specificity of the humoral response, they are restricted by the limited number of viral strains that can be studied and often present significant biological safety concerns to the operator. In this study, we describe the adaptation of a replication-defective vesicular stomatitis virus (VSVΔG) based pseudotyping system for the measurement of morbillivirus neutralising antibodies. By expressing the haemagglutinin (H) and fusion (F) proteins of canine distemper virus (CDV) on VSVΔG pseudotypes bearing a luciferase marker gene, neutralising antibody titres could be measured rapidly and with high sensitivity. Further, by exchanging the glycoprotein expression construct, responses against distinct viral strains or species may be measured. Using this technique, we demonstrate cross neutralisation between CDV and peste des petits ruminants virus (PPRV). As an example of the value of the technique, we demonstrate that UK dogs vary in the breadth of immunity induced by CDV vaccination; in some dogs the neutralising response is CDV-specific while, in others, the neutralising response extends to the ruminant morbillivirus PPRV. This technique will facilitate a comprehensive comparison of cross-neutralisation to be conducted across the morbilliviruses. PMID:26706278

  12. Efficient generation of vesicular stomatitis virus (VSV)-pseudotypes bearing morbilliviral glycoproteins and their use in quantifying virus neutralising antibodies.

    PubMed

    Logan, Nicola; McMonagle, Elizabeth; Drew, Angharad A; Takahashi, Emi; McDonald, Michael; Baron, Michael D; Gilbert, Martin; Cleaveland, Sarah; Haydon, Daniel T; Hosie, Margaret J; Willett, Brian J

    2016-02-01

    Morbillivirus neutralising antibodies are traditionally measured using either plaque reduction neutralisation tests (PRNTs) or live virus microneutralisation tests (micro-NTs). While both test formats provide a reliable assessment of the strength and specificity of the humoral response, they are restricted by the limited number of viral strains that can be studied and often present significant biological safety concerns to the operator. In this study, we describe the adaptation of a replication-defective vesicular stomatitis virus (VSVΔG) based pseudotyping system for the measurement of morbillivirus neutralising antibodies. By expressing the haemagglutinin (H) and fusion (F) proteins of canine distemper virus (CDV) on VSVΔG pseudotypes bearing a luciferase marker gene, neutralising antibody titres could be measured rapidly and with high sensitivity. Further, by exchanging the glycoprotein expression construct, responses against distinct viral strains or species may be measured. Using this technique, we demonstrate cross neutralisation between CDV and peste des petits ruminants virus (PPRV). As an example of the value of the technique, we demonstrate that UK dogs vary in the breadth of immunity induced by CDV vaccination; in some dogs the neutralising response is CDV-specific while, in others, the neutralising response extends to the ruminant morbillivirus PPRV. This technique will facilitate a comprehensive comparison of cross-neutralisation to be conducted across the morbilliviruses. PMID:26706278

  13. Characterization of epitopes on the rabies virus glycoprotein by selection and analysis of escape mutants.

    PubMed

    Fallahi, Firouzeh; Wandeler, Alexander I; Nadin-Davis, Susan A

    2016-07-15

    The glycoprotein (G) is the only surface protein of the lyssavirus particle and the only viral product known to be capable of eliciting the production of neutralizing antibodies. In this study, the isolation of escape mutants resistant to monoclonal antibody (Mab) neutralization was attempted by a selection strategy employing four distinct rabies virus strains: the extensively passaged Evelyn Rokitnicki Abelseth (ERA) strain and three field isolates representing two bat-associated variants and the Western Canada skunk variant (WSKV). No escape mutants were generated from either of the bat-associated viral variants but two neutralization mutants were derived from the WSKV isolate. Seven independent ERA mutants were recovered using Mabs directed against antigenic sites I (four mutants) and IIIa (three mutants) of the glycoprotein. The cross-neutralization patterns of these viral mutants were used to determine the precise location and nature of the G protein epitopes recognized by these Mabs. Nucleotide sequencing of the G gene indicated that those mutants derived using Mabs directed to antigenic site (AS) III all contained amino acid substitutions in this site. However, of the four mutants selected with AS I Mabs, two bore mutations within AS I as expected while the remaining two carried mutations in AS II. WSKV mutants exhibited mutations at the sites appropriate for the Mabs used in their selection. All ERA mutant preparations were more cytopathogenic than the parental virus when propagated in cell culture; when in vivo pathogenicity in mice was examined, three of these mutants exhibited reduced pathogenicity while the remaining four mutants exhibited comparable pathogenic properties to those of the parent virus. PMID:27132040

  14. Immunization with recombinant varicella-zoster virus expressing herpes simplex virus type 2 glycoprotein D reduces the severity of genital herpes in guinea pigs.

    PubMed Central

    Heineman, T C; Connelly, B L; Bourne, N; Stanberry, L R; Cohen, J

    1995-01-01

    Varicella-zoster virus (VZV) is an attractive candidate for a live-virus vector for the delivery of foreign antigens. The Oka vaccine strain of VZV is safe and effective in humans, and recombinant Oka VZV (ROka) can be generated by transfecting cells with a set of overlapping cosmid DNAs. By this method, the herpes simplex virus type 2 (HSV-2) glycoprotein D (gD2) gene was inserted into an intergenic site in the unique short region of the Oka VZV genome. Expression of gD2 in cells infected with the recombinant Oka strain VZV (ROka-gD2) was confirmed by antibody staining of fixed cells and by immunoblot analysis. Immune electron microscopy demonstrated the presence of gD2 in the envelope of ROka-gD2 virions. The ability of ROka-gD2 to protect guinea pigs against HSV-2 challenge was assessed by inoculating animals with three doses of uninfected human fibroblasts, fibroblasts infected with ROka VZV, or fibroblasts infected with ROka-gD2. Neutralizing antibodies specific for HSV-2 developed in animals immunized with ROka-gD2. Forty days after the third inoculation, animals were challenged intravaginally with HSV-2. Inoculation of guinea pigs with ROka-gD2 significantly reduced the severity of primary HSV-2 infection (P < 0.001). These experiments demonstrate that the Oka strain of VZV can be used as a live virus vector to protect animals from disease with a heterologous virus. PMID:7494331

  15. Quantitative serology assays for determination of antibody responses to Ebola virus glycoprotein and matrix protein in nonhuman primates and humans.

    PubMed

    Vu, Hong; Shulenin, Sergey; Grolla, Allen; Audet, Jonathan; He, Shihua; Kobinger, Gary; Unfer, Robert C; Warfield, Kelly L; Aman, M Javad; Holtsberg, Frederick W

    2016-02-01

    The West Africa Ebola virus disease (EVD) outbreak has reached unprecedented magnitude and caused worldwide concerns for the spread of this deadly virus. Recent findings in nonhuman primates (NHPs) demonstrate that antibodies can be protective against EVD. However, the role of antibody response in vaccine-mediated protection is not fully understood. To address these questions quantitative serology assays are needed for measurement of the antibody response to key Ebola virus (EBOV) proteins. Serology enzyme-linked immunosorbent assays (ELISA's), using a reference detection antibody, were developed in order to standardize the quantitation of antibody levels in vaccinated NHPs or in humans exposed to EBOV or immunized with an EBOV vaccine. Critical reagents were generated to support the development of the serology ELISAs. Recombinant EBOV matrix protein (VP40) was expressed in Escherichia coli and purified. Two variants of the glycoprotein (GP), the ectodomain lacking the transmembrane domain (GPΔTM), and an engineered GP lacking the mucin-like domain (GPΔmuc) were expressed and purified from mammalian cell systems. Using these proteins, three ELISA methods were developed and optimized for reproducibility and robustness, including stability testing of critical reagents. The assay was used to determine the antibody response against VP40, GPΔTM, and GPΔmuc in a NHP vaccine study using EBOV virus-like particles (VLP) vaccine expressing GP, VP40 and the nucleoprotein. Additionally, these ELISAs were used to successfully detect antibody responses to VP40, GPΔTM and GPΔmuc in human sera from EBOV infected individuals. PMID:26681387

  16. Transduction of motor neurons and muscle fibers by intramuscular injection of HIV-1-based vectors pseudotyped with select rabies virus glycoproteins.

    PubMed

    Mentis, George Z; Gravell, Maneth; Hamilton, Rebecca; Shneider, Neil A; O'Donovan, Michael J; Schubert, Manfred

    2006-10-30

    For studies of motor neuron function or for therapeutic purposes, novel pseudotype HIV-1-based vectors were developed that are capable of expressing transgenes in motor neurons following injection into mouse hind limb muscles. To specifically target motor neurons, glycoproteins from two rabies virus (RV) isolates, the mouse-brain adapted challenge virus 24 (CVS-24) variants, CVS-N2c and CVS-B2c were evaluated for pseudotype formation with an HIV-1-based vector. Both RV glycoproteins incorporated into vector envelopes, and both pseudotypes yielded high titers with Hek293T and cortical plate neuron cultures. Increased neuronotropism by the CVS-N2c pseudotype was not observed, suggesting that vector tropism is not solely determined by the fusogenic viral glycoprotein. Vector injection into hind limb muscles resulted in EYFP reporter gene expression in the injected muscle fibers and in spinal cord motor neurons innervating the same muscle, indicating retrograde vector transport. Intramuscular vector injections into the soleus and tibialis anterior muscles transduced 26% and 16% of all motor neurons in each motor nucleus, respectively. These transduction efficiencies may allow novel approaches to functional studies of the motor system and the treatment of neuromuscular disease. PMID:16725205

  17. A Substitution in the Transmembrane Region of the Glycoprotein Leads to an Unstable Attenuation of Machupo Virus

    PubMed Central

    Patterson, Michael; Koma, Takaaki; Seregin, Alexey; Huang, Cheng; Miller, Milagros; Smith, Jennifer; Yun, Nadezhda; Smith, Jeanon

    2014-01-01

    Machupo virus (MACV) is the etiologic agent of Bolivian hemorrhagic fever (BHF). Utilizing a reverse-genetics system recently developed, we report the rescue of a rationally modified recombinant MACV containing a single mutation in the transmembrane region of the glycoprotein. Following challenge of susceptible mice, we identified a significant reduction in virulence in the novel virus. We also identified an instability leading to reversion of the single mutation to a wild-type genotype. PMID:25031335

  18. New monoclonal antibodies to the Ebola virus glycoprotein: Identification and analysis of the amino acid sequence of the variable domains.

    PubMed

    Panina, A A; Aliev, T K; Shemchukova, O B; Dement'yeva, I G; Varlamov, N E; Pozdnyakova, L P; Bokov, M N; Dolgikh, D A; Sveshnikov, P G; Kirpichnikov, M P

    2016-03-01

    We determined the nucleotide and amino acid sequences of variable domains of three new monoclonal antibodies to the glycoprotein of Ebola virus capsid. The framework and hypervariable regions of immunoglobulin heavy and light chains were identified. The primary structures were confirmed using massspectrometry analysis. Immunoglobulin database search showed the uniqueness of the sequences obtained. PMID:27193713

  19. A recombinant measles vaccine virus expressing wild-type glycoproteins: consequences for viral spread and cell tropism.

    PubMed

    Johnston, I C; ter Meulen, V; Schneider-Schaulies, J; Schneider-Schaulies, S

    1999-08-01

    Wild-type, lymphotropic strains of measles virus (MV) and tissue culture-adapted MV vaccine strains possess different cell tropisms. This observation has led to attempts to identify the viral receptors and to characterize the functions of the MV glycoproteins. We have functionally analyzed the interactions of MV hemagglutinin (H) and fusion (F) proteins of vaccine (Edmonston) and wild-type (WTF) strains in different combinations in transfected cells. Cell-cell fusion occurs when both Edmonston F and H proteins are expressed in HeLa or Vero cells. The expression of WTF glycoproteins in HeLa cells did not result in syncytia, yet they fused efficiently with cells of lymphocytic origin. To further investigate the role of the MV glycoproteins in virus cell entry and also the role of other viral proteins in cell tropism, we generated recombinant vaccine MVs containing one or both glycoproteins from WTF. These viruses were viable and grew similarly in lymphocytic cells. Recombinant viruses expressing the WTFH protein showed a restricted spread in HeLa cells but spread efficiently in Vero cells. Parental WTF remained restricted in both cell types. Therefore, not only differential receptor usage but also other cell-specific factors are important in determining MV cell tropism. PMID:10400788

  20. N-linked Glycosylation of Classical Swine Fever Virus Strain Brescia Erns Glycoprotein Alters Virulence in Swine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Erns is one of the three envelope glycoproteins of Classical Swine Fever Virus (CSFV). We recently reported the influence of glycosylation of E2 in the virulence of CSFV strain Brescia. Here, we studied the effect of Erns N-linked glycosylation pattern on virulence of CSFV strain Brescia in swine. ...

  1. Herpes simplex viruses lacking glycoprotein D are unable to inhibit virus penetration: quantitative evidence for virus-specific cell surface receptors

    SciTech Connect

    Johnson, D.C.; Ligas, M.W.

    1988-12-01

    Herpes simplex virus (HSV) glycoprotein D (gD) plays an essential role in the entry of virus into cells. HSV mutants unable to express gD were constructed. The mutants can be propagated on VD60 cells, which supply the viruses with gD; however, virus particles lacking gD were produced in mutant-infected Vero cells. Virus particles with or without gD adsorbed to a large number of sites on the cell surface; however, virions lacking gD did not enter cells. Cells pretreated with UV-inactivated virions containing gD were resistant to infection with HSV type 1 (HSV-1) and HSV-2. In contrast, cell pretreated with UV-inactivated virions lacking gD could be infected with HSV-1 and HSV-2. If infectious HSV-1 was added prior to UV-inactivated virus particles containing gD, the infectious virus entered cells and replicated. Therefore, virus particles containing gD appear to block specific cell surface receptors which are very limited in number. Particles lacking gD are presumably unable to interact with these receptors, suggesting that gD is an essential receptor-binding polypeptide.

  2. Proteolytic cleavage of glycoprotein B is dispensable for in vitro replication, but required for syncytium formation of pseudorabies virus.

    PubMed

    Okazaki, Katsunori

    2007-07-01

    Glycoprotein B (gB) is the most conserved glycoprotein among herpesviruses and it plays important roles in virus infectivity. In most herpesviruses, including pseudorabies virus (PRV), gB is cleaved by a cellular protease into two disulfide-linked subunits. In the present study, I found that the PRV gB generated in human colon carcinoma LoVo cells, which lack the ubiquitous protease furin, remained in the uncleaved form and the virus replicated in these cells without cell fusion. The uncleaved gB was converted into its subunits after furin digestion. The virus also replicated in Madin-Darby bovine kidney cells without cell fusion in the presence of a furin inhibitor, whereas distinct syncytia were formed in the absence of the inhibitor. LoVo cells constitutively expressing furin showed cell fusion when they were infected with the virus. Penetration kinetics assays revealed that the virus carrying uncleaved gB penetrated the cells at the same rate as the virus carrying cleaved gB. These results indicate that PRV gB is cleaved by furin and that the cleavage is dispensable for virus replication in vitro. Furthermore, gB cleavage is involved in syncytium formation but not in penetration kinetics, suggesting that different mechanisms operate between cell-cell fusion and virus-cell fusion by PRV. PMID:17554016

  3. Molecular characterization of E2 glycoprotein of classical swine fever virus: adaptation and propagation in porcine kidney cells.

    PubMed

    Kumar, Rakesh; Barman, Nagendra N; Khatoon, Elina; Rajbongshi, Gitika; Deka, Nipu; Morla, Sudhir; Kumar, Sachin

    2015-05-01

    Classical swine fever virus (CSFV) is the causative agent of a highly contagious disease, hog cholera in pigs. The disease is endemic in many parts of the world, and vaccination is the only way to protect the animals from CSFV infection. The lapinized vaccine strains are occasionally not protective because of animal to animal passage, inadequate vaccination strategy, suboptimal vaccine dose, and emergence of new variants. The surface glycoprotein E2 of CSFV is a major antigenic determinant and can modulate the disease outcome in pigs. In the present study, we characterized the CSFV in porcine kidney cells. The CSFV vaccine strains showed enhanced replication following 15 passages in porcine kidney cells. Nucleotide sequence analysis of the E2 protein gene of the cell culture-adapted vaccine strain of CSFV showed a mutation in putative amino acid sequences that are identical to its virulent counterpart. The study suggests the possibility of exaltation in vaccine strains following its adaptation in host cells and paves the way for a further exploration of the biology of its outbreak. PMID:25552311

  4. Detection of asymptomatic initial herpes simplex virus (HSV) infections in animals immunized with subunit HSV glycoprotein vaccines.

    PubMed Central

    Bernstein, D I; Ashley, R L; Stanberry, L R; Myers, M G

    1990-01-01

    The evaluation of herpes simplex virus (HSV) vaccine efficacy will require methods to detect asymptomatic acquisition of HSV infection and to assess the risk of recurrences in these patients. HSV-infected vaccinees should develop antibodies to HSV polypeptides not included in subunit vaccines. Sera from 57 HSV glycoprotein-vaccinated guinea pigs that had asymptomatic initial infections after genital HSV type 2 challenge were collected after vaccination but before HSV challenge and again 30 days after HSV challenge to determine the antibody response to HSV polypeptides. Antibodies to nonvaccine HSV polypeptides were detected in sera collected after viral challenge from 32 (56%) of these 57 animals. Twenty-six (81%) of the 32 animals with detectable antibody developed recurrent disease; however, recurrences also developed in 11 (44%) of the remaining 25 that did not show detectable antibody to nonvaccine HSV polypeptides. The magnitude of vaginal viral shedding during the initial disease period following challenge was significantly lower in animals that did not develop antibody to nonvaccine polypeptides compared with those that did develop antibody (area under the viral shedding curve, 5.2 +/- 3.2 versus 18.1 +/- 5.8; P less than 0.0001) . These data suggest that detection of antibody to nonvaccine HSV polypeptides will identify the majority (70%) of initially asymptomatic vaccinees that develop recurrent disease but that latency can be established even with markedly reduced levels of viral replication that did not induce a detectable antibody response. Images PMID:2153698

  5. Single-dose Live-attenuated Vesicular Stomatitis Virus-based Vaccine Protects African Green Monkeys from Nipah Virus Disease

    PubMed Central

    Prescott, Joseph; DeBuysscher, Blair L.; Feldmann, Friederike; Gardner, Donald J.; Haddock, Elaine; Martellaro, Cynthia; Scott, Dana; Feldmann, Heinz

    2015-01-01

    Nipah virus is a zoonotic paramyxovirus that causes severe respiratory and/or encephalitic disease in humans, often resulting in death. It is transmitted from pteropus fruit bats, which serve as the natural reservoir of the virus, and outbreaks occur on an almost annual basis in Bangladesh or India. Outbreaks are small and sporadic, and several cases of human-to-human transmission have been documented as an important feature of the epidemiology of Nipah virus disease. There are no approved countermeasures to combat infection and medical intervention is supportive. We recently generated a recombinant replication-competent vesicular stomatitis virus-based vaccine that encodes a Nipah virus glycoprotein as an antigen and is highly efficacious in the hamster model of Nipah virus disease. Herein, we show that this vaccine protects African green monkeys, a well-characterized model of Nipah virus disease, from disease one month after a single intramuscular administration of the vaccine. Vaccination resulted in a rapid and strong virus-specific immune response which inhibited virus shedding and replication. This vaccine platform provides a rapid means to afford protection from Nipah virus in an outbreak situation. PMID:25865472

  6. Single-dose live-attenuated vesicular stomatitis virus-based vaccine protects African green monkeys from Nipah virus disease.

    PubMed

    Prescott, Joseph; DeBuysscher, Blair L; Feldmann, Friederike; Gardner, Donald J; Haddock, Elaine; Martellaro, Cynthia; Scott, Dana; Feldmann, Heinz

    2015-06-01

    Nipah virus is a zoonotic paramyxovirus that causes severe respiratory and/or encephalitic disease in humans, often resulting in death. It is transmitted from pteropus fruit bats, which serve as the natural reservoir of the virus, and outbreaks occur on an almost annual basis in Bangladesh or India. Outbreaks are small and sporadic, and several cases of human-to-human transmission have been documented as an important feature of the epidemiology of Nipah virus disease. There are no approved countermeasures to combat infection and medical intervention is supportive. We recently generated a recombinant replication-competent vesicular stomatitis virus-based vaccine that encodes a Nipah virus glycoprotein as an antigen and is highly efficacious in the hamster model of Nipah virus disease. Herein, we show that this vaccine protects African green monkeys, a well-characterized model of Nipah virus disease, from disease one month after a single intramuscular administration of the vaccine. Vaccination resulted in a rapid and strong virus-specific immune response which inhibited virus shedding and replication. This vaccine platform provides a rapid means to afford protection from Nipah virus in an outbreak situation. PMID:25865472

  7. Immunogenicity and functional characterization of Leishmania-derived hepatitis C virus envelope glycoprotein complex.

    PubMed

    Grzyb, Katarzyna; Czarnota, Anna; Brzozowska, Agnieszka; Cieślik, Anna; Rąbalski, Łukasz; Tyborowska, Jolanta; Bieńkowska-Szewczyk, Krystyna

    2016-01-01

    Hepatitis C virus (HCV) envelope glycoproteins E1 and E2 are the main inducers of a cross-neutralizing antibody response which plays an important role in the early phase of viral infection. Correctly folded and immunologically active E1E2 complex can be expressed in mammalian cells, though the production process might still prove restrictive, even if the immunological response of a vaccine candidate is positive. Here, we report a characterization and immunogenicity study of a full-length (fE1E2) and soluble version of the E1E2 complex (tE1E2) from genotype 1a, successfully expressed in the cells of Leishmania tarentolae. In a functional study, we confirmed the binding of both Leishmania-derived E1E2 complexes to the CD-81 receptor and the presence of the major epitopes participating in a neutralizing antibody response. Both complexes were proved to be highly immunogenic in mice and elicited neutralizing antibody response. Moreover, cross-reactivity of the mouse sera was detected for all tested HCV genotypes with the highest signal intensity observed for genotypes 1a, 1b, 5 and 6. Since the development of a prophylactic vaccine against HCV is still needed to control the global infection, our Leishmania-derived E1E2 glycoproteins could be considered a potential cost-effective vaccine candidate. PMID:27481352

  8. Immunogenicity and functional characterization of Leishmania-derived hepatitis C virus envelope glycoprotein complex

    PubMed Central

    Grzyb, Katarzyna; Czarnota, Anna; Brzozowska, Agnieszka; Cieślik, Anna; Rąbalski, Łukasz; Tyborowska, Jolanta; Bieńkowska-Szewczyk, Krystyna

    2016-01-01

    Hepatitis C virus (HCV) envelope glycoproteins E1 and E2 are the main inducers of a cross-neutralizing antibody response which plays an important role in the early phase of viral infection. Correctly folded and immunologically active E1E2 complex can be expressed in mammalian cells, though the production process might still prove restrictive, even if the immunological response of a vaccine candidate is positive. Here, we report a characterization and immunogenicity study of a full-length (fE1E2) and soluble version of the E1E2 complex (tE1E2) from genotype 1a, successfully expressed in the cells of Leishmania tarentolae. In a functional study, we confirmed the binding of both Leishmania-derived E1E2 complexes to the CD-81 receptor and the presence of the major epitopes participating in a neutralizing antibody response. Both complexes were proved to be highly immunogenic in mice and elicited neutralizing antibody response. Moreover, cross-reactivity of the mouse sera was detected for all tested HCV genotypes with the highest signal intensity observed for genotypes 1a, 1b, 5 and 6. Since the development of a prophylactic vaccine against HCV is still needed to control the global infection, our Leishmania-derived E1E2 glycoproteins could be considered a potential cost-effective vaccine candidate. PMID:27481352

  9. Co-translational Processing of Glycoprotein 3 from Equine Arteritis Virus

    PubMed Central

    Matczuk, Anna Karolina; Kunec, Dušan; Veit, Michael

    2013-01-01

    Signal peptide cleavage and N-glycosylation of proteins are co-translational processes, but little is known about their interplay if they compete for adjacent sites. Here we report two unique findings for processing of glycoprotein 3 of equine arteritis virus. Glycoprotein 3 (Gp3) contains an N-terminal signal peptide, which is not removed, although bioinformatics predicts cleavage with high probability. There is an overlapping sequon, NNTT, adjacent to the signal peptide that we show to be glycosylated at both asparagines. Exchanging the overlapping sequon and blocking glycosylation allows signal peptide cleavage, indicating that carbohydrate attachment inhibits processing of a potentially cleavable signal peptide. Bioinformatics analyses suggest that a similar processing scheme may exist for some cellular proteins. Membrane fractionation and secretion experiments revealed that the signal peptide of Gp3 does not act as a membrane anchor, indicating that it is completely translocated into the lumen of the endoplasmic reticulum. Membrane attachment is caused by the hydrophobic C terminus of Gp3, which, however, does not span the membrane but rather attaches the protein peripherally to endoplasmic reticulum membranes. PMID:24142700

  10. Stable rescue of a glycoprotein gII deletion mutant of pseudorabies virus by glycoprotein gI of bovine herpesvirus 1.

    PubMed

    Kopp, A; Mettenleiter, T C

    1992-05-01

    Glycoproteins homologous to glycoprotein B (gB) of herpes simplex virus constitute the most highly conserved group of herpesvirus glycoproteins. This strong conservation of amino acid sequences might be indicative of a common functional role. Indeed, gB homologs have been implicated in the processes of viral entry and virus-mediated cell-cell fusion. Recently, we showed that pseudorabies virus (PrV) lacking the essential gB-homologous glycoprotein gII could be propagated on a cell line expressing the gB homolog of bovine herpesvirus 1, gI(BHV-1), leading to a phenotypic complementation of the gII defect (I. Rauh, F. Weiland, F. Fehler, G. Keil, and T.C. Mettenleiter, J. Virol. 65:621-631, 1991). However, this pseudotypic virus could still replicate only on complementing cell lines, thereby limiting experimental approaches to analyze the effects of the gB exchange in detail. We describe here the construction and isolation of a PrV recombinant, 9112C2, that lacks gII(PrV) but instead stably carries and expresses the gene encoding gI(BHV-1). The recombinant is able to replicate on noncomplementing cells with growth kinetics and final titers similar to those of its gII-positive wild-type PrV parent. Neutralization tests and immunoprecipitation analyses demonstrated incorporation of gI(BHV-1) into 9112C2 virions with concomitant absence of gII(PrV). Analysis of in vitro host ranges of wild-type PrV, BHV-1, and recombinant 9112C2 showed that in cells of pig, rabbit, canine, monkey, or human origin, the plating efficiency of 9112C2 was similar to that of its PrV parent. Exchange of gII(PrV) for gI(BHV-1) in recombinant 9112C2 or by phenotypic complementation of gII- PrV propagated on gI(BHV-1)-expressing cell lines resulted in penetration kinetics intermediate between those of wild-type PrV and BHV-1. In conclusion, we report the first isolation of a viral recombinant in which a lethal glycoprotein mutation has been rescued by a homologous glycoprotein of a different

  11. Glycoprotein J of infectious laryngotracheitis virus is required for efficient egress of infectious virions from cells.

    PubMed

    Mundt, Alice; Mundt, Egbert; Hogan, Robert J; García, Maricarmen

    2011-11-01

    Glycoprotein J (gJ) of infectious laryngotracheitis virus (ILTV) represents a major viral antigen and is dispensable for replication in cell culture and chickens. We generated gJ deletion mutants derived from the United States Department of Agriculture standard challenge strain (USDA-ch), a GFP-expressing mutant GΔgJ, a gJ deletion mutant void of any foreign DNA insertion (BΔgJ) and a gJ rescue mutant gJR with US5 restored. GΔgJ, BΔgJ and gJR were characterized in cell culture and embryonated eggs. Entry kinetic assays showed that the gJ deletion mutants did not differ in their entry kinetics from gJR. Replication kinetics strongly indicated that gJ plays an important role during egress of the virus. Differences in the abilities of the mutants to replicate in chorioallantoic membranes of chicken embryos and to release infectious virus into the allantoic fluid supported a function of gJ during the egress of ILTV from infected cells. PMID:21752963

  12. Expression in bacteria of gB-glycoprotein-coding sequences of Herpes simplex virus type 2.

    PubMed

    Person, S; Warner, S C; Bzik, D J; Debroy, C; Fox, B A

    1985-01-01

    A plasmid with an insert that encodes the glycoprotein B(gB) gene of Herpes simplex virus type 2 (HSV-2) has been isolated. DNA sequences coding for a portion of the HSV-2 gB peptide were cloned into a bacterial lacZ alpha expression vector and used to transform Escherichia coli. Upon induction of lacZpo-promoted transcription, some of the bacteria became filamentous and produced inclusion bodies containing a large amount of a 65-kDal peptide that was shown to be precipitated by broad-spectrum antibodies to HSV-2 and HSV-1. The HSV-2 insert of one of these clones specifies amino acid residues corresponding to 135 through 629 of the gB of HSV-1 [Bzik et al., Virology 133 (1984) 301-314]. PMID:2412940

  13. Human immunodeficiency virus type 1 and 2 envelope glycoproteins oligomerize through conserved sequences.

    PubMed Central

    Center, R J; Kemp, B E; Poumbourios, P

    1997-01-01

    Hetero-oligomerization between human immunodeficiency virus type 2 (HIV-2) envelope glycoprotein (Env) truncation mutants and epitope-tagged gp160 is dependent on the presence of gp41 transmembrane protein (TM) amino acids 552 to 589, a putative amphipathic alpha-helical sequence. HIV-2 Env truncation mutants containing this sequence were also able to form cross-type hetero-oligomers with HIV-1 Env. HIV-2/HIV-1 hetero-oligomerization was, however, more sensitive to disruption by mutagenesis or increased temperature. The conservation of the Env oligomerization function of the HIV-1 and HIV-2 alpha-helical sequences suggests that retroviral TM alpha-helical motifs may have a universal role in oligomerization. PMID:9188654

  14. Functional processing and secretion of Chikungunya virus E1 and E2 glycoproteins in insect cells

    PubMed Central

    2011-01-01

    Background Chikungunya virus (CHIKV) is a mosquito-borne, arthrogenic Alphavirus that causes large epidemics in Africa, South-East Asia and India. Recently, CHIKV has been transmitted to humans in Southern Europe by invading and now established Asian tiger mosquitoes. To study the processing of envelope proteins E1 and E2 and to develop a CHIKV subunit vaccine, C-terminally his-tagged E1 and E2 envelope glycoproteins were produced at high levels in insect cells with baculovirus vectors using their native signal peptides located in CHIKV 6K and E3, respectively. Results Expression in the presence of either tunicamycin or furin inhibitor showed that a substantial portion of recombinant intracellular E1 and precursor E3E2 was glycosylated, but that a smaller fraction of E3E2 was processed by furin into mature E3 and E2. Deletion of the C-terminal transmembrane domains of E1 and E2 enabled secretion of furin-cleaved, fully processed E1 and E2 subunits, which could then be efficiently purified from cell culture fluid via metal affinity chromatography. Confocal laser scanning microscopy on living baculovirus-infected Sf21 cells revealed that full-length E1 and E2 translocated to the plasma membrane, suggesting similar posttranslational processing of E1 and E2, as in a natural CHIKV infection. Baculovirus-directed expression of E1 displayed fusogenic activity as concluded from syncytia formation. CHIKV-E2 was able to induce neutralizing antibodies in rabbits. Conclusions Chikungunya virus glycoproteins could be functionally expressed at high levels in insect cells and are properly glycosylated and cleaved by furin. The ability of purified, secreted CHIKV-E2 to induce neutralizing antibodies in rabbits underscores the potential use of E2 in a subunit vaccine to prevent CHIKV infections. PMID:21762510

  15. Chimeric human parainfluenza virus bearing the Ebola virus glycoprotein as the sole surface protein is immunogenic and highly protective against Ebola virus challenge

    PubMed Central

    Bukreyev, Alexander; Marzi, Andrea; Feldmann, Friederike; Zhang, Liqun; Yang, Lijuan; Ward, Jerrold M.; Dorward, David W.; Pickles, Raymond J.; Murphy, Brian R.; Feldmann, Heinz; Collins, Peter L.

    2009-01-01

    We generated a new live-attenuated vaccine against Ebola virus (EBOV) based on a chimeric virus HPIV3/ΔF-HN/EboGP that contains the EBOV glycoprotein (GP) as the sole transmembrane envelope protein combined with the internal proteins of human parainfluenza virus type 3 (HPIV3). Electron microscopy analysis of the virus particles showed that they have an envelope and surface spikes resembling those of EBOV and a particle size and shape resembling those of HPIV3. When HPIV3/ΔF-HN/EboGP was inoculated via apical surface of an in vitro model of human ciliated airway epithelium, the virus was released from the apical surface; when applied to basolateral surface, the virus infected basolateral cells but did not spread through the tissue. Following intranasal (IN) inoculation of guinea pigs, scattered infected cells were detected in the lungs by immunohistochemistry, but infectious HPIV3/ΔF-HN/EboGP could not be recovered from the lungs, blood, or other tissues. Despite the attenuation, the virus was highly immunogenic, and a single IN dose completely protected the animals against a highly lethal intraperitoneal challenge of guinea pig-adapted EBOV. PMID:19010509

  16. Chimeric human parainfluenza virus bearing the Ebola virus glycoprotein as the sole surface protein is immunogenic and highly protective against Ebola virus challenge

    SciTech Connect

    Bukreyev, Alexander Marzi, Andrea; Feldmann, Friederike; Zhang Liqun; Dorward, David W.; Pickles, Raymond J.; Feldmann, Heinz; Collins, Peter L.

    2009-01-20

    We generated a new live-attenuated vaccine against Ebola virus (EBOV) based on a chimeric virus HPIV3/{delta}F-HN/EboGP that contains the EBOV glycoprotein (GP) as the sole transmembrane envelope protein combined with the internal proteins of human parainfluenza virus type 3 (HPIV3). Electron microscopy analysis of the virus particles showed that they have an envelope and surface spikes resembling those of EBOV and a particle size and shape resembling those of HPIV3. When HPIV3/{delta}F-HN/EboGP was inoculated via apical surface of an in vitro model of human ciliated airway epithelium, the virus was released from the apical surface; when applied to basolateral surface, the virus infected basolateral cells but did not spread through the tissue. Following intranasal (IN) inoculation of guinea pigs, scattered infected cells were detected in the lungs by immunohistochemistry, but infectious HPIV3/{delta}F-HN/EboGP could not be recovered from the lungs, blood, or other tissues. Despite the attenuation, the virus was highly immunogenic, and a single IN dose completely protected the animals against a highly lethal intraperitoneal challenge of guinea pig-adapted EBOV.

  17. Influenza virus pyrogenicity: central role of structural orientation of virion components and involvement of viral lipid and glycoproteins.

    PubMed

    Pickering, J M; Smith, H; Sweet, C

    1992-06-01

    Ultraviolet light-inactivated, non-infectious influenza virus is pyrogenic; virion components are probably responsible for this pyrogenicity. To try to identify the pyrogenic component, influenza virions were disrupted with either bromelain or sodium deoxycholate (DOC). Treatment of infectious virions with bromelain, under conditions that removed the surface glycoproteins (spikes), destroyed their pyrogenicity. The supernatant, containing non-aggregated and modified glycoproteins, was also non-pyrogenic. Disruption of virions with DOC considerably reduced pyrogenicity; however, some was retained by the sub-viral cores. Viral nucleoprotein and matrix protein, purified from the supernatant, were non-pyrogenic. Aggregated stellate clusters of surface glycoproteins separated on sucrose gradients were pyrogenic in half of numerous tests performed with different batches of material. Treatment of virus with ether resulted in complete loss of pyrogenicity. Liposomes made from extracted viral lipid were non-pyrogenic. In contrast, virosomes made from the viral lipid and the aggregated stellate clusters of surface glycoproteins were pyrogenic. Hence, optimum pyrogenicity depends upon the integrity of the virus particle, but haemagglutinin and/or neuraminidase appear essential, and lipid may be involved. PMID:1607857

  18. G glycoprotein amino acid residues required for human monoclonal antibody RAB1 neutralization are conserved in rabies virus street isolates.

    PubMed

    Wang, Yang; Rowley, Kirk J; Booth, Brian J; Sloan, Susan E; Ambrosino, Donna M; Babcock, Gregory J

    2011-08-01

    Replacement of polyclonal anti-rabies immunoglobulin (RIG) used in rabies post-exposure prophylaxis (PEP) with a monoclonal antibody will eliminate cost and availability constraints that currently exist using RIG in the developing world. The human monoclonal antibody RAB1 has been shown to neutralize all rabies street isolates tested; however for the laboratory-adapted fixed strain, CVS-11, mutation in the G glycoprotein of amino acid 336 from asparagine (N) to aspartic acid (D) resulted in resistance to neutralization. Interestingly, this same mutation in the G glycoprotein of a second laboratory-adapted fixed strain (ERA) did not confer resistance to RAB1 neutralization. Using cell surface staining and lentivirus pseudotyped with rabies virus G glycoprotein (RABVpp), we identified an amino acid alteration in CVS-11 (K346), not present in ERA (R346), which was required in combination with D336 to confer resistance to RAB1. A complete analysis of G glycoprotein sequences from GenBank demonstrated that no identified rabies isolates contain the necessary combination of G glycoprotein mutations for resistance to RAB1 neutralization, consistent with the broad neutralization of RAB1 observed in direct viral neutralization experiments with street isolates. All combinations of amino acids 336 and 346 reported in the sequence database were engineered into the ERA G glycoprotein and RAB1 was able to neutralize RABVpp bearing ERA G glycoprotein containing all known combinations at these critical residues. These data demonstrate that RAB1 has the capacity to neutralize all identified rabies isolates and a minimum of two distinct mutations in the G glycoprotein are required for abrogation of RAB1 neutralization. PMID:21693135

  19. Herpes simplex virus glycoprotein C: molecular mimicry of complement regulatory proteins by a viral protein.

    PubMed

    Huemer, H P; Wang, Y; Garred, P; Koistinen, V; Oppermann, S

    1993-08-01

    Herpes simplex virus (HSV) encodes a protein, glycoprotein C (gC), which binds to the third complement component, the central mediator of complement activation. In this study the structural and functional relationships of gC from HSV type 1 (HSV-1) and known human complement regulatory proteins factor H, properdin, factor B, complement receptor 1 (CR1) and 2 (CR2) were investigated. The interaction of gC with C3b was studied using purified complement components, synthetic peptides, antisera against different C3 fragments and anti-C3 monoclonal antibodies (mAb) with known inhibitory effects on C3-ligand interactions. All the mAb that inhibited gC/C3b interactions, in a differential manner, also prevented binding of C3 fragments to factors H, B, CR1 or CR2. No blocking was observed with synthetic peptides representing different C3 regions or with factor B and C3d, whereas C3b, C3c and factor H were inhibitory, as well as purified gC. There was no binding of gC to cobra venom factor (CVF), a C3c-like fragment derived from cobra gland. Purified gC bound to iC3, iC3b and C3c, but failed to bind to C3d. Glycoprotein C bound only weakly to iC3 derived from bovine and porcine plasma, thus indicating a preference of the viral protein for the appropriate host. Binding of gC was also observed to proteolytic C3 fragments, especially to the beta-chain, thus suggesting the importance of the C3 region as a binding site. Purified gC from HSV-1, but not HSV-2, inhibited the binding of factor H and properdin but not of CR1 to C3b. The binding of iC3b to CR2, a molecule involved in B-cell activation and binding of the Epstein-Barr virus, was also inhibited by the HSV-1 protein. As factor H and properdin, the binding of which was inhibited by gC, are important regulators of the alternative complement pathway, these data further support a role of gC in the evasion of HSV from a major first-line host defence mechanism, i.e. the complement system. In addition, the inhibition of the C3/CR

  20. [The occurrence of antibodies to the glycoprotein antigen (gp70) of the enzootic leukemia virus in a leukemic herd].

    PubMed

    Hofírek, B

    1980-08-01

    The serological examination of 175 head of cattle in a herd suffering from leucosis included the study of the occurrence of antibodies to the glycoprotein antigen (gp70) of the virus of enzootic leucosis (BLV). At the same time, these antibodies were studied, as occurring in the F1 generation of the progeneis of 51 positive and 38 negative cows. The results demonstrate two important facts: increasing age brings about a higher percentage of animals having precipitating antibodies in the leucosis-affected herd; the other important finding is that the positive reaction of a cow has no significant influence on the occurrence of the antibodies in the progeny. It was found that under the actual conditions of the mentioned herd, the horizontal transmission of enzootic leucosis was predominant and that precipitating antibodies were detected by the immunodiffusion method in animals at the age from 9 to 12 months. It is desirable from the viewpoint of diagnostics and eradication of enzootic bovine leucosis to apply with utmost consistency the serological methods of examination and individual diagnostics. There is no reason for destroying whole families of animals in which the disease occurred, because no genetically conditioned occurrence of enzootic leucosis was demonstrated in the progenies of cows suffering from the disease. These facts should be respected in amending the instructions for controlling enzootic bovine leucosis. PMID:6252677

  1. Marek's disease virus latency.

    PubMed

    Morgan, R W; Xie, Q; Cantello, J L; Miles, A M; Bernberg, E L; Kent, J; Anderson, A

    2001-01-01

    MDV latency is defined as the persistence of the viral genome in the absence of production of infectious virus except during reactivation. A number of systems for studying MDV latency exist, and most involve the use of lymphoblastoid cells or tumors. It has been difficult to divorce latency and transformation. Understanding the relationship between these two states remains a major challenge for the MDV system. Based on their patterns of expression, the MDV LATs are apt to be important in the balance between latent and lytic infections. The LATs are a complex group of transcripts. The profile of gene expression that characterizes latency differs among all herpesviruses, and MDV is no exception. MDV LATs bear little resemblance to LATs of other alphaherpesviruses or to the LATs of other lymphotropic herpesviruses. LAT splicing patterns are complex and the relationships among various spliced species or between these species and the large 10-kb transcript are unknown. In addition, the existence of any protein gene products of significance is unknown at this time. More work is needed to further investigate the significance and function of these RNAs. Better technology to construct mutants in the MDV system is badly needed, since the analysis of mutants in the chicken is a powerful and unique advantage of the MDV system. PMID:11217424

  2. Detection of Autoantibodies Against Myelin Oligodendrocyte Glycoprotein in Multiple Sclerosis and Related Diseases.

    PubMed

    Spadaro, Melania; Meinl, Edgar

    2016-01-01

    Autoantibodies against myelin oligodendrocyte glycoprotein (MOG) occur in a proportion of patients with different inflammatory demyelinating diseases of the central nervous system, such as childhood multiple sclerosis (MS), acute disseminated encephalomyelitis (ADEM), and neuromyelitis optica spectrum disorders (NMOSD). We describe here in detail a sensitive cell-based assay that allows the identification of autoantibodies against MOG in serum. PMID:25814289

  3. Novel human 3-domain disulfide-stabilized antibody fragment against glycoprotein of rabies virus.

    PubMed

    Cai, Kun; Wang, Hui; Bao, Shizhong; Shi, Jing; Hou, Xiaojun; Gao, Xiang; Liu, Hao; Yin, Jun

    2008-04-01

    Mutated disulfide bond sites VH (Cys44) and VL (Cys100) were constructed in variable domains (Fvs) of the human anti-glycoprotein antigen of the rabies virus (anti-GPRV), and the light chain variable (VL) and heavy chain variable (VH) fragments were linked using the heavy chain constant region 1 (CH1) of the human immunoglobulin (Ig) to successfully construct a 3-domain disulfide-stabilized fragment of variables (3d-dsFv). 3d-dsFv was mainly expressed as an inclusion body. After refolding by the conventional dilution method, 3d-dsFv was purified using a nickel-nitrilotriacetic acid (Ni-NTA) column. Enyzme-linked immunosorbent assay (ELISA) was used to determine the binding activity of 3d-dsFv to GPRV. Flow cytometry studies and rapid fluorescent focus inhibition test were used to evaluate the function of 3d-dsFv. The results showed that the stability of 3d-dsFv was improved notably in some aspects such as thermal kinetics, ability to withstand urea denaturation, etc. 3d-dsFv could bind specially to infective cells and the GPRV. The titration of 3d-dsFv to RV-CVS is 83.3 IU/mg, and it can easily reach 2.5IU/mL, which is the value suggested by the WHO as effective for neutralization titration of the rabies virus. PMID:18424153

  4. Glycoprotein D actively induces rapid internalization of two nectin-1 isoforms during herpes simplex virus entry

    SciTech Connect

    Stiles, Katie M.; Krummenacher, Claude

    2010-03-30

    Entry of herpes simplex virus (HSV) occurs either by fusion at the plasma membrane or by endocytosis and fusion with an endosome. Binding of glycoprotein D (gD) to a receptor such as nectin-1 is essential in both cases. We show that virion gD triggered the rapid down-regulation of nectin-1 with kinetics similar to those of virus entry. In contrast, nectin-1 was not constitutively recycled from the surface of uninfected cells. Both the nectin-1alpha and beta isoforms were internalized in response to gD despite having different cytoplasmic tails. However, deletion of the nectin-1 cytoplasmic tail slowed down-regulation of nectin-1 and internalization of virions. These data suggest that nectin-1 interaction with a cytoplasmic protein is not required for its down-regulation. Overall, this study shows that gD binding actively induces the rapid internalization of various forms of nectin-1. We suggest that HSV activates a nectin-1 internalization pathway to use for endocytic entry.

  5. Functional Characterization of Glycoprotein H Chimeras Composed of Conserved Domains of the Pseudorabies Virus and Herpes Simplex Virus 1 Homologs

    PubMed Central

    Böhm, Sebastian W.; Backovic, Marija; Klupp, Barbara G.; Rey, Felix A.; Fuchs, Walter

    2015-01-01

    ABSTRACT Membrane fusion is indispensable for entry of enveloped viruses into host cells. The conserved core fusion machinery of the Herpesviridae consists of glycoprotein B (gB) and the gH/gL complex. Recently, crystal structures of gH/gL of herpes simplex virus 2 (HSV-2) and Epstein-Barr virus and of a core fragment of pseudorabies virus (PrV) gH identified four structurally conserved gH domains. To investigate functional conservation, chimeric genes encoding combinations of individual domains of PrV and herpes simplex virus 1 (HSV-1) gH were expressed in rabbit kidney cells, and their processing and transport to the cell surface, as well as activity in fusion assays including gB, gD, and gL of PrV or HSV-1, were analyzed. Chimeric gH containing domain I of HSV-1 and domains II to IV of PrV exhibited limited fusion activity in the presence of PrV gB and gD and HSV-1 gL, but not of PrV gL. More strikingly, chimeric gH consisting of PrV domains I to III and HSV-1 domain IV exhibited considerable fusion activity together with PrV gB, gD, and gL. Replacing PrV gB with the HSV-1 protein significantly enhanced this activity. A cell line stably expressing this chimeric gH supported replication of gH-deleted PrV. Our results confirm the specificity of domain I for gL binding, demonstrate functional conservation of domain IV in two alphaherpesviruses from different genera, and indicate species-specific interactions of this domain with gB. They also suggest that gH domains II and III might form a structural and functional unit which does not tolerate major substitutions. IMPORTANCE Envelope glycoprotein H (gH) is essential for herpesvirus-induced membrane fusion, which is required for host cell entry and viral spread. Although gH is structurally conserved within the Herpesviridae, its precise role and its interactions with other components of the viral fusion machinery are not fully understood. Chimeric proteins containing domains of gH proteins from different

  6. A recombinant rabies virus encoding two copies of the glycoprotein gene confers protection in dogs against a virulent challenge.

    PubMed

    Liu, Xiaohui; Yang, Youtian; Sun, Zhaojin; Chen, Jing; Ai, Jun; Dun, Can; Fu, Zhen F; Niu, Xuefeng; Guo, Xiaofeng

    2014-01-01

    The rabies virus (RABV) glycoprotein (G) is the principal antigen responsible for the induction of virus neutralizing antibodies (VNA) and is the major modality of protective immunity in animals. A recombinant RABV HEP-Flury strain was generated by reverse genetics to encode two copies of the G-gene (referred to as HEP-dG). The biological properties of HEP-dG were compared to those of the parental virus (HEP-Flury strain). The HEP-dG recombinant virus grew 100 times more efficiently in BHK-21 cell than the parental virus, yet the virulence of the dG recombinant virus in suckling mice was lower than the parental virus. The HEP-dG virus can improve the expression of G-gene mRNA and the G protein and produce more offspring viruses in cells. The amount of G protein revealed a positive relationship with immunogenicity in mice and dogs. The inactivated HEP-dG recombinant virus induced higher levels of VNA and conferred better protection against virulent RABV in mice and dogs than the inactivated parental virus and a commercial vaccine. The protective antibody persisted for at least 12 months. These data demonstrate that the HEP-dG is stable, induces a strong VNA response and confers protective immunity more effectively than the RABV HEP-Flury strain. HEP-dG could be a potential candidate in the development of novel inactivated rabies vaccines. PMID:24498294

  7. Virion incorporation of envelope glycoproteins with long but not short cytoplasmic tails is blocked by specific, single amino acid substitutions in the human immunodeficiency virus type 1 matrix.

    PubMed Central

    Freed, E O; Martin, M A

    1995-01-01

    Incorporation of envelope glycoproteins into a budding retrovirus is an essential step in the formation of an infectious virus particle. By using site-directed mutagenesis, we identified specific amino acid residues in the matrix domain of the human immunodeficiency virus type 1 (HIV-1) Gag protein that are critical to the incorporation of HIV-1 envelope glycoproteins into virus particles. Pseudotyping analyses were used to demonstrate that two heterologous envelope glycoproteins with short cytoplasmic tails (the envelope of the amphotropic murine leukemia virus and a naturally truncated HIV-2 envelope) are efficiently incorporated into HIV-1 particles bearing the matrix mutations. Furthermore, deletion of the cytoplasmic tail of HIV-1 transmembrane envelope glycoprotein gp41 from 150 to 7 or 47 residues reversed the incorporation block imposed by the matrix mutations. These results suggest the existence of a specific functional interaction between the HIV-1 matrix and the gp41 cytoplasmic tail. PMID:7853546

  8. Site occupancy and glycan compositional analysis of two soluble recombinant forms of the attachment glycoprotein of Hendra virus.

    PubMed

    Colgrave, Michelle L; Snelling, Hayley J; Shiell, Brian J; Feng, Yan-Ru; Chan, Yee-Peng; Bossart, Katharine N; Xu, Kai; Nikolov, Dimitar B; Broder, Christopher C; Michalski, Wojtek P

    2012-04-01

    Hendra virus (HeV) continues to cause morbidity and mortality in both humans and horses with a number of sporadic outbreaks. HeV has two structural membrane glycoproteins that mediate the infection of host cells: the attachment (G) and the fusion (F) glycoproteins that are essential for receptor binding and virion-host cell membrane fusion, respectively. N-linked glycosylation of viral envelope proteins are critical post-translation modifications that have been implicated in roles of structural integrity, virus replication and evasion of the host immune response. Deciphering the glycan composition and structure on these glycoproteins may assist in the development of glycan-targeted therapeutic intervention strategies. We examined the site occupancy and glycan composition of recombinant soluble G (sG) glycoproteins expressed in two different mammalian cell systems, transient human embryonic kidney 293 (HEK293) cells and vaccinia virus (VV)-HeLa cells, using a suite of biochemical and biophysical tools: electrophoresis, lectin binding and tandem mass spectrometry. The N-linked glycans of both VV and HEK293-derived sG glycoproteins carried predominantly mono- and disialylated complex-type N-glycans and a smaller population of high mannose-type glycans. All seven consensus sequences for N-linked glycosylation were definitively found to be occupied in the VV-derived protein, whereas only four sites were found and characterized in the HEK293-derived protein. We also report, for the first time, the existence of O-linked glycosylation sites in both proteins. The striking characteristic of both proteins was glycan heterogeneity in both N- and O-linked sites. The structural features of G protein glycosylation were also determined by X-ray crystallography and interactions with the ephrin-B2 receptor are discussed. PMID:22171062

  9. Sialic Acids on Varicella-Zoster Virus Glycoprotein B Are Required for Cell-Cell Fusion*

    PubMed Central

    Suenaga, Tadahiro; Matsumoto, Maki; Arisawa, Fuminori; Kohyama, Masako; Hirayasu, Kouyuki; Mori, Yasuko; Arase, Hisashi

    2015-01-01

    Varicella-zoster virus (VZV) is a member of the human Herpesvirus family that causes varicella (chicken pox) and zoster (shingles). VZV latently infects sensory ganglia and is also responsible for encephalomyelitis. Myelin-associated glycoprotein (MAG), a member of the sialic acid (SA)-binding immunoglobulin-like lectin family, is mainly expressed in neural tissues. VZV glycoprotein B (gB) associates with MAG and mediates membrane fusion during VZV entry into host cells. The SA requirements of MAG when associating with its ligands vary depending on the specific ligand, but it is unclear whether the SAs on gB are involved in the association with MAG. In this study, we found that SAs on gB are essential for the association with MAG as well as for membrane fusion during VZV infection. MAG with a point mutation in the SA-binding site did not bind to gB and did not mediate cell-cell fusion or VZV entry. Cell-cell fusion and VZV entry mediated by the gB-MAG interaction were blocked by sialidase treatment. N-glycosylation or O-glycosylation inhibitors also inhibited the fusion and entry mediated by gB-MAG interaction. Furthermore, gB with mutations in N-glycosylation sites, i.e. asparagine residues 557 and 686, did not associate with MAG, and the cell-cell fusion efficiency was low. Fusion between the viral envelope and cellular membrane is essential for host cell entry by herpesviruses. Therefore, these results suggest that SAs on gB play important roles in MAG-mediated VZV infection. PMID:26105052

  10. Glycoprotein E of Varicella-Zoster Virus Enhances Cell-Cell Contact in Polarized Epithelial Cells

    PubMed Central

    Mo, Chengjun; Schneeberger, Eveline E.; Arvin, Ann M.

    2000-01-01

    Varicella-zoster virus (VZV) infection involves the cell-cell spread of virions, but how viral proteins interact with the host cell membranes that comprise intercellular junctions is not known. Madin-Darby canine kidney (MDCK) cells were constructed to express the glycoproteins gE, gI, or gE/gI constitutively and were used to examine the effects of these VZV glycoproteins in polarized epithelial cells. At low cell density, VZV gE induced partial tight junction (TJ) formation under low-calcium conditions, whether expressed alone or with gI. Although most VZV gE was intracellular, gE was also shown to colocalize with the TJ protein ZO-1 with or without concomitant expression of gI. Freeze fracture electron microscopy revealed normal TJ strand morphology in gE-expressing MDCK cells. Functionally, the expression of gE was associated with a marked acceleration in the establishment of maximum transepithelial electrical resistance (TER) in MDCK-gE cells; MDCK-gI and MDCK-gE/gI cells exhibited a similar pattern of early TER compared to MDCK cells, although peak resistances were lower than those of gE alone. VZV gE expression altered F-actin organization and lipid distribution, but coexpression of gI modulated these effects. Two regions of the gE ectodomain, amino acids (aa) 278 to 355 and aa 467 to 498, although lacking Ca2+ binding motifs, exhibit similarities with corresponding regions of the cell adhesion molecules, E-cadherin and desmocollin. These observations suggest that VZV gE and gE/gI may contribute to viral pathogenesis by facilitating epithelial cell-cell contacts. PMID:11070038

  11. Sialic Acids on Varicella-Zoster Virus Glycoprotein B Are Required for Cell-Cell Fusion.

    PubMed

    Suenaga, Tadahiro; Matsumoto, Maki; Arisawa, Fuminori; Kohyama, Masako; Hirayasu, Kouyuki; Mori, Yasuko; Arase, Hisashi

    2015-08-01

    Varicella-zoster virus (VZV) is a member of the human Herpesvirus family that causes varicella (chicken pox) and zoster (shingles). VZV latently infects sensory ganglia and is also responsible for encephalomyelitis. Myelin-associated glycoprotein (MAG), a member of the sialic acid (SA)-binding immunoglobulin-like lectin family, is mainly expressed in neural tissues. VZV glycoprotein B (gB) associates with MAG and mediates membrane fusion during VZV entry into host cells. The SA requirements of MAG when associating with its ligands vary depending on the specific ligand, but it is unclear whether the SAs on gB are involved in the association with MAG. In this study, we found that SAs on gB are essential for the association with MAG as well as for membrane fusion during VZV infection. MAG with a point mutation in the SA-binding site did not bind to gB and did not mediate cell-cell fusion or VZV entry. Cell-cell fusion and VZV entry mediated by the gB-MAG interaction were blocked by sialidase treatment. N-glycosylation or O-glycosylation inhibitors also inhibited the fusion and entry mediated by gB-MAG interaction. Furthermore, gB with mutations in N-glycosylation sites, i.e. asparagine residues 557 and 686, did not associate with MAG, and the cell-cell fusion efficiency was low. Fusion between the viral envelope and cellular membrane is essential for host cell entry by herpesviruses. Therefore, these results suggest that SAs on gB play important roles in MAG-mediated VZV infection. PMID:26105052

  12. Native oligomeric human immunodeficiency virus type 1 envelope glycoprotein elicits diverse monoclonal antibody reactivities.

    PubMed Central

    Earl, P L; Broder, C C; Long, D; Lee, S A; Peterson, J; Chakrabarti, S; Doms, R W; Moss, B

    1994-01-01

    We synthesized and purified a recombinant human immunodeficiency virus type 1 (HIV-1) envelope (Env) glycoprotein, lacking the gp120/gp41 cleavage site as well as the transmembrane domain, that is secreted principally as a stable oligomer. Mice were immunized with separated monomeric and oligomeric HIV-1 Env glycoproteins to analyze the repertoire of antibody responses to the tertiary and quaternary structure of the protein. Hybridomas were generated and assayed for reactivity by immunoprecipitation of nondenatured Env protein. A total of 138 monoclonal antibodies (MAbs) were generated and cloned, 123 of which were derived from seven animals immunized with oligomeric Env. Within this group, a significant response was obtained against the gp41 ectodomain; 49 MAbs recognized epitopes in gp41, 82% of which were conformational. The influence of conformation on gp120 antigenicity was less pronounced, with 40% of the anti-gp120 MAbs binding to conformational epitopes, many of which blocked CD4 binding. Surprisingly, less than 7% of the MAbs derived from mice immunized with oligomeric Env recognized the V3 loop. In addition, MAbs to linear epitopes in the C-terminal domain of gp120 were not obtained, suggesting that this region of the protein may be partially masked in the oligomeric molecule. A total of 15 MAbs were obtained from two mice immunized with monomeric Env. Nearly half of these recognized the V3 loop, suggesting that this region may be a less predominant epitope in the context of oligomeric Env than in monomeric protein. Thus, immunization with oligomeric Env generates a large proportion of antibodies to conformational epitopes in both gp120 and gp41, many of which may be absent from monomeric Env. Images PMID:7512157

  13. Phosphorylation of varicella-zoster virus glycoprotein gpI by mammalian casein kinase II and casein kinase I

    SciTech Connect

    Grose, C.; Jackson, W. ); Traugh, J.A. )

    1989-09-01

    Varicella-zoster virus (VZV) glycoprotein gpI is the predominant viral glycoprotein within the plasma membranes of infected cells. This viral glycoprotein is phosphorylated on its polypeptide backbone during biosynthesis. In this report, the authors investigated the protein kinases which participate in the phosphorylation events. Under in vivo conditions, VZV gpI was phosphorylated on its serine and threonine residues by protein kinases present within lysates of either VZV-infected or uninfected cells. Because this activity was diminished by heparin, a known inhibitor of casein kinase II, isolated gpI was incubated with purified casein kinase II and shown to be phosphorylated in an in vitro assay containing ({gamma}-{sup 32}P)ATP. The same glycoprotein was phosphorylated when ({sup 32}P)GTP was substituted for ({sup 32}P)ATP in the protein kinase assay. They also tested whether VZV gpI was phosphorylated by two other ubiquitous mammalian protein kinases--casein kinase I and cyclic AMP-dependent kinase--and found that only casein kinase I modified gpI. When the predicted 623-amino-acid sequence of gpI was examined, two phosphorylation sites known to be optimal for casein kinase II were observed. In summary, this study showed that VZV gpI was phosphorylated by each of two mammalian protein kinases (casein kinase I and casein kinase II) and that potential serine-threonine phosphorylation sites for each of these two kinases were present in the viral glycoprotein.

  14. Characterization of duck enteritis virus UL53 gene and glycoprotein K

    PubMed Central

    2011-01-01

    Background Most of the previous research work had focused on the epidemiology and prevention of duck enteritis virus (DEV). Whilst with the development of protocols in molecular biology, nowadays more and more information about the genes of DEV was reported. But little information about DEV UL53 gene and glycoprotein K(gK) was known except our reported data. Results In our paper, the fluorescent quantitative real-time PCR(FQ-RT-PCR) assay and nucleic acid inhibition test were used to study the transcription characteristic of the DEV UL53 gene. Except detecting the mRNA of DEV UL53 gene, the product gK encoded by UL53 gene was detected through the expression kinetics of UL53 gene by the purified rabbit anti-UL53 protein polyclonal antibodies. Western-blotting and indirect immunofluorescence assays were used to detect gK. From the results of these experiments, the UL53 gene and gK were respectively identified as a late gene and a really late protein. On the other hand, the indirect immunofluorescence assay provided another information that the intracellular localization of DEV gK was mainly distributed in cytoplasm. Conclusions By way of conclusions, we conceded that DEV UL53 gene is a really late gene, which is coincident with properties of UL53 homologs from other herpesvirus, such as ILTV(Infectious Laryngotracheitis virus) and HSV-1(Herpes simplex virus type 1). The properties of intracellular localization about gK protein provided a foundation for further functional analysis and further studies will be focused on constructing of the UL53 gene DEV mutant. PMID:21586146

  15. Characterization of the soluble glycoprotein released from vesicular stomatitis virus-infected cells.

    PubMed Central

    Chatis, P A; Morrison, T G

    1983-01-01

    Vesicular stomatitis virus-infected Chinese hamster ovary cells release into the extracellular medium a soluble form of the vesicular stomatitis virus glycoprotein (G protein) termed Gs (Kang and Prevec, Virology 46:678-680, 1971). The properties of this molecule and the cellular site at which it is generated were characterized. By comparing the sizes and the peptide maps of the unglycosylated forms of G and Gs, we found that between 5,000 and 6,000 daltons of the carboxy-terminal end of the G protein is cleaved to generate the Gs molecule. This truncated molecule contains no fatty acid. Gs released from cells grown at 39 degrees C migrated on polyacrylamide gels slightly slower than Gs released at 30 degrees C. The unglycosylated form of Gs also showed this size difference. Furthermore, unglycosylated Gs was resolved into two species upon isoelectric focusing: the relative amounts of the two species depended upon the temperature at which infected cells were incubated. Full-sized unglycosylated virus-associated G also was resolved into two species, but the more basic form predominated at both 30 and 39 degrees C. The appearance of Gs in the extracellular medium depended upon the presence of stable, full-sized G at the cell surface. The amount of Gs released was quantitated in seven different situations in which the migration of G to the cell surface was inhibited. In all cases, the amount of Gs released was also decreased. In addition, incubation of cells surface labeled with 125I resulted in the release of 125I-labeled Gs protein, as well as full-sized G protein. These results suggest that Gs is generated primarily by proteolytic cleavage of plasma membrane-associated G at a site in the molecule just amino terminal to the membrane-spanning region of the molecule. Images PMID:6296461

  16. Structure-Based Design of Head-Only Fusion Glycoprotein Immunogens for Respiratory Syncytial Virus.

    PubMed

    Boyington, Jeffrey C; Joyce, M Gordon; Sastry, Mallika; Stewart-Jones, Guillaume B E; Chen, Man; Kong, Wing-Pui; Ngwuta, Joan O; Thomas, Paul V; Tsybovsky, Yaroslav; Yang, Yongping; Zhang, Baoshan; Chen, Lei; Druz, Aliaksandr; Georgiev, Ivelin S; Ko, Kiyoon; Zhou, Tongqing; Mascola, John R; Graham, Barney S; Kwong, Peter D

    2016-01-01

    Respiratory syncytial virus (RSV) is a significant cause of severe respiratory illness worldwide, particularly in infants, young children, and the elderly. Although no licensed vaccine is currently available, an engineered version of the metastable RSV fusion (F) surface glycoprotein-stabilized in the pre-fusion (pre-F) conformation by "DS-Cav1" mutations-elicits high titer RSV-neutralizing responses. Moreover, pre-F-specific antibodies, often against the neutralization-sensitive antigenic site Ø in the membrane-distal head region of trimeric F glycoprotein, comprise a substantial portion of the human response to natural RSV infection. To focus the vaccine-elicited response to antigenic site Ø, we designed a series of RSV F immunogens that comprised the membrane-distal head of the F glycoprotein in its pre-F conformation. These "head-only" immunogens formed monomers, dimers, and trimers. Antigenic analysis revealed that a majority of the 70 engineered head-only immunogens displayed reactivity to site Ø-targeting antibodies, which was similar to that of the parent RSV F DS-Cav1 trimers, often with increased thermostability. We evaluated four of these head-only immunogens in detail, probing their recognition by antibodies, their physical stability, structure, and immunogenicity. When tested in naïve mice, a head-only trimer, half the size of the parent RSV F trimer, induced RSV titers, which were statistically comparable to those induced by DS-Cav1. When used to boost DS-Cav1-primed mice, two head-only RSV F immunogens, a dimer and a trimer, boosted RSV-neutralizing titers to levels that were comparable to those boosted by DS-Cav1, although with higher site Ø-directed responses. Our results provide proof-of-concept for the ability of the smaller head-only RSV F immunogens to focus the vaccine-elicited response to antigenic site Ø. Decent primary immunogenicity, enhanced physical stability, potential ease of manufacture, and potent immunogenicity upon boosting

  17. Expression and solubilization of insect cell-based rabies virus glycoprotein and assessment of its immunogenicity and protective efficacy in mice.

    PubMed

    Ramya, R; Mohana Subramanian, B; Sivakumar, V; Senthilkumar, R L; Sambasiva Rao, K R S; Srinivasan, V A

    2011-10-01

    Rabies is a fatal zoonotic disease of serious public health and economic significance worldwide. The rabies virus glycoprotein (RVG) has been the major target for subunit vaccine development, since it harbors domains responsible for induction of virus-neutralizing antibodies, infectivity, and neurovirulence. The glycoprotein (G) was cloned using the baculovirus expression vector system (BEVS) and expressed in Spodoptera frugiperda (Sf-9) cells. In order to obtain a soluble form of G suitable for experimentation in mice, 18 different combinations of buffers and detergents were evaluated for their ability to solubilize the insect cell membrane-associated G. The combination that involved 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) detergent in lysis buffer 1, formulated with Tris, NaCl, 10% dimethyl sulfoxide (DMSO), and EDTA, gave the highest yield of soluble G, as evidenced by the experimental data. Subsequently, several other parameters, such as the concentration of CHAPS and the duration and temperature of the treatment for the effective solubilization of G, were optimized. The CHAPS detergent, buffered at a concentration of 0.4% to 0.7% (wt/vol) at room temperature (23 to 25°C) for 30 min to 1 h using buffer 1, containing 10% DMSO, resulted in consistently high yields. The G solubilized using CHAPS detergent was found to be immunogenic when tested in mice, as evidenced by high virus-neutralizing antibody titers in sera and 100% protection upon virulent intracerebral challenge with the challenge virus standard (CVS) strain of rabies virus. The results of the mice study indicated that G solubilized with CHAPS detergent retained the immunologically relevant domains in the native conformation, thereby paving the way for producing a cell-free and efficacious subunit vaccine. PMID:21813661

  18. Expression and Solubilization of Insect Cell-Based Rabies Virus Glycoprotein and Assessment of Its Immunogenicity and Protective Efficacy in Mice ▿

    PubMed Central

    Ramya, R.; Mohana Subramanian, B.; Sivakumar, V.; Senthilkumar, R. L.; Sambasiva Rao, K. R. S.; Srinivasan, V. A.

    2011-01-01

    Rabies is a fatal zoonotic disease of serious public health and economic significance worldwide. The rabies virus glycoprotein (RVG) has been the major target for subunit vaccine development, since it harbors domains responsible for induction of virus-neutralizing antibodies, infectivity, and neurovirulence. The glycoprotein (G) was cloned using the baculovirus expression vector system (BEVS) and expressed in Spodoptera frugiperda (Sf-9) cells. In order to obtain a soluble form of G suitable for experimentation in mice, 18 different combinations of buffers and detergents were evaluated for their ability to solubilize the insect cell membrane-associated G. The combination that involved 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) detergent in lysis buffer 1, formulated with Tris, NaCl, 10% dimethyl sulfoxide (DMSO), and EDTA, gave the highest yield of soluble G, as evidenced by the experimental data. Subsequently, several other parameters, such as the concentration of CHAPS and the duration and temperature of the treatment for the effective solubilization of G, were optimized. The CHAPS detergent, buffered at a concentration of 0.4% to 0.7% (wt/vol) at room temperature (23 to 25°C) for 30 min to 1 h using buffer 1, containing 10% DMSO, resulted in consistently high yields. The G solubilized using CHAPS detergent was found to be immunogenic when tested in mice, as evidenced by high virus-neutralizing antibody titers in sera and 100% protection upon virulent intracerebral challenge with the challenge virus standard (CVS) strain of rabies virus. The results of the mice study indicated that G solubilized with CHAPS detergent retained the immunologically relevant domains in the native conformation, thereby paving the way for producing a cell-free and efficacious subunit vaccine. PMID:21813661

  19. Functional Analysis of the 60-Nucleotide Duplication in the Respiratory Syncytial Virus Buenos Aires Strain Attachment Glycoprotein

    PubMed Central

    Hotard, Anne L.; Laikhter, Elizabeth; Brooks, Kelsie; Hartert, Tina V.

    2015-01-01

    ABSTRACT There are two subgroups of respiratory syncytial virus (RSV), A and B, and within each subgroup, isolates are further divided into clades. Several years ago, multiple subgroup B isolates which contained a duplication of 60 nucleotides in the glycoprotein (G) gene were described. These isolates were given a new clade designation of BA based on the site of isolation, Buenos Aires, Argentina. BA RSV strains have since become the predominant circulating clade of RSV B viruses. We hypothesized that the duplicated region in G serves to enhance the function of G in the virus life cycle. We generated recombinant viruses that express a consensus BA G gene or a consensus BA G gene lacking the duplication (GΔdup). We determined that the duplicated region functions during virus attachment to cells. Additionally, we showed that in vitro, the virus containing the duplication has a fitness advantage compared to the virus without the duplication. Our data demonstrate that the duplicated region in the BA strain G protein augments virus attachment and fitness. IMPORTANCE Respiratory syncytial virus (RSV) is an important pathogen for infants for which there is no vaccine. Different strains of RSV circulate from year to year, and the predominating strains change over time. Subgroup B RSV strains with a duplication in the attachment glycoprotein (G) emerged and then became the dominant B genotype. We found that a recombinant virus harboring the duplication bound more efficiently to cells and was more fit than a recombinant strain lacking the duplication. Our work advances a mechanism for an important natural RSV mutation. PMID:26018171

  20. Pathogenicity of different rabies virus variants inversely correlates with apoptosis and rabies virus glycoprotein expression in infected primary neuron cultures.

    PubMed

    Morimoto, K; Hooper, D C; Spitsin, S; Koprowski, H; Dietzschold, B

    1999-01-01

    The mouse-adapted rabies virus strain CVS-24 has stable variants, CVS-B2c and CVS-N2c, which differ greatly in their pathogenicity for normal adult mice and in their ability to infect nonneuronal cells. The glycoprotein (G protein), which has previously been implicated in rabies virus pathogenicity, shows substantial structural differences between these variants. Although prior studies have identified antigenic site III of the G protein as the major pathogenicity determinant, CVS-B2c and CVS-N2c do not vary at this site. The possibility that pathogenicity is inversely related to G protein expression levels is suggested by the finding that CVS-B2c, the less pathogenic variant, expresses at least fourfold-higher levels of G protein than CVS-N2c in infected neurons. Although there is some difference between CVS-B2c- and CVS-N2c-infected neurons in G protein mRNA expression levels, the differential expression of G protein appears to be largely determined by posttranslational mechanisms that affect G protein stability. Pulse-chase experiments indicated that the G protein of CVS-B2c is degraded more slowly than that of CVS-N2c. The accumulation of G protein correlated with the induction of programmed cell death in CVS-B2c-infected neurons. The extent of apoptosis was considerably lower in CVS-N2c-infected neurons, where G protein expression was minimal. While nucleoprotein (N protein) expression levels were similar in neurons infected with either variant, the transport of N protein into neuronal processes was strongly inhibited in CVS-B2c-infected cells. Thus, downregulation of G protein expression in neuronal cells evidently contributes to rabies virus pathogenesis by preventing apoptosis and the apparently associated failure of the axonal transport of N protein. PMID:9847357

  1. Pathogenicity of Different Rabies Virus Variants Inversely Correlates with Apoptosis and Rabies Virus Glycoprotein Expression in Infected Primary Neuron Cultures

    PubMed Central

    Morimoto, Kinjiro; Hooper, D. Craig; Spitsin, Sergei; Koprowski, Hilary; Dietzschold, Bernhard

    1999-01-01

    The mouse-adapted rabies virus strain CVS-24 has stable variants, CVS-B2c and CVS-N2c, which differ greatly in their pathogenicity for normal adult mice and in their ability to infect nonneuronal cells. The glycoprotein (G protein), which has previously been implicated in rabies virus pathogenicity, shows substantial structural differences between these variants. Although prior studies have identified antigenic site III of the G protein as the major pathogenicity determinant, CVS-B2c and CVS-N2c do not vary at this site. The possibility that pathogenicity is inversely related to G protein expression levels is suggested by the finding that CVS-B2c, the less pathogenic variant, expresses at least fourfold-higher levels of G protein than CVS-N2c in infected neurons. Although there is some difference between CVS-B2c- and CVS-N2c-infected neurons in G protein mRNA expression levels, the differential expression of G protein appears to be largely determined by posttranslational mechanisms that affect G protein stability. Pulse-chase experiments indicated that the G protein of CVS-B2c is degraded more slowly than that of CVS-N2c. The accumulation of G protein correlated with the induction of programmed cell death in CVS-B2c-infected neurons. The extent of apoptosis was considerably lower in CVS-N2c-infected neurons, where G protein expression was minimal. While nucleoprotein (N protein) expression levels were similar in neurons infected with either variant, the transport of N protein into neuronal processes was strongly inhibited in CVS-B2c-infected cells. Thus, downregulation of G protein expression in neuronal cells evidently contributes to rabies virus pathogenesis by preventing apoptosis and the apparently associated failure of the axonal transport of N protein. PMID:9847357

  2. Hepatitis C Virus Envelope Glycoprotein E1 Forms Trimers at the Surface of the Virion

    PubMed Central

    Falson, Pierre; Bartosch, Birke; Alsaleh, Khaled; Tews, Birke Andrea; Loquet, Antoine; Ciczora, Yann; Riva, Laura; Montigny, Cédric; Montpellier, Claire; Duverlie, Gilles; Pécheur, Eve-Isabelle; le Maire, Marc; Cosset, François-Loïc

    2015-01-01

    ABSTRACT In hepatitis C virus (HCV)-infected cells, the envelope glycoproteins E1 and E2 assemble as a heterodimer. To investigate potential changes in the oligomerization of virion-associated envelope proteins, we performed SDS-PAGE under reducing conditions but without thermal denaturation. This revealed the presence of SDS-resistant trimers of E1 in the context of cell-cultured HCV (HCVcc) as well as in the context of HCV pseudoparticles (HCVpp). The formation of E1 trimers was found to depend on the coexpression of E2. To further understand the origin of E1 trimer formation, we coexpressed in bacteria the transmembrane (TM) domains of E1 (TME1) and E2 (TME2) fused to reporter proteins and analyzed the fusion proteins by SDS-PAGE and Western blotting. As expected for strongly interacting TM domains, TME1–TME2 heterodimers resistant to SDS were observed. These analyses also revealed homodimers and homotrimers of TME1, indicating that such complexes are stable species. The N-terminal segment of TME1 exhibits a highly conserved GxxxG sequence, a motif that is well documented to be involved in intramembrane protein-protein interactions. Single or double mutations of the glycine residues (Gly354 and Gly358) in this motif markedly decreased or abrogated the formation of TME1 homotrimers in bacteria, as well as homotrimers of E1 in both HCVpp and HCVcc systems. A concomitant loss of infectivity was observed, indicating that the trimeric form of E1 is essential for virus infectivity. Taken together, these results indicate that E1E2 heterodimers form trimers on HCV particles, and they support the hypothesis that E1 could be a fusion protein. IMPORTANCE HCV glycoproteins E1 and E2 play an essential role in virus entry into liver cells as well as in virion morphogenesis. In infected cells, these two proteins form a complex in which E2 interacts with cellular receptors, whereas the function of E1 remains poorly understood. However, recent structural data suggest that E1

  3. Vaccination with the Secreted Glycoprotein G of Herpes Simplex Virus 2 Induces Protective Immunity after Genital Infection.

    PubMed

    Önnheim, Karin; Ekblad, Maria; Görander, Staffan; Bergström, Tomas; Liljeqvist, Jan-Åke

    2016-04-01

    Herpes simplex virus 2 (HSV-2) infects the genital mucosa and establishes a life-long infection in sensory ganglia. After primary infection HSV-2 may reactivate causing recurrent genital ulcerations. HSV-2 infection is prevalent, and globally more than 400 million individuals are infected. As clinical trials have failed to show protection against HSV-2 infection, new vaccine candidates are warranted. The secreted glycoprotein G (sgG-2) of HSV-2 was evaluated as a prophylactic vaccine in mice using two different immunization and adjuvant protocols. The protocol with three intramuscular immunizations combining sgG-2 with cytosine-phosphate-guanine dinucleotide (CpG) motifs and alum induced almost complete protection from genital and systemic disease after intra-vaginal challenge with HSV-2. Robust immunoglobulin G (IgG) antibody titers were detected with no neutralization activity. Purified splenic CD4+ T cells proliferated and produced interferon-γ (IFN-γ) when re-stimulated with the antigen in vitro. sgG-2 + adjuvant intra-muscularly immunized mice showed a significant reduction of infectious HSV-2 and increased IFN-γ levels in vaginal washes. The HSV-2 DNA copy numbers were significantly reduced in dorsal root ganglia, spinal cord, and in serum at day six or day 21 post challenge. We show that a sgG-2 based vaccine is highly effective and can be considered as a novel candidate in the development of a prophylactic vaccine against HSV-2 infection. PMID:27110813

  4. Vaccination with the Secreted Glycoprotein G of Herpes Simplex Virus 2 Induces Protective Immunity after Genital Infection

    PubMed Central

    Önnheim, Karin; Ekblad, Maria; Görander, Staffan; Bergström, Tomas; Liljeqvist, Jan-Åke

    2016-01-01

    Herpes simplex virus 2 (HSV-2) infects the genital mucosa and establishes a life-long infection in sensory ganglia. After primary infection HSV-2 may reactivate causing recurrent genital ulcerations. HSV-2 infection is prevalent, and globally more than 400 million individuals are infected. As clinical trials have failed to show protection against HSV-2 infection, new vaccine candidates are warranted. The secreted glycoprotein G (sgG-2) of HSV-2 was evaluated as a prophylactic vaccine in mice using two different immunization and adjuvant protocols. The protocol with three intramuscular immunizations combining sgG-2 with cytosine-phosphate-guanine dinucleotide (CpG) motifs and alum induced almost complete protection from genital and systemic disease after intra-vaginal challenge with HSV-2. Robust immunoglobulin G (IgG) antibody titers were detected with no neutralization activity. Purified splenic CD4+ T cells proliferated and produced interferon-γ (IFN-γ) when re-stimulated with the antigen in vitro. sgG-2 + adjuvant intra-muscularly immunized mice showed a significant reduction of infectious HSV-2 and increased IFN-γ levels in vaginal washes. The HSV-2 DNA copy numbers were significantly reduced in dorsal root ganglia, spinal cord, and in serum at day six or day 21 post challenge. We show that a sgG-2 based vaccine is highly effective and can be considered as a novel candidate in the development of a prophylactic vaccine against HSV-2 infection. PMID:27110813

  5. Secreted herpes simplex virus-2 glycoprotein G alters thermal pain sensitivity by modifying NGF effects on TRPV1.

    PubMed

    Cabrera, Jorge Rubén; Viejo-Borbolla, Abel; Alcamí, Antonio; Wandosell, Francisco

    2016-01-01

    Genital herpes is a painful disease frequently caused by the neurotropic pathogen herpes simplex virus type 2 (HSV-2). We have recently shown that HSV-2-secreted glycoprotein G (SgG2) interacts with and modulates the activity of the neurotrophin nerve growth factor (NGF). This interaction modifies the response of the NGF receptor TrkA, increasing NGF-dependent axonal growth. NGF is not only an axonal growth modulator but also an important mediator of pain and inflammation regulating the amount, localization, and activation of the thermal pain receptor transient receptor potential vanilloid 1 (TRPV1). In this work, we addressed whether SgG2 could contribute to HSV-2-induced pain. Injection of SgG2 in the mouse hindpaw produced a rapid and transient increase in thermal pain sensitivity. At the molecular level, this acute increase in thermal pain induced by SgG2 injection was dependent on differential NGF-induced phosphorylation and in changes in the amount of TrkA and TRPV1 in the dermis. These results suggest that SgG2 alters thermal pain sensitivity by modulating TRPV1 receptor. PMID:27576911

  6. A "virus" disease of chinook salmon

    USGS Publications Warehouse

    Ross, A.J.; Rucker, R.R.

    1960-01-01

    Epizootics among chinook salmon fingerlings at the Coleman National Fish Hatchery have occurred periodically since 1941. A virus or virus-like filterable agent has been demonstrated to be the causative agent of this disease.

  7. Immunization with vaccinia virus recombinants that express the surface glycoproteins of human parainfluenza virus type 3 (PIV3) protects patas monkeys against PIV3 infection.

    PubMed Central

    Spriggs, M K; Collins, P L; Tierney, E; London, W T; Murphy, B R

    1988-01-01

    Patas monkeys (Eryphrocebus patas) were immunized intradermally with two vaccinia virus recombinants that individually express the hemagglutinin-neuraminidase glycoprotein or the fusion glycoprotein of human parainfluenza virus type 3 (PIV3). These immunizations induced a high titer of PIV3 serum-neutralizing antibodies. At 1 month after immunization, monkeys were challenged intratracheally with PIV3. Subsequent virus replication was reduced in these monkeys by 3.2 log10 and 1.9 log10 (mean peak virus titers) in the upper and lower respiratory tracts, respectively, compared with control animals. The average duration of virus shedding was also reduced from 9.0 to 3.4 days in the upper respiratory tract and from 5.3 to 1.2 days in the lower respiratory tract. These findings demonstrate that a single intradermal dose of live recombinant vaccinia viruses can significantly restrict the replication of a virus which primarily infects the epithelial cells of the respiratory tract. PMID:2831389

  8. Detecting molecular adaptation at individual codons in the glycoprotein gene of the geographically diversified infectious hematopoietic necrosis virus, a fish rhabdovirus.

    PubMed

    Padhi, Abinash; Verghese, Bindhu

    2008-03-01

    Salmonid fishes, the principal hosts of the infectious hematopoietic necrosis virus (IHNV), are a candidate species for aquaculture in many countries. IHNV causes an acute disease resulting in severe economic loss in salmonid fish farming. Previous phylogenetic analyses revealed the existence of multiple genogroups of this virus throughout the geographical range of its host. Here, we report the importance of natural selection in shaping the evolution of certain codons at the surface glycoprotein (G-protein) gene of this virus. Maximum likelihood (ML)-based codon substitution analyses revealed that approximately 2.8% of the codons for the entire G-protein are shown to have higher nonsynonymous substitution per nonsynonymous site (dn) than the synonymous substitutions per synonymous site (ds) (dn/ds=omega>4.335). Thus, the data suggest that positive selection (omega>1) is the major driving force in the evolution of certain codons. However, majority of these positively selected sites cannot be mapped to the regions of antigenic determinants of IHNV. Based on the reports of previous studies, epitopes with positively selected sites are immunodominant and viruses can escape from immune responses by producing antigenic variation at positively selected sites, therefore, vaccines directed against these neutralizing epitopes of IHNV that consist of no positively selected sites will be more effective. Some of the positively selected sites showed radical change in amino acids with respect to their charge and polarity; however, it is unclear how these changes affect the fitness of the virus. PMID:18178282

  9. Comparative in vivo safety and efficacy of a glycoprotein G-deficient candidate vaccine strain of infectious laryngotracheitis virus delivered via eye drop.

    PubMed

    Coppo, Mauricio J C; Noormohammadi, Amir H; Hartley, Carol A; Gilkerson, James R; Browning, Glenn F; Devlin, Joanne M

    2011-08-01

    Infectious laryngotracheitis (ILT) is an acute respiratory disease in poultry that is commonly controlled by vaccination with conventionally attenuated virus strains. Despite the use of these vaccines, ILT remains a threat to the intensive poultry industry. Our laboratory has developed a novel candidate vaccine strain of infectious laryngotracheitis virus (ILTV) lacking glycoprotein G (ΔgG-ILTV). The aim of the present study was to directly compare this candidate vaccine with three currently available commercial vaccines in vivo. Five groups of specific-pathogen-free chickens were eye-drop inoculated with one of the three commercial vaccine strains (SA2-ILTV, A20-ILTV or Serva-ILTV), or ΔgG-ILTV, or sterile medium. Vaccine safety was assessed by examining clinical signs, weight gain and persistence of virus in the trachea. Vaccine efficacy was assessed by scoring clinical signs and conducting post-mortem analyses following challenge with virulent virus. Following vaccination, birds that received ΔgG-ILTV had the highest weight gain among the vaccinated groups and had clinical scores that were significantly lower than birds vaccinated with SA2-ILTV or A20-ILTV, but not significantly different from those of birds vaccinated with Serva-ILTV. Analysis of clinical scores, weight gain, tracheal pathology and virus replication after challenge revealed a comparable level of efficacy for all vaccines. Findings from this study further demonstrate the suitability of ΔgG-ILTV as a vaccine to control ILT. PMID:21812721

  10. Novel Respiratory Syncytial Virus-Like Particle Vaccine Composed of the Postfusion and Prefusion Conformations of the F Glycoprotein

    PubMed Central

    Boigard, Hélène; Bhatia, Bipin; Fallon, John T.; Alimova, Alexandra; Gottlieb, Paul

    2016-01-01

    Respiratory syncytial virus (RSV) is the leading cause of severe respiratory disease in infants and children and represents an important global health burden for the elderly and the immunocompromised. Despite decades of research efforts, no licensed vaccine for RSV is available. We have developed virus-like particle (VLP)-based RSV vaccines assembled with the human metapneumovirus (hMPV) matrix protein (M) as the structural scaffold and the RSV fusion glycoprotein (F) in either the postfusion or prefusion conformation as its prime surface immunogen. Vaccines were composed of postfusion F, prefusion F, or a combination of the two conformations and formulated with a squalene-based oil emulsion as adjuvant. Immunization with these VLP vaccines afforded full protection against RSV infection and prevented detectable viral replication in the mouse lung after challenge. Analyses of lung cytokines and chemokines showed that VLP vaccination mostly induced the production of gamma interferon (IFN-γ), a marker of the Th1-mediated immune response, which is predominantly required for viral protection. Conversely, immunization with a formalin-inactivated RSV (FI-RSV) vaccine induced high levels of inflammatory chemokines and cytokines of the Th2- and Th17-mediated types of immune responses, as well as severe lung inflammation and histopathology. The VLP vaccines showed restricted production of these immune mediators and did not induce severe bronchiolitis or perivascular infiltration as seen with the FI-RSV vaccine. Remarkably, analysis of the serum from immunized mice showed that the VLP vaccine formulated using a combination of postfusion and prefusion F elicited the highest level of neutralizing antibody and enhanced the Th1-mediated immune response. PMID:27030590

  11. Safety and vaccine efficacy of a glycoprotein G deficient strain of infectious laryngotracheitis virus delivered in ovo.

    PubMed

    Legione, Alistair R; Coppo, Mauricio J C; Lee, Sang-Won; Noormohammadi, Amir H; Hartley, Carol A; Browning, Glenn F; Gilkerson, James R; O'Rourke, Denise; Devlin, Joanne M

    2012-11-26

    Infectious laryngotracheitis virus (ILTV), an alphaherpesvirus, causes respiratory disease in chickens and is commonly controlled by vaccination with conventionally attenuated vaccines. Glycoprotein G (gG) is a virulence factor in ILTV and a gG deficient strain of ILTV (ΔgG-ILTV) has shown potential for use as a vaccine. In the poultry industry vaccination via drinking water is common, but technology is now available to allow quicker and more accurate in ovo vaccination of embryos at 18 days of incubation. In this study ΔgG-ILTV was delivered to chicken embryos at three different doses (10(2), 10(3) and 10(4) plaque forming units per egg) using manual in ovo vaccination. At 20 days after hatching, birds were challenged intra-tracheally with wild type ILTV and protection was measured. In ovo vaccination was shown to be safe, as there were no developmental differences between birds from hatching up to 20 days of age, as measured by weight gain. The highest dose of vaccine was the most efficacious, resulting in a weight gain not significantly different from unvaccinated/unchallenged birds seven days after challenge. In contrast, birds vaccinated with the lowest dose showed weight gains not significantly different from unvaccinated/challenged birds. Gross pathology and histopathology of the trachea reflected these observations, with birds vaccinated with the highest dose having less severe lesions. However, qPCR results suggested the vaccine did not prevent the challenge virus replicating in the trachea. This study is the first to assess in ovo delivery of a live attenuated ILTV vaccine and shows that in ovo vaccination with ΔgG-ILTV can be both safe and efficacious. PMID:23084851

  12. Impact of Valency of a Glycoprotein B-Specific Monoclonal Antibody on Neutralization of Herpes Simplex Virus

    PubMed Central

    Krawczyk, Adalbert; Krauss, Jürgen; Eis-Hübinger, Anna M.; Däumer, Martin P.; Schwarzenbacher, Robert; Dittmer, Ulf; Schneweis, Karl E.; Jäger, Dirk; Roggendorf, Michael; Arndt, Michaela A. E.

    2011-01-01

    Herpes simplex virus (HSV) glycoprotein B (gB) is an integral part of the multicomponent fusion system required for virus entry and cell-cell fusion. Here we investigated the mechanism of viral neutralization by the monoclonal antibody (MAb) 2c, which specifically recognizes the gB of HSV type 1 (HSV-1) and HSV-2. Binding of MAb 2c to a type-common discontinuous epitope of gB resulted in highly efficient neutralization of HSV at the postbinding/prefusion stage and completely abrogated the viral cell-to-cell spread in vitro. Mapping of the antigenic site recognized by MAb 2c to the recently solved crystal structure of the HSV-1 gB ectodomain revealed that its discontinuous epitope is only partially accessible within the observed multidomain trimer conformation of gB, likely representing its postfusion conformation. To investigate how MAb 2c may interact with gB during membrane fusion, we characterized the properties of monovalent (Fab and scFv) and bivalent [IgG and F(ab′)2] derivatives of MAb 2c. Our data show that the neutralization capacity of MAb 2c is dependent on cross-linkage of gB trimers. As a result, only bivalent derivatives of MAb 2c exhibited high neutralizing activity in vitro. Notably, bivalent MAb 2c not only was capable of preventing mucocutaneous disease in severely immunodeficient NOD/SCID mice upon vaginal HSV-1 challenge but also protected animals even with neuronal HSV infection. We also report for the first time that an anti-gB specific monoclonal antibody prevents HSV-1-induced encephalitis entirely independently from complement activation, antibody-dependent cellular cytotoxicity, and cellular immunity. This indicates the potential for further development of MAb 2c as an anti-HSV drug. PMID:21123390

  13. Novel Respiratory Syncytial Virus-Like Particle Vaccine Composed of the Postfusion and Prefusion Conformations of the F Glycoprotein.

    PubMed

    Cimica, Velasco; Boigard, Hélène; Bhatia, Bipin; Fallon, John T; Alimova, Alexandra; Gottlieb, Paul; Galarza, Jose M

    2016-06-01

    Respiratory syncytial virus (RSV) is the leading cause of severe respiratory disease in infants and children and represents an important global health burden for the elderly and the immunocompromised. Despite decades of research efforts, no licensed vaccine for RSV is available. We have developed virus-like particle (VLP)-based RSV vaccines assembled with the human metapneumovirus (hMPV) matrix protein (M) as the structural scaffold and the RSV fusion glycoprotein (F) in either the postfusion or prefusion conformation as its prime surface immunogen. Vaccines were composed of postfusion F, prefusion F, or a combination of the two conformations and formulated with a squalene-based oil emulsion as adjuvant. Immunization with these VLP vaccines afforded full protection against RSV infection and prevented detectable viral replication in the mouse lung after challenge. Analyses of lung cytokines and chemokines showed that VLP vaccination mostly induced the production of gamma interferon (IFN-γ), a marker of the Th1-mediated immune response, which is predominantly required for viral protection. Conversely, immunization with a formalin-inactivated RSV (FI-RSV) vaccine induced high levels of inflammatory chemokines and cytokines of the Th2- and Th17-mediated types of immune responses, as well as severe lung inflammation and histopathology. The VLP vaccines showed restricted production of these immune mediators and did not induce severe bronchiolitis or perivascular infiltration as seen with the FI-RSV vaccine. Remarkably, analysis of the serum from immunized mice showed that the VLP vaccine formulated using a combination of postfusion and prefusion F elicited the highest level of neutralizing antibody and enhanced the Th1-mediated immune response. PMID:27030590

  14. Analysis of the role of antibody-dependent cellular cytotoxic antibody activity in murine neonatal herpes simplex virus infection with antibodies to synthetic peptides of glycoprotein D and monoclonal antibodies to glycoprotein B.

    PubMed Central

    Kohl, S; Strynadka, N C; Hodges, R S; Pereira, L

    1990-01-01

    The role of antibody in neonatal herpes simplex virus (HSV) infection remains controversial. A battery of well-characterized monoclonal antibodies to HSV glycoprotein B (gB), and polyclonal antibodies against synthetic peptides of predicted epitopes of HSV glycoprotein D (gD) were used to determine in vitro functional activity and association with protection against lethal infection in a murine model of neonatal HSV disease. Antiviral neutralization activity of HSV was not associated with antibody-dependent cellular cytotoxicity (ADCC) activity to HSV-infected cells in vitro. In a model of high dose challenge (10(4) PFU), protection was not afforded by any antibody alone, but was by antibody plus human mononuclear cells, and highly associated with ADCC functional activity (P less than 0.001). In a low dose challenge model, neutralizing activity of antibody alone was associated with protection in vivo (P less than 0.001). Of the nine neutralizing epitopes of gD in vitro, eight were predicted surface regions. Four of the five epitopic sites of gD (2-21, 267-276, 288-297, and 303-312) that were determined to be important targets of ADCC and in vivo protection were also predicted to be surface regions. The only exception was the antiserum to region 52-61 which was predicted to be buried and also showed these activities. ADCC as well as neutralizing antibody activity are important in protection against neonatal HSV infection. PMID:2164044

  15. The glycoprotein genes and gene junctions of the fish rhabdoviruses spring viremia of carp virus and hirame rhabdovirus: Analysis of relationships with other rhabdoviruses

    USGS Publications Warehouse

    Bjorklund, H.V.; Higman, K.H.; Kurath, G.

    1996-01-01

    The nucleotide sequences of the glycoprotein genes and all of the internal gene junctions of the fish pathogenic rhabdoviruses spring viremia of carp virus (SVCV) and hirame rhabdovirus (HIRRV) have been determined from cDNA clones generated from viral genomic RNA. The SVCV glycoprotein gene sequence is 1588 nucleotides (nt) long and encodes a 509 amino acid (aa) protein. The HIRRV glycoprotein gene sequence comprises 1612 nt, coding for a 508 aa protein. In sequence comparisons of 15 rhabdovirus glycoproteins, the SVCV glycoprotein gene showed the highest amino acid sequence identity (31.2-33.2%) with vesicular stomatitis New Jersey virus (VSNJV), Chandipura virus (CHPV) and vesicular stomatitis Indiana virus (VSIV). The HIRRV glycoprotein gene showed a very high amino acid sequence identity (74.3%) with the glycoprotein gene of another fish pathogenic rhabdovirus, infectious hematopoietic necrosis virus (IHNV), but no significant similarity with glycoproteins of VSIV or rabies virus (RABV). In phylogenetic analyses SVCV was grouped consistently with VSIV, VSNJV and CHPV in the Vesiculovirus genus of Rhabdoviridae. The fish rhabdoviruses HIRRV, IHNV and viral hemorrhagic septicemia virus (VHSV) showed close relationships with each other, but only very distant relationships with mammalian rhabdoviruses. The gene junctions are highly conserved between SVCV and VSIV, well conserved between IHNV and HIRRV, but not conserved between HIRRV/IHNV and RABV. Based on the combined results we suggest that the fish lyssa-type rhabdoviruses HIRRV, IHNV and VHSV may be grouped in their own genus within the family Rhabdoviridae. Aquarhabdovirus has been proposed for the name of this new genus.

  16. Control of virus diseases of berry crops.

    PubMed

    Martin, Robert R; Tzanetakis, Ioannis E

    2015-01-01

    Virus control in berry crops starts with the development of plants free of targeted pathogens, usually viruses, viroids, phytoplasmas, and systemic bacteria, through a combination of testing and therapy. These then become the top-tier plants in certification programs and are the source from which all certified plants are produced, usually after multiple cycles of propagation. In certification schemes, efforts are made to produce plants free of the targeted pathogens to provide plants of high health status to berry growers. This is achieved using a systems approach to manage virus vectors. Once planted in fruit production fields, virus control shifts to disease control where efforts are focused on controlling viruses or virus complexes that result in disease. In fruiting fields, infection with a virus that does not cause disease is of little concern to growers. Virus control is based on the use of resistance and tolerance, vector management, and isolation. PMID:25591882

  17. Development of an enzyme-linked immunosorbent assay to detect chicken serum antibody to glycoprotein G of infectious laryngotracheitis virus.

    PubMed

    Shil, Niraj K; Markham, Philip F; Noormohammadi, Amir H; O'Rourke, Denise; Devlin, Joanne M

    2012-09-01

    Infectious laryngotracheitis (ILT) is a significant upper respiratory tract disease of chickens and has a worldwide distribution. Diagnostic enzyme-linked immunosorbent assays (ELISAs) are commonly used in ILT disease control programs. These ELISAs generally detect serum antibody to infectious laryngotracheitis virus (ILTV) and frequently utilize whole virus as the ELISA antigen. This study investigated the use of recombinant glycoprotein G (gG) of ILTV as an alterative to the use of whole virus antigen. Codon-optimized ILTV gG was expressed in Escherichia coli as a fusion protein with a maltose binding protein tag (gG-MBP). Another gG fusion protein with a 6-histidine tag (gG-His) was expressed in a baculovirus expression system. Following purification, the proteins were assessed for their suitability to be used as an antigen in an ELISA to detect ILTV-specific antibodies in sera from commercial and specific-pathogen-free (SPF) birds. The gG-MBP antigen showed some nonspecific reactions with chicken sera, but the gG-HIS antigen was found to be suitable for differentiating between sera collected from ILTV-vaccinated and unvaccinated chickens. The highest levels of agreement between the results from the gG-HIS ELISA and the commercial Trop-ILT ELISA were achieved using a cut-off value for positivity equal to the geometric mean antibody concentration of the sera from the unvaccinated birds plus 1 SD. This produced a very good level of agreement (kappa [kappa] value of 0.821) using sera from commercial birds and a moderate level of agreement (kappa value of 0.506) using sera from SPF birds. Importantly, this ELISA was also tested for its ability to discriminate between sera collected from SPF chickens vaccinated with a gG deletion mutant candidate vaccine strain of ILTV (gG-ve ILTV) and sera collected from SPF chickens vaccinated with other ILTV strains. The results showed that the gG-His ELISA has the potential to serve as a companion diagnostic tool in conjunction

  18. Three-Dimensional Structure of Herpes Simplex Virus Type 1 Glycoprotein D at 2.4-Nanometer Resolution

    PubMed Central

    Pilling, Andrew; Rosenberg, Mark F.; Willis, Sharon H.; Jäger, Joachim; Cohen, Gary H.; Eisenberg, Roselyn J.; Meredith, David M.; Holzenburg, Andreas

    1999-01-01

    Herpes simplex virus type 1 glycoprotein D (gD) is essential for virus infectivity and is responsible for binding to cellular membrane proteins and subsequently promoting fusion between the virus envelope and the cell. No structural data are available for gD or for any other herpesvirus envelope protein. Here we present a three-dimensional model for the baculovirus-expressed truncated protein gD1(306t) based on electron microscopic data. We demonstrate that gD1(306t) appears as a homotetramer containing a pronounced pocket in the center of the molecule. Monoclonal antibody binding demonstrates that the molecule is oriented such that the pocket protrudes away from the virus envelope. PMID:10438875

  19. INDUCTION OF NEUTRALIZING ANTIBODIES TO HENDRA AND NIPAH GLYCOPROTEINS USING A VENEZUELAN EQUINE ENCEPHALITIS VIRUS IN VIVO EXPESSION SYSTEM

    PubMed Central

    Defang, Gabriel N.; Khetawat, Dimple; Broder, Christopher C.; Quinnan, Gerald V.

    2010-01-01

    The emergence of Hendra Virus (HeV) and Nipah Virus (NiV) which can cause fatal infections in both animals and humans has triggered a search for an effective vaccine. Here, we have explored the potential for generating an effective humoral immune response to these zoonotic pathogens using an alphavirus-based vaccine platform. Groups of mice were immunized with Venezuelan equine encephalitis virus replicon particles (VRP) encoding the attachment or fusion glycoproteins of either HeV or NiV. We demonstrate the induction of highly potent cross-reactive neutralizing antibodies to both viruses using this approach. Preliminary study suggested early enhancement in the antibody response with use of a modified version of VRP. Overall, these data suggest that the use of an alphavirus-derived vaccine platform might serve as a viable approach for development of an effective vaccine against the henipaviruses. PMID:21050901

  20. Structure-function analysis of herpes simplex virus glycoprotein B with fusion-from-without activity

    SciTech Connect

    Roller, Devin G.; Dollery, Stephen J.; Doyle, James L.; Nicola, Anthony V.

    2008-12-20

    Fusion-from-without (FFWO) is the rapid induction of cell fusion by virions in the absence of viral protein synthesis. The combination of two amino acid mutations in envelope glycoprotein B (gB), one in the ectodomain and one in the cytoplasmic tail, can confer FFWO activity to wild type herpes simplex virus (HSV). In this report, we analyzed the entry and cell fusion phenotypes of HSV that contains FFWO gB, with emphasis on the cellular receptors for HSV, nectin-1, nectin-2 and HVEM. The ability of an HSV strain with FFWO gB to efficiently mediate FFWO via a specific gD-receptor correlated with its ability to mediate viral entry by that receptor. A FFWO form of gB was not sufficient to switch the entry of HSV from a pH-dependent, endocytic pathway to a direct fusion, pH-independent pathway. The conformation of gB with FFWO activity was not globally altered relative to wild type. However, distinct monoclonal antibodies had reduced reactivity with FFWO gB, suggesting an altered antigenic structure relative to wild type. FFWO was blocked by preincubation of virions with neutralizing antibodies to gB or gD. Together with previous studies, the results indicate that the roles of gB in FFWO and in virus-cell fusion during entry are related but not identical. This study also suggests that the FFWO function of gB is not a specific determinant for the selection of HSV entry pathway and that antigenic differences in FFWO gB may reflect its enhanced fusion activity.

  1. Antigenic and Mutational Analyses of Herpes Simplex Virus Glycoprotein B Reveal Four Functional Regions▿

    PubMed Central

    Bender, Florent C.; Samanta, Minu; Heldwein, Ekaterina E.; de Leon, Manuel Ponce; Bilman, Elina; Lou, Huan; Whitbeck, J. Charles; Eisenberg, Roselyn J.; Cohen, Gary H.

    2007-01-01

    Glycoprotein B (gB), along with gD, gH, and gL, is essential for herpes simplex virus (HSV) entry. The crystal structure of the gB ectodomain revealed it to be an elongated multidomain trimer. We generated and characterized a panel of 67 monoclonal antibodies (MAbs). Eleven of the MAbs had virus-neutralizing activity. To organize gB into functional regions within these domains, we localized the epitopes recognized by the entire panel of MAbs and mapped them onto the crystal structure of gB. Most of the MAbs were directed to continuous or discontinuous epitopes, but several recognized discontinuous epitopes that showed some resistance to denaturation, and we refer to them as pseudo-continuous. Each category contained some MAbs with neutralizing activity. To map continuous epitopes, we used overlapping peptides that spanned the gB ectodomain and measured binding by enzyme-linked immunosorbent assay. To identify discontinuous and pseudocontinuous epitopes, a purified form of the ectodomain of gB, gB(730t), was cleaved by α-chymotrypsin into two major fragments comprising amino acids 98 to 472 (domains I and II) and amino acids 473 to 730 (major parts of domains III, IV, and V). We also constructed a series of gB truncations to augment the other mapping strategies. Finally, we used biosensor analysis to assign the MAbs to competition groups. Together, our results identified four functional regions: (i) one formed by residues within domain I and amino acids 697 to 725 of domain V; (ii) a second formed by residues 391 to 410, residues 454 to 475, and a less-defined region within domain II; (iii) a region containing residues of domain IV that lie close to domain III; and (iv) the first 12 residues of the N terminus that were not resolved in the crystal structure. Our data suggest that multiple domains are critical for gB function. PMID:17267495

  2. Varicella-zoster virus glycoprotein expression differentially induces the unfolded protein response in infected cells.

    PubMed

    Carpenter, John E; Grose, Charles

    2014-01-01

    Varicella-zoster virus (VZV) is a human herpesvirus that spreads to children as varicella or chicken pox. The virus then establishes latency in the nervous system and re-emerges, typically decades later, as zoster or shingles. We have reported previously that VZV induces autophagy in infected cells as well as exhibiting evidence of the Unfolded Protein Response (UPR): XBP1 splicing, a greatly expanded Endoplasmic Reticulum (ER) and CHOP expression. Herein we report the results of a UPR specific PCR array that measures the levels of mRNA of 84 different components of the UPR in VZV infected cells as compared to tunicamycin treated cells as a positive control and uninfected, untreated cells as a negative control. Tunicamycin is a mixture of chemicals that inhibits N-linked glycosylation in the ER with resultant protein misfolding and the UPR. We found that VZV differentially induces the UPR when compared to tunicamycin treatment. For example, tunicamycin treatment moderately increased (8-fold) roughly half of the array elements while downregulating only three (one ERAD and two FOLD components). VZV infection on the other hand upregulated 33 components including a little described stress sensor CREB-H (64-fold) as well as ER membrane components INSIG and gp78, which modulate cholesterol synthesis while downregulating over 20 components mostly associated with ERAD and FOLD. We hypothesize that this expression pattern is associated with an expanding ER with downregulation of active degradation by ERAD and apoptosis as the cell attempts to handle abundant viral glycoprotein synthesis. PMID:25071735

  3. Development of an infectious surrogate hepatitis C virus based on a recombinant vesicular stomatitis virus expressing hepatitis C virus envelope glycoproteins and green fluorescent protein.

    PubMed

    Okuma, Kazu; Fukagawa, Koji; Tateyama, Seiji; Kohma, Takuya; Mochida, Keiko; Hiyoshi, Masateru; Takahama, Youichi; Hamaguchi, Yukio; Hirose, Kunitaka; Buonocore, Linda; Rose, John K; Mizuochi, Toshiaki; Hamaguchi, Isao

    2015-01-01

    To develop surrogate viruses for hepatitis C virus (HCV), we previously produced recombinant vesicular stomatitis viruses (rVSVs) lacking glycoprotein G but instead expressing chimeric HCV E1/E2 fused to G. These rVSVs were not infectious in HCV-susceptible hepatoma cells. In this study, to develop an infectious surrogate HCV based on an rVSV (vesicular stomatitis virus [VSV]/HCV), we generated a novel rVSV encoding the native E1/E2 (H77 strain) and green fluorescent protein (GFP) instead of G. Here, we showed that this VSV/HCV efficiently infected human hepatoma cells, including Huh7 human hepatoma cells, expressed GFP in these cells, and propagated, but did not do so in nonsusceptible BHK-21 cells. The infectivity of VSV/HCV, measured as the number of foci of GFP-positive cells, was specifically reduced by the addition of chimpanzee anti-HCV serum, anti-E2 antibody, or anti-CD81 antibody to the cultures. When sera obtained from HCV-infected or uninfected patients were added, infection was selectively inhibited only by the sera of HCV-infected patients. These data together suggest that this infectious GFP-expressing VSV/HCV could be a useful tool for studying the mechanisms of HCV entry into cells and for assessing potential inhibitors of viral entry, including neutralizing antibodies. PMID:25672345

  4. Marek’s disease virus genomics

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Marek’s disease virus (MDV) is one of the most oncogenic herpesviruses known and induces a rapid onset T-cell lymphoma and demyelinating disease in chickens. It represents the first of three neoplastic diseases (including hepatocellular carcinoma: hepatitis B virus; and cervical carcinoma: human pap...

  5. Low-pH conformational changes of rabies virus glycoprotein and their role in membrane fusion.

    PubMed Central

    Gaudin, Y; Ruigrok, R W; Knossow, M; Flamand, A

    1993-01-01

    Fusion of rabies virus with membranes occurs at acidic pH and is mediated by the viral spike glycoprotein (G). In this paper, we provide the basis for a description of structural transitions associated with exposure to low pH and of their role in membrane fusion. First, we have extended previous studies of fusion kinetics and we have shown that low-pH inhibition of fusion is detectable at 0.5 pH units higher than fusion. Second, low-pH-induced conformational changes were analyzed by using electron microscopy and monoclonal antibody binding assays. The existence of a pH-dependent equilibrium between the native and a low-pH inactive conformation was demonstrated. Third, besides these two conformations, we, using the fluorescent probe ANS (8-anilino-1-naphthalenesulfonic acid), provide evidence for the existence of a transient third state which appears to be more hydrophobic than the native state. Our results suggest that this transient state is responsible for viral aggregation at low pH and could play a role in the first steps of the fusion mechanism. Images PMID:8437221

  6. Immunogenicity of synthetic peptides representing neutralizing epitopes on the glycoprotein of infectious hematopoietic necrosis virus

    USGS Publications Warehouse

    Emmenegger, E.; Landolt, M.; LaPatra, S.; Winton, J.

    1997-01-01

    Three peptides, P76, P226, and P268 representing 3 putative antigen~c determinants on the glycoprotein of infectious hematopoietic necrosis virus (IHNV), were synthesized and injected into rainbow trout Oncorhynchus mykiss to assess their immunogen~city. Antisera extracted from the immunized trout were analyzed uslng an enzyme linked imrnunosorbent assay (ELISA) for the presence of antibodies that could bind to the peptides or to intact virions of IHNV. The antisera were also tested for neutralizing activity against IHNV by a complement-mediated neutralization assay. In general, recognition of the peptides and IHNV was low and only a few antibody binding patterns were demonstrated. Antisera from fish injected with P76 constructs recognized the homologous peptide more than the heterologous peptides, whereas antisera from fish inoculated with either P226 or P268 constructs recognized P76 equally, or better, than the homologous peptide; however, there was a high degree of individual variation within each treatment group. Neutralization actlvlty was demonstrated by serum from a single flsh lnlected with one of the pept~des (P268) and from 7 of 10 positive control f~sh Infected with an attenuated strain of IHNV Possible explanations for the dichotomous immune responses are discussed. These results indicate we need to improve our overall understanding of the

  7. Structure of the Ebola Virus Glycoprotein Bound to An Antibody From a Human Survivor

    SciTech Connect

    Lee, J.E.; Fusco, M.L.; Hessell, A.J.; Oswald, W.B.; Burton, D.R.; Saphire, E.O.

    2009-05-20

    Ebola virus (EBOV) entry requires the surface glycoprotein (GP) to initiate attachment and fusion of viral and host membranes. Here we report the crystal structure of EBOV GP in its trimeric, pre-fusion conformation (GP1+GP2) bound to a neutralizing antibody, KZ52, derived from a human survivor of the 1995 Kikwit outbreak. Three GP1 viral attachment subunits assemble to form a chalice, cradled by the GP2 fusion subunits, while a novel glycan cap and projected mucin-like domain restrict access to the conserved receptor-binding site sequestered in the chalice bowl. The glycocalyx surrounding GP is likely central to immune evasion and may explain why survivors have insignificant neutralizing antibody titres. KZ52 recognizes a protein epitope at the chalice base where it clamps several regions of the pre-fusion GP2 to the amino terminus of GP1. This structure provides a template for unraveling the mechanism of EBOV GP-mediated fusion and for future immunotherapeutic development.

  8. Identification of glycosaminoglycan-binding sites within hepatitis C virus envelope glycoprotein E2*.

    PubMed

    Olenina, L V; Kuzmina, T I; Sobolev, B N; Kuraeva, T E; Kolesanova, E F; Archakov, A I

    2005-11-01

    Heparan sulphate is one of the candidate receptors for hepatitis C virus (HCV). Envelope glycoproteins of HCV have been proposed to be responsible for recognition and binding with cell receptors. They are characterized by great genetic polymorphism. In this study the mapping of regions with glycosaminoglycan-binding properties within HCV envelope proteins has been undertaken. We prepared a set of overlapping peptides corresponding to conserved regions of these envelope proteins and analysed them by solid phase heparin-binding assay. The search for established glycosaminoglycan-binding motifs in the HCV envelope proteins showed the absence of the sites corresponding to the glycosaminoglycan-binding patterns in consensus sequence. We identified one highly conserved and two less conserved heparin-binding sequences within the envelope protein E2 based on solid phase assay results. We did not find any differences in binding efficiency of these peptides with heparin, heparan sulphate or dextran sulphate. Our data supported the specific association between HCV envelope protein E2 and cell surface glycosaminoglycans. We hypothesize that identified regions from E2 can contribute to HCV binding to cell surface glycosaminoglycans. PMID:16255759

  9. Overexpression of the Rabies Virus Glycoprotein Results in Enhancement of Apoptosis and Antiviral Immune Response

    PubMed Central

    Faber, Milosz; Pulmanausahakul, Rojjanaporn; Hodawadekar, Suchita S.; Spitsin, Sergei; McGettigan, James P.; Schnell, Matthias J.; Dietzschold, Bernhard

    2002-01-01

    A recombinant rabies virus (RV) carrying two identical glycoprotein (G) genes (SPBNGA-GA) was constructed and used to determine the effect of RV G overexpression on cell viability and immunity. Immunoprecipitation analysis and flow cytometry showed that tissue culture cells infected with SPBNGA-GA produced, on average, twice as much RV G as cells infected with RV carrying only a single RV G gene (SPBNGA). The overexpression of RV G in SPBNGA-GA-infected NA cells was paralleled by a significant increase in caspase 3 activity followed by a marked decrease in mitochondrial respiration, neither of which was observed in SPBNGA-infected cells. Furthermore, fluorescence staining and confocal microscopy revealed an increased extent of apoptosis and markedly reduced neurofilament and F actin in SPBNGA-GA-infected primary neuron cultures compared with neuronal cells infected with SPBNGA, supporting the concept that RV G or motifs of the RV G gene trigger the apoptosis cascade. Mice immunized with SPBNGA-GA showed substantially higher antibody titers against the RV G and against the nucleoprotein than SPBNGA-immunized mice, suggesting that the speed or extent of apoptosis directly determines the magnitude of the antibody response. PMID:11884563

  10. Sialylated oligosaccharides O-glycosidically linked to glycoprotein C from herpes simplex virus type 1.

    PubMed

    Dall'Olio, F; Malagolini, N; Speziali, V; Campadelli-Fiume, G; Serafini-Cessi, F

    1985-10-01

    Glycoprotein C (gC) was purified by immunoabsorbent from herpes simplex virus type-1-infected BHK cells labeled with [14C]glucosamine for 11 h and chased for 3 h. Glycopeptides obtained by pronase digestion of gC were fractionated by Bio-Gel filtration and concanavalin A-Sepharose chromatography. Each glycopeptide fraction was analyzed for amino sugar composition by thin-layer chromatography. The majority of radioactivity was recovered as N-acetylglucosamine, but a significant amount of labeled N-acetylgalactosamine was detected and recovered preferentially in some glycopeptide species. Mild alkaline borohydride treatment of the glycopeptides resulted in the release of small degradation products which contained N-acetylgalactosaminitol as the major labeled component and a drastic reduction of N-acetylgalactosamine in the residual glycopeptides. These results demonstrated that gC carries O-glycosidically linked oligosaccharides in addition to the N-linked di- and triantennary glycans previously described (F. Serafini-Cessi, F. Dall'Olio, L. Pereira, and G. Campadelli-Fiume, J. Virol. 51:838-844, 1984). Chromatographic behavior on DEAE-Sephacel chromatography and neuraminidase digestion of O-linked oligosaccharides indicated the presence of two major sialylated species carrying one and two sialic acid residues, respectively. The characterization of a peculiar glycopeptide species supported the notion that some of the O-linked oligosaccharides are bound to a cluster of hydroxyamino acids located near an N-glycosylation site which carries one N-linked diantennary oligosaccharide. PMID:2993643

  11. C3d enhanced DNA vaccination induced humoral immune response to glycoprotein C of pseudorabies virus

    SciTech Connect

    Tong Tiezhu; Fan Huiying; Tan Yadi; Xiao Shaobo; Ling Jieyu; Chen Huanchun; Guo Aizhen . E-mail: aizhen@mail.hzau.edu.cn

    2006-09-08

    Murine C3d were utilized to enhance immunogenicity of pseudorabies virus (PrV) gC DNA vaccination. Three copies of C3d and four copies of CR2-binding domain M28{sub 4} were fused, respectively, to truncated gC gene encoding soluble glycoprotein C (sgC) in pcDNA3.1. BALB/c mice were, respectively, immunized with recombinant plasmids, blank vector, and inactivated vaccine. The antibody ELISA titer for sgC-C3d{sub 3} DNA was 49-fold more than that for sgC DNA, and the neutralizing antibody obtained 8-fold rise. Protection of mice from death after lethal PrV (316 LD{sub 5}) challenge was augmented from 25% to 100%. Furthermore, C3d fusion increased Th2-biased immune response by inducing IL-4 production. The IL-4 level for sgC-C3d{sub 3} DNA immunization approached that for the inactivated vaccine. Compared to C3d, M28 enhanced sgC DNA immunogenicity to a lesser extent. In conclusion, we demonstrated that murine C3d fusion significantly enhanced gC DNA immunity by directing Th1-biased to a balanced and more effective Th1/Th2 response.

  12. Human immunodeficiency virus glycoprotein (gp120) induction of monocyte arachidonic acid metabolites and interleukin 1.

    PubMed Central

    Wahl, L M; Corcoran, M L; Pyle, S W; Arthur, L O; Harel-Bellan, A; Farrar, W L

    1989-01-01

    This study reports on the direct effect of the envelope glycoprotein (gp120) of the human immunodeficiency virus type 1 (HIV-1) on human monocyte function. Addition of preparations of purified gp120 from the HIV-1 to human monocytes resulted in the production of interleukin 1 (IL-1) and arachidonic acid metabolites from the cyclooxygenase and lipoxygenase pathways. Quantification of prostaglandin E2 (PGE2) and IL-1 revealed an increase in both mediators with 50 ng of gp120 per ml and an increase of 12- and 30- to 40-fold with 200-400 ng of gp120 per ml, respectively. Unlike native gp120, the recombinant nonglycosylated gp120 fragments PB1-RF and PB1-IIIB, as well as one of the core structural proteins of HIV-1, p24, did not increase arachidonic acid metabolism or IL-1 activity. Cytofluorometric analysis revealed that gp120 blocked the binding of OKT4A to the CD4 on monocytes, whereas OKT4 binding was unaffected. Involvement of the CD4 in signal transduction was further demonstrated by the ability of OKT4 and OKT4A monoclonal antibodies to increase monocyte PGE2, IL-1 activity, and nanogram amounts of IL-1 beta. PMID:2536171

  13. Pseudotyping of lentiviral vector with novel vesiculovirus envelope glycoproteins derived from Chandipura and Piry viruses.

    PubMed

    Hu, Shuang; Mohan Kumar, Dipu; Sax, Chelsea; Schuler, Clayton; Akkina, Ramesh

    2016-01-15

    While the envelope glycoprotein of vesicular stomatitis virus (VSV-G) is widely used for pseudotyping of lentiviral vectors, sub-optimal gene transfer into certain cell types and its sensitivity to inactivation by human complement hinders its broader applications. To find alternative candidates, here we evaluated two serologically distinct novel viral envelopes derived from Chandipura (CNV-G) and Piry (PRV-G) vesiculoviruses. Both permitted generation of high titer psuedotyped lentiviral vectors with a capacity for high efficiency gene transfer into various cell types from different species. In human lymphoid and hematopoietic stem cells, their transduction efficiency was significantly lower than that of VSV-G. However, both novel envelopes were found to be more resistant to inactivation by human serum complement compared to VSV-G. Thus CNV-G and PRV-G envelopes can be harnessed for multiple uses in the future based on the cell type that needs to be gene transduced and possibly for in vivo gene transfer. PMID:26650691

  14. Functional Implications of the Human T-Lymphotropic Virus Type 1 Transmembrane Glycoprotein Helical Hairpin Structure

    PubMed Central

    Maerz, Anne L.; Center, Rob J.; Kemp, Bruce E.; Kobe, Bostjan; Poumbourios, Pantelis

    2000-01-01

    Retrovirus entry into cells follows receptor binding by the surface-exposed envelope glycoprotein (Env) subunit (SU), which triggers the membrane fusion activity of the transmembrane (TM) protein. TM protein fragments expressed in the absence of SU adopt helical hairpin structures comprising a central coiled coil, a region of chain reversal containing a disulfide-bonded loop, and a C-terminal segment that packs onto the exterior of the coiled coil in an antiparallel manner. Here we used in vitro mutagenesis to test the functional role of structural elements observed in a model helical hairpin, gp21 of human T-lymphotropic virus type 1. Membrane fusion activity requires the stabilization of the N and C termini of the central coiled coil by a hydrophobic N cap and a small hydrophobic core, respectively. A conserved Gly-Gly hinge motif preceding the disulfide-bonded loop, a salt bridge that stabilizes the chain reversal region, and interactions between the C-terminal segment and the coiled coil are also critical for fusion activity. Our data support a model whereby the chain reversal region transmits a conformational signal from receptor-bound SU to induce the fusion-activated helical hairpin conformation of the TM protein. PMID:10864675

  15. A recombinant pseudotyped lentivirus expressing the envelope glycoprotein of Hantaan virus induced protective immunity in mice

    PubMed Central

    2013-01-01

    Background Hantaviruses cause acute hemorrhagic fever with renal syndrome (HFRS). Currently, several types of inactivated HFRS vaccines are widely used, however the limited ability of these immunogen to elicit neutralizing antibodies restricts vaccine efficacy. Development of an effective vaccine to overcome this weakness is must. Methods In the present study, a recombinant pseudotyped lentivirus bearing the hantaan virus (HTNV) envelope glycoproteins (GP), rLV-M, was constructed. C57BL/6 mice were immunized with the rLV-M and a series of immunological assays were conducted to determine the immunogenicity of the recombinant pseudotyped lentivirus. The humoral and cell-mediated immune responses induced by rLV-M were compared with those of the inactivated HFRS vaccine. Results Indirect immunofluorescence assay (IFA) showed the rLV-M expressed target proteins in HEK-293cells. In mice, the rLV-M efficiently induced GP-specific humoral responses and protection against HTNV infection. Furthermore, the rLV-M induced higher neutralizing antibody titers than the inactivated HFRS vaccine control. Conclusions The results indicated the potential of using a pseudotyped lentivirus as a delivery vector for a hantavirus vaccine immunogen. PMID:24093752

  16. Structure of the Ebola virus glycoprotein bound to a human survivor antibody

    PubMed Central

    Lee, Jeffrey E.; Fusco, Marnie L.; Hessell, Ann J.; Oswald, Wendelien B.; Burton, Dennis R.; Saphire, Erica Ollmann

    2008-01-01

    Ebola virus (EBOV) entry requires the surface glycoprotein, GP, to initiate attachment and fusion of viral and host membranes. Here, we report the crystal structure of EBOV GP in its trimeric, pre-fusion conformation (GP1+GP2) bound to a neutralizing antibody, KZ52, derived from a human survivor of the 1995 Kikwit outbreak. Three GP1 viral attachment subunits assemble to form a chalice, cradled by the GP2 fusion subunits, while a novel glycan cap and projected mucin-like domain restrict access to the conserved receptor-binding site sequestered in the chalice bowl. The glycocalyx surrounding GP is likely central to immune evasion and may explain why survivors have insignificant neutralizing antibody titres. KZ52 recognizes a protein epitope at the chalice base where it clamps several regions of the pre-fusion GP2 to the N terminus of GP1. This structure now provides a template for unraveling the mechanism of EBOV GP-mediated fusion and for future immunotherapeutic development. PMID:18615077

  17. Characterization of hepatitis C virus envelope glycoprotein complexes expressed by recombinant vaccinia viruses.

    PubMed

    Ralston, R; Thudium, K; Berger, K; Kuo, C; Gervase, B; Hall, J; Selby, M; Kuo, G; Houghton, M; Choo, Q L

    1993-11-01

    We constructed recombinant vaccinia virus vectors for expression of the structural region of hepatitis C virus (HCV). Infection of mammalian cells with a vector (vv/HCV1-906) encoding C-E1-E2-NS2 generated major protein species of 22 kDa (C), 33 to 35 kDa (E1), and 70 to 72 kDa (E2), as observed previously with other mammalian expression systems. The bulk of the E1 and E2 expressed by vv/HCV1-906 was found integrated into endoplasmic reticulum membranes as core-glycosylated species, suggesting that these E1 and E2 species represent intracellular forms of the HCV envelope proteins. HCV E1 and E2 formed E1-E2 complexes which were precipitated by either anti-E1 or anti-E2 serum and which sedimented at approximately 15 S on glycerol density gradients. No evidence of intermolecular disulfide bonding between E1 and E2 was detected. E1 and E2 were copurified to approximately 90% purity by mild detergent extraction followed by chromatography on Galanthus nivalus lectin-agarose and DEAE-Fractogel. Immunization of chimpanzees with purified E1-E2 generated high titers of anti-E1 and anti-E2 antibodies. Further studies, to be reported separately, demonstrated that purified E1-E2 complexes were recognized at high frequency by HCV+ human sera (D. Y. Chien, Q.-L. Choo, R. Ralston, R. Spaete, M. Tong, M. Houghton, and G. Kuo, Lancet, in press) and generated protective immunity in chimpanzees (Q.-L. Choo, G. Kuo, R. Ralston, A. Weiner, D. Chien, G. Van Nest, J. Han, K. Berger, K. Thudium, J. Kansopon, J. McFarland, A. Tabrizi, K. Ching, B. Mass, L. B. Cummins, E. Muchmore, and M. Houghton, submitted for publication), suggesting that these purified HCV envelope proteins display native HCV epitopes. PMID:8411378

  18. Structure of Epstein-Barr Virus Glycoprotein 42 Suggests a Mechanism for Triggering Receptor-Activated Virus Entry

    SciTech Connect

    Kirschner, Austin N.; Sorem, Jessica; Longnecker, Richard; Jardetzky, Theodore S.

    2009-05-26

    Epstein-Barr virus requires glycoproteins gH/gL, gB, and gp42 to fuse its lipid envelope with B cells. Gp42 is a type II membrane protein consisting of a flexible N-terminal region, which binds gH/gL, and a C-terminal lectin-like domain that binds to the B-cell entry receptor human leukocyte antigen (HLA) class II. Gp42 triggers membrane fusion after HLA binding, a process that requires simultaneous binding to gH/gL and a functional hydrophobic pocket in the lectin domain adjacent to the HLA binding site. Here we present the structure of gp42 in its unbound form. Comparisons to the previously determined structure of a gp42:HLA complex reveals additional N-terminal residues forming part of the gH/gL binding site and structural changes in the receptor binding domain. Although the core of the lectin domain remains similar, significant shifts in two loops and an {alpha} helix bordering the essential hydrophobic pocket suggest a structural mechanism for triggering fusion.

  19. Induction of protective immunity in animals vaccinated with recombinant vaccinia viruses that express PreM and E glycoproteins of Japanese encephalitis virus.

    PubMed Central

    Yasuda, A; Kimura-Kuroda, J; Ogimoto, M; Miyamoto, M; Sata, T; Sato, T; Takamura, C; Kurata, T; Kojima, A; Yasui, K

    1990-01-01

    A cDNA clone representing the genome of structural proteins of Japanese encephalitis virus (JEV) was inserted into the thymidine kinase gene of vaccinia virus strains LC16mO and WR under the control of a strong early-late promoter for the vaccinia virus 7.5-kilodalton polypeptide. Indirect immunofluorescence and fluorescence-activated flow cytometric analysis revealed that the recombinant vaccinia viruses expressed JEV E protein on the membrane surface, as well as in the cytoplasm, of recombinant-infected cells. In addition, the E protein expressed from the JEV recombinants reacted to nine different characteristic monoclonal antibodies, some of which have hemagglutination-inhibiting and JEV-neutralizing activities. Radioimmunoprecipitation analysis demonstrated that two major proteins expressed in recombinant-infected cells were processed and glycosylated as the authentic PreM and E glycoproteins of JEV. Inoculation of rabbits with the infectious recombinant vaccinia virus resulted in rapid production of antiserum specific for the PreM and E glycoproteins of JEV. This antiserum had both hemagglutination-inhibiting and virus-neutralizing activities against JEV. Furthermore, mice vaccinated with the recombinant also produced JEV-neutralizing antibodies and were resistant to challenge with JEV. Images PMID:2159544

  20. Comparison of the safety and protective efficacy of vaccination with glycoprotein-G-deficient infectious laryngotracheitis virus delivered via eye-drop, drinking water or aerosol.

    PubMed

    Devlin, J M; Browning, G F; Gilkerson, J R; Fenton, S P; Hartley, C A

    2008-02-01

    Infectious laryngotracheitis virus (ILTV), an alphaherpesvirus, causes respiratory disease in chickens and is commonly controlled by vaccination with conventionally attenuated virus strains. These vaccines have limitations due to residual pathogenicity and reversion to virulence. To avoid these problems and to better control disease, attention has recently turned towards developing a novel vaccine strain that lacks virulence gene(s). Glycoprotein G (gG) is a virulence factor in ILTV. A gG-deficient strain of ILTV has been shown to be less pathogenic than currently available vaccine strains following intratracheal inoculation of specific pathogen free chickens. Intratracheal inoculation of gG-deficient ILTV has also been shown to induce protection against disease following challenge with virulent virus. Intratracheal inoculation, however, is not suitable for large-scale vaccination of commercial poultry flocks. In this study, inoculation of gG-deficient ILTV via eye-drop, drinking water and aerosol were investigated. Aerosol inoculation resulted in undesirably low levels of safety and protective efficacy. Inoculation via eye-drop and drinking water was safe, and the levels of protective efficacy were comparable with intratracheal inoculation. Thus, gG-deficient ILTV appears to have potential for use in large-scale poultry vaccination programmes when administered via eye-drop or in drinking water. PMID:18202954

  1. Blackberry (Rubus spp.)-Virus Diseases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Many viruses have been found in blackberries in the Pacific Northwest. Blackberry calico virus (a carlavirus) is universally present in older commercial 'Thornless Loganberry' fields. Similar calico diseases occur in field-run 'Marion', 'Chehalem', 'Olallie', and 'Waldo' blackberries. Other virus di...

  2. Reinvestigation of the role of the rabies virus glycoprotein in viral pathogenesis using a reverse genetics approach.

    PubMed

    Morimoto, K; Foley, H D; McGettigan, J P; Schnell, M J; Dietzschold, B

    2000-10-01

    The rabies virus glycoprotein (G) gene of the highly neuroinvasive and neurotropic strains SHBRV-18, CVS-N2c, and CVS-B2c was introduced into the non-neuroinvasive and less neurotropic SN-10 strain to provide further insight into the role of G in the pathogenesis of rabies. Phenotypic analyses of the recombinant viruses revealed, as expected, that the neurotropism of a particular rabies virus strain was a function of its G. Nevertheless, the pathogenicity of the recombinant viruses was, in every case, markedly lower than that of the wild-type viruses suggesting that while the G dictates neurotropism, other viral attributes are also important in pathogenesis. The low pathogenicity of the recombinant viruses is at least in part due to a strong increase in transcription activity. On the other hand, the production of infectious virus by the R-SHB18 recombinant virus-infected cells was significantly delayed by comparison with SHBRV-18 wild-type virus infected-cells. Replacement of the R-SHB18 G cytoplasmic domain, transmembrane domain, and stem region with its SN-10 G counterparts neither results in a significant increase in budding efficiency nor an increase in pathogenicity. These results suggest that an optimal match of the cytoplasmic domain of G with the matrix protein may not be sufficient for maximal virus budding efficiency, which is evidently a major factor of virus pathogenicity. Our studies indicate that to maintain pathogenicity, the interactions between various structural elements of rabies virus must be highly conserved and the expression of viral proteins, in particular the G protein, must be strictly controlled. PMID:11031690

  3. The Transmembrane Domain of Hepatitis C Virus Glycoprotein E1 Is a Signal for Static Retention in the Endoplasmic Reticulum

    PubMed Central

    Cocquerel, Laurence; Duvet, Sandrine; Meunier, Jean-Christophe; Pillez, André; Cacan, René; Wychowski, Czeslaw; Dubuisson, Jean

    1999-01-01

    Hepatitis C virus (HCV) glycoproteins E1 and E2 assemble to form a noncovalent heterodimer which, in the cell, accumulates in the endoplasmic reticulum (ER). Contrary to what is observed for proteins with a KDEL or a KKXX ER-targeting signal, the ER localization of the HCV glycoprotein complex is due to a static retention in this compartment rather than to its retrieval from the cis-Golgi region. A static retention in the ER is also observed when E2 is expressed in the absence of E1 or for a chimeric protein containing the ectodomain of CD4 in fusion with the transmembrane domain (TMD) of E2. Although they do not exclude the presence of an intracellular localization signal in E1, these data do suggest that the TMD of E2 is an ER retention signal for HCV glycoprotein complex. In this study chimeric proteins containing the ectodomain of CD4 or CD8 fused to the C-terminal hydrophobic sequence of E1 were shown to be localized in the ER, indicating that the TMD of E1 is also a signal for ER localization. In addition, these chimeric proteins were not processed by Golgi enzymes, indicating that the TMD of E1 is responsible for true retention in the ER, without recycling through the Golgi apparatus. Together, these data suggest that at least two signals (TMDs of E1 and E2) are involved in ER retention of the HCV glycoprotein complex. PMID:10074109

  4. Rabies virulence: effect on pathogenicity and sequence characterization of rabies virus mutations affecting antigenic site III of the glycoprotein.

    PubMed Central

    Seif, I; Coulon, P; Rollin, P E; Flamand, A

    1985-01-01

    Using four neutralizing monoclonal antibodies which presumably bind to the same antigenic site on the CVS glycoprotein (antigenic site III as defined by cross-neutralization tests), we isolated 58 mutants of the CVS strain of rabies virus. These mutants were highly resistant to the selecting antibodies and grew efficiently in cell cultures. We classified them into five groups on the basis of the pattern of resistance to the four antibodies. We determined pathogenicities of the mutants for adult mice by intracerebral inoculation. Group 2 mutants were nonpathogenic or had attenuated pathogenicity. On the contrary, mutants from the other groups were pathogenic, causing paralysis and death as does CVS. We determined the nucleotide alterations of representative mutants from each group by using the dideoxy method of RNA sequencing. In the glycoproteins of eight nonpathogenic or attenuated mutants, we identified an amino acid substitution at position 333. Arginine 333 was replaced by either glutamine or glycine. In the glycoprotein of eight pathogenic mutants, we identified an amino acid substitution at lysine 330, asparagine 336, or isoleucine 338. Thus, although all substitutions affected neutralization and were located close to each other in the glycoprotein sequence, only substitutions at position 333 affected pathogenicity. Images PMID:2579247

  5. Structure-Based Design of Head-Only Fusion Glycoprotein Immunogens for Respiratory Syncytial Virus

    PubMed Central

    Boyington, Jeffrey C.; Chen, Man; Kong, Wing-Pui; Ngwuta, Joan O.; Thomas, Paul V.; Tsybovsky, Yaroslav; Yang, Yongping; Zhang, Baoshan; Chen, Lei; Druz, Aliaksandr; Georgiev, Ivelin S.; Ko, Kiyoon; Zhou, Tongqing; Mascola, John R.; Graham, Barney S.; Kwong, Peter D.

    2016-01-01

    Respiratory syncytial virus (RSV) is a significant cause of severe respiratory illness worldwide, particularly in infants, young children, and the elderly. Although no licensed vaccine is currently available, an engineered version of the metastable RSV fusion (F) surface glycoprotein—stabilized in the pre-fusion (pre-F) conformation by “DS-Cav1” mutations—elicits high titer RSV-neutralizing responses. Moreover, pre-F-specific antibodies, often against the neutralization-sensitive antigenic site Ø in the membrane-distal head region of trimeric F glycoprotein, comprise a substantial portion of the human response to natural RSV infection. To focus the vaccine-elicited response to antigenic site Ø, we designed a series of RSV F immunogens that comprised the membrane-distal head of the F glycoprotein in its pre-F conformation. These “head-only” immunogens formed monomers, dimers, and trimers. Antigenic analysis revealed that a majority of the 70 engineered head-only immunogens displayed reactivity to site Ø-targeting antibodies, which was similar to that of the parent RSV F DS-Cav1 trimers, often with increased thermostability. We evaluated four of these head-only immunogens in detail, probing their recognition by antibodies, their physical stability, structure, and immunogenicity. When tested in naïve mice, a head-only trimer, half the size of the parent RSV F trimer, induced RSV titers, which were statistically comparable to those induced by DS-Cav1. When used to boost DS-Cav1-primed mice, two head-only RSV F immunogens, a dimer and a trimer, boosted RSV-neutralizing titers to levels that were comparable to those boosted by DS-Cav1, although with higher site Ø-directed responses. Our results provide proof-of-concept for the ability of the smaller head-only RSV F immunogens to focus the vaccine-elicited response to antigenic site Ø. Decent primary immunogenicity, enhanced physical stability, potential ease of manufacture, and potent immunogenicity

  6. Structure-Based Functional Analyses of Domains II and III of Pseudorabies Virus Glycoprotein H

    PubMed Central

    Böhm, Sebastian W.; Eckroth, Elisa; Backovic, Marija; Klupp, Barbara G.; Rey, Felix A.; Fuchs, Walter

    2014-01-01

    ABSTRACT Enveloped viruses utilize membrane fusion for entry into, and release from, host cells. For entry, members of the Herpesviridae require at least three envelope glycoproteins: the homotrimeric gB and a heterodimer of gH and gL. The crystal structures of three gH homologues, including pseudorabies virus (PrV) gH, revealed four conserved domains. Domain II contains a planar β-sheet (“fence”) and a syntaxin-like bundle of three α-helices (SLB), similar to those found in eukaryotic fusion proteins, potentially executing an important role in gH function. To test this hypothesis, we introduced targeted mutations into the PrV gH gene, which either disrupt the helices of the SLB by introduction of proline residues or covalently join them by artificial intramolecular disulfide bonds between themselves, to the adjacent fence region, or to domain III. Disruption of either of the three α-helices of the SLB (A250P, V275P, V298P) severely affected gH function in in vitro fusion assays and replication of corresponding PrV mutants. Considerable defects in fusion activity of gH, as well as in penetration kinetics and cell-to-cell spread of PrV mutants, were also observed after disulfide linkage of two α-helices within the SLB (A284C-S291C) or between SLB and domain III (H251C-L432C), as well as by insertions of additional cysteine pairs linking fence, SLB, and domain III. In vitro fusion activity of mutated gH could be partly restored by reduction of the artificial disulfide bonds. Our results indicate that the structure and flexibility of the SLB are relevant for the function of PrV gH in membrane fusion. IMPORTANCE Mutational analysis based on crystal structures of proteins is a powerful tool to understand protein function. Here, we continued our study of pseudorabies virus gH, a part of the core fusion machinery of herpesviruses. We previously showed that the “flap” region in domain IV of PrV gH is important for its function. We now demonstrate that mutations

  7. Identification of glycoprotein storage diseases by lectins: a new diagnostic method.

    PubMed

    Alroy, J; Orgad, U; Ucci, A A; Pereira, M E

    1984-12-01

    The specific diagnosis of glycoprotein storage diseases is made by demonstrating a deficiency in enzyme activity or an elevation of undegraded oligosaccharides in cells or body fluids. Prospective sampling and expensive specialized biochemistry, which is also time consuming, are required for such studies. We used lectin reagents on paraffin-embedded tissue sections to identify the specific sugars in undegraded stored substances. We studied 22 cases of glycoprotein storage diseases and differentiated histochemically between alpha- and beta-mannosidosis, fucosidosis, and sialisidosis. Cells affected with alpha-mannosidosis stained with Concanavalia ensiformis (Con A), Triticum vulgaris (WGA), and succinyl-WGA (S-WGA), while beta-mannosidosis cells did not stain with any of the lectins used. In fucosidosis the affected cells stained with Ulex europeus-I (UEA-I), while sialisidosis-affected cells stained with WGA, and in three cases with Arachis hypogea (PNA). This study indicates that lectin histochemistry provides a reliable specific diagnostic pattern for some glycoprotein storage diseases using a simple and inexpensive method. PMID:6501863

  8. [Epidemiological characteristics of Zika virus disease].

    PubMed

    Li, Jiandong; Li, Dexin

    2016-03-01

    Zika virus disease is an emerging mosquito-borne acute infectious disease caused by Zika virus, so far there have been no available vaccine or specific treatment. Currently, the outbreaks of Zika virus disease mainly occurs in the Americas, but the regional distribution of the disease is in rapid expansion, 34 countries and territories have reported autochthonous transmission of the virus. The illness is usually mild with very rarely death, but increased reports of birth defects and neurologic disorders in the areas affected by Zika virus has caused extensive concern worldwide. In China, the competent vectors for Zika virus are widely distributed, imported viraemic cases may become a source of local transmission of the virus. However, Zika virus disease is preventable, the spread of virus could be stopped when the effective prevention measures are taken. This paper summarizes the retrieval results from Medline database and the information from the reports of the governments of countries affected or health organizations about the epidemiological characteristics of Zika virus disease. PMID:27005530

  9. Enveloped Virus-Like Particle Expression of Human Cytomegalovirus Glycoprotein B Antigen Induces Antibodies with Potent and Broad Neutralizing Activity

    PubMed Central

    Kirchmeier, Marc; Fluckiger, Anne-Catherine; Soare, Catalina; Bozic, Jasminka; Ontsouka, Barthelemy; Ahmed, Tanvir; Diress, Abebaw; Pereira, Lenore; Schödel, Florian; Plotkin, Stanley; Dalba, Charlotte; Klatzmann, David

    2014-01-01

    A prophylactic vaccine to prevent the congenital transmission of human cytomegalovirus (HCMV) in newborns and to reduce life-threatening disease in immunosuppressed recipients of HCMV-infected solid organ transplants is highly desirable. Neutralizing antibodies against HCMV confer significant protection against infection, and glycoprotein B (gB) is a major target of such neutralizing antibodies. However, one shortcoming of past HCMV vaccines may have been their failure to induce high-titer persistent neutralizing antibody responses that prevent the infection of epithelial cells. We used enveloped virus-like particles (eVLPs), in which particles were produced in cells after the expression of murine leukemia virus (MLV) viral matrix protein Gag, to express either full-length CMV gB (gB eVLPs) or the full extracellular domain of CMV gB fused with the transmembrane and cytoplasmic domains from vesicular stomatitis virus (VSV)-G protein (gB-G eVLPs). gB-G-expressing eVLPs induced potent neutralizing antibodies in mice with a much greater propensity toward epithelial cell-neutralizing activity than that induced with soluble recombinant gB protein. An analysis of gB antibody binding titers and T-helper cell responses demonstrated that high neutralizing antibody titers were not simply due to enhanced immunogenicity of the gB-G eVLPs. The cells transiently transfected with gB-G but not gB plasmid formed syncytia, consistent with a prefusion gB conformation like those of infected cells and viral particles. Two of the five gB-G eVLP-induced monoclonal antibodies we examined in detail had neutralizing activities, one of which possessed particularly potent epithelial cell-neutralizing activity. These data differentiate gB-G eVLPs from gB antigens used in the past and support their use in a CMV vaccine candidate with improved neutralizing activity against epithelial cell infection. PMID:24334684

  10. Enveloped virus-like particle expression of human cytomegalovirus glycoprotein B antigen induces antibodies with potent and broad neutralizing activity.

    PubMed

    Kirchmeier, Marc; Fluckiger, Anne-Catherine; Soare, Catalina; Bozic, Jasminka; Ontsouka, Barthelemy; Ahmed, Tanvir; Diress, Abebaw; Pereira, Lenore; Schödel, Florian; Plotkin, Stanley; Dalba, Charlotte; Klatzmann, David; Anderson, David E

    2014-02-01

    A prophylactic vaccine to prevent the congenital transmission of human cytomegalovirus (HCMV) in newborns and to reduce life-threatening disease in immunosuppressed recipients of HCMV-infected solid organ transplants is highly desirable. Neutralizing antibodies against HCMV confer significant protection against infection, and glycoprotein B (gB) is a major target of such neutralizing antibodies. However, one shortcoming of past HCMV vaccines may have been their failure to induce high-titer persistent neutralizing antibody responses that prevent the infection of epithelial cells. We used enveloped virus-like particles (eVLPs), in which particles were produced in cells after the expression of murine leukemia virus (MLV) viral matrix protein Gag, to express either full-length CMV gB (gB eVLPs) or the full extracellular domain of CMV gB fused with the transmembrane and cytoplasmic domains from vesicular stomatitis virus (VSV)-G protein (gB-G eVLPs). gB-G-expressing eVLPs induced potent neutralizing antibodies in mice with a much greater propensity toward epithelial cell-neutralizing activity than that induced with soluble recombinant gB protein. An analysis of gB antibody binding titers and T-helper cell responses demonstrated that high neutralizing antibody titers were not simply due to enhanced immunogenicity of the gB-G eVLPs. The cells transiently transfected with gB-G but not gB plasmid formed syncytia, consistent with a prefusion gB conformation like those of infected cells and viral particles. Two of the five gB-G eVLP-induced monoclonal antibodies we examined in detail had neutralizing activities, one of which possessed particularly potent epithelial cell-neutralizing activity. These data differentiate gB-G eVLPs from gB antigens used in the past and support their use in a CMV vaccine candidate with improved neutralizing activity against epithelial cell infection. PMID:24334684

  11. Augmented immune responses in pigs immunized with an inactivated porcine reproductive and respiratory syndrome virus containing the deglycosylated glycoprotein 5 under field conditions

    PubMed Central

    2016-01-01

    Purpose Porcine reproductive and respiratory syndrome virus (PRRSV) leads to major economic losses in the swine industry. Vaccination is the most effective method to control the disease by PRRSV. Materials and Methods In this study, the efficacy of a glycoprotein (GP) 5-modified inactivated vaccine was investigated in pigs. The study was performed in three farms: farm A, which was porcine reproductive and respiratory syndrome (PRRS)-negative, farm B (PRRS-active), which showed clinical signs of PRRS but had not used vaccines, and farm C (PRRS-stable), which had a history of endemic PRRS over the past years, but showed no more clinical signs after periodic administration of modified live virus vaccine. Results The inactivated vaccine induced great enhancement in serum neutralizing antibody titer, which was sufficient to protect pigs from further infections of PRRSV in a farm where pre-existing virus was circulating. Conclusion These results indicated that vaccination with the inactivated vaccine composed of viruses possessing deglycosylated GP5 would provide enhanced protection to pigs from farms suffering from endemic PRRSV. PMID:26866026

  12. Absence of cytotoxic antibody to human immunodeficiency virus-infected cells in humans and its induction in animals after infection or immunization with purified envelope glycoprotein gp120

    SciTech Connect

    Nara, P.L.; Robey, W.G.; Gonda, M.A.; Carter, S.G.; Fischinger, P.J.

    1987-06-01

    The presence of antibody-dependent complement-mediated cytotoxicity (ACC) was assessed in humans and chimpanzees, which are capable of infection with human immunodeficiency virus isolate HTLV-IIIb, and examined in the goat after immunization with the major viral glycoprotein (gp120) of HTLV-IIIb. In infected humans no antibody mediating ACC was observed regardless of the status of disease. Even healthy individuals with high-titer, broadly reactive, neutralizing antibodies has no ACC. In contrast, chimpanzees infected with HTLV-IIIb, from whom virus could be isolated, not only had neutralizing antibody but also antibodies broadly reactive in ACC, even against distantly related human immunodeficiency virus isolates, as well as against their own reisolated virus. In the goat, the gp120 of HTLV-IIIb induced a highly type-specific response as measured by both ACC and flow cytofluorometry of live infected H9 cells. Normal human cells were not subject to ACC by animal anti-HTLV-III gp120-specific sera. Induction of ACC and neutralizing antibody were closely correlated in the animal experimental models but not in humans. The presence of ACC in gp120-inoculated goats and HTLV-III-infected chimpanzees represent a qualitative difference that may be important in the quest for the elicitation of a protective immunity in humans.

  13. Evolutionary pattern of human respiratory syncytial virus (subgroup A): cocirculating lineages and correlation of genetic and antigenic changes in the G glycoprotein.

    PubMed Central

    García, O; Martín, M; Dopazo, J; Arbiza, J; Frabasile, S; Russi, J; Hortal, M; Perez-Breña, P; Martínez, I; García-Barreno, B

    1994-01-01

    The genetic and antigenic variability of the G glycoproteins from 76 human respiratory syncytial (RS) viruses (subgroup A) isolated during six consecutive epidemics in either Montevideo, Uruguay, or Madrid, Spain, have been analyzed. Genetic diversity was evaluated for all viruses by the RNase A mismatch cleavage method and for selected strains by dideoxy sequencing. The sequences reported here were added to those published for six isolates from Birmingham, United Kingdom, and for two reference strains (A2 and Long), to derive a phylogenetic tree of subgroup A viruses that contained two main branches and several subbranches. During the same epidemic, viruses from different branches were isolated. In addition, closely related viruses were isolated in distant places and in different years. These results illustrate the capacity of the virus to spread worldwide, influencing its mode of evolution. The antigenic analysis of all isolates was carried out with a panel of anti-G monoclonal antibodies that recognized strain-specific (or variable) epitopes. A close correlation between genetic relatedness and antigenic relatedness in the G protein was observed. These results, together with an accumulation of amino acid changes in a major antigenic area of the G glycoprotein, suggest that immune selection may be a factor influencing the generation of RS virus diversity. The pattern of RS virus evolution is thus similar to that described for influenza type B viruses, expect that the level of genetic divergence among the G glycoproteins of RS virus isolates is the highest reported for an RNA virus gene product. Images PMID:8057427

  14. Alteration of the N-linked Glycosylation Condition of E1 Glycoprotein of Classical Swine Fever Virus Strain Brescia Alters Virulence in Swine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    E1, along with Erns and E2 is one of the three envelope glycoproteins of Classical Swine Fever Virus (CSFV). Previously we showed that glycosylation status of virulent CSFV strain Brescia E2 or Erns affects virus virulence. Here, the three putative glycosylation sites of E1 were serially removed by ...

  15. Removal of a N-linked Glycosylation Site on the Classical Swine Fever Virus Strain Brescia E(rns) Glycoprotein Affects Virulence in Swine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Classical Swine Fever Virus (CSFV) E(rns) glycoprotein is involved in several functions; including virus attachment and entry to target cells, production of antibodies, and virulence. Here, we describe the role of CSFV strain Brescia E(rns) glycosylation on virulence in swine. Amino acid residue N...

  16. Effect of specific amino acid substitutions in the putative fusion peptide of structural glycoprotein E2 on Classical Swine Fever Virus replication

    Technology Transfer Automated Retrieval System (TEKTRAN)

    E2, along with E^rns and E1, is an envelope glycoprotein of Classical Swine Fever Virus (CSFV). E2 is involved in several virus functions including cell attachment, host range susceptibility and virulence in natural hosts. In infected cells, E2 forms homodimers as well as heterodimers with E1, media...

  17. Evaluation of bivalent Newcastle disease virus (NDV) vectored infectious laryngotracheitis vaccines in broiler chickens in the presence of NDV maternally derived antibody

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Previously we have demonstrated that Newcastle disease virus (NDV) recombinants expressing the infectious laryngotracheitis virus (ILTV) glycoproteins B (gB) or D (gD) protein conferred complete clinical protection against ILTV and NDV challenges in specific pathogen free (SPF) and 3 week old commer...

  18. Herpes Simplex Virus Type 2 Glycoprotein H Interacts with Integrin αvβ3 To Facilitate Viral Entry and Calcium Signaling in Human Genital Tract Epithelial Cells

    PubMed Central

    Cheshenko, Natalia; Trepanier, Janie B.; González, Pablo A.; Eugenin, Eliseo A.; Jacobs, William R.

    2014-01-01

    ABSTRACT Herpes simplex virus (HSV) entry requires multiple interactions at the cell surface and activation of a complex calcium signaling cascade. Previous studies demonstrated that integrins participate in this process, but their precise role has not been determined. These studies were designed to test the hypothesis that integrin αvβ3 signaling promotes the release of intracellular calcium (Ca2+) stores and contributes to viral entry and cell-to-cell spread. Transfection of cells with small interfering RNA (siRNA) targeting integrin αvβ3, but not other integrin subunits, or treatment with cilengitide, an Arg-Gly-Asp (RGD) mimetic, impaired HSV-induced Ca2+ release, viral entry, plaque formation, and cell-to-cell spread of HSV-1 and HSV-2 in human cervical and primary genital tract epithelial cells. Coimmunoprecipitation studies and proximity ligation assays indicated that integrin αvβ3 interacts with glycoprotein H (gH). An HSV-2 gH-null virus was engineered to further assess the role of gH in the virus-induced signaling cascade. The gH-2-null virus bound to cells and activated Akt to induce a small Ca2+ response at the plasma membrane, but it failed to trigger the release of cytoplasmic Ca2+ stores and was impaired for entry and cell-to-cell spread. Silencing of integrin αvβ3 and deletion of gH prevented phosphorylation of focal adhesion kinase (FAK) and the transport of viral capsids to the nuclear pore. Together, these findings demonstrate that integrin signaling is activated downstream of virus-induced Akt signaling and facilitates viral entry through interactions with gH by activating the release of intracellular Ca2+ and FAK phosphorylation. These findings suggest a new target for HSV treatment and suppression. IMPORTANCE Herpes simplex viruses are the leading cause of genital disease worldwide, the most common infection associated with neonatal encephalitis, and a major cofactor for HIV acquisition and transmission. There is no effective vaccine

  19. Immune Modulating Effect by a Phosphoprotein-deleted Rabies Virus Vaccine Vector Expressing Two Copies of the Rabies Virus Glycoprotein Gene

    PubMed Central

    Cenna, Jonathan; Tan, Gene S.; Papaneri, Amy B.; Dietzschold, Bernhard; Schnell, Matthias J.; McGettigan, James P.

    2009-01-01

    The type of immune response induced by a vaccine is a critical factor that determines its effectiveness in preventing infection or disease. Inactivated and live rabies virus (RV) vaccine strains elicit an IgG1-biased and IgG1/IgG2a-balanced antibody response, respectively. However, IgG2a antibodies are potent inducers of anti-viral effector functions, and therefore, a viral vaccine vector that can elicit an IgG2a-biased antibody response may be more effective against RV infection. Here we describe the humoral immune response of a live replication-deficient phosphoprotein (P)-deleted RV vector (SPBN-ΔP), or a recombinant P-deleted virus that expresses two copies of the RV glycoprotein (G) gene (SPBN-ΔP-RVG), and compare it to a UV-inactivated RV. Mice inoculated with UV-inactivated RV induced predominantly an IgG1-specific antibody response, while live recombinant SPBN-ΔP exhibited a mixed IgG1/IgG2a antibody response, which is consistent with the isotype profiles from the replication-competent parental viruses. Survivorship in mice after pathogenic RV challenge indicates a ten-fold higher efficiency of live SPBN-ΔP compared to UV-inactivated SPBN-ΔP. In addition, SPBN-ΔP-RVG induced a more rapid and robust IgG2a response that protected mice more effectively than SPBN-ΔP. Of note, 103 ffu of SPBN-ΔP-RVG induced anti-RV antibodies that were 100% protective in mice against pathogenic RV challenge. The increased immune response was directed not only against RV G but also against the ribonucleoprotein (RNP), indicating that the expression of two RV G genes from SPBN-ΔP-RVG enhances the immune response to other RV antigens as well. In addition, Rag2 mice inoculated intramuscularly with 105 ffu/mouse of SPBN-ΔP showed no clinical signs of rabies, and no viral RNA was detected in the spinal cord or brain of inoculated mice. Therefore, the safety of the P-deleted vectors along with the onset and magnitude of the IgG2a-induced immune response by SPBN

  20. Human monoclonal antibodies targeting the haemagglutinin glycoprotein can neutralize H7N9 influenza virus.

    PubMed

    Chen, Zhe; Wang, Jianmin; Bao, Linlin; Guo, Li; Zhang, Weijia; Xue, Ying; Zhou, Hongli; Xiao, Yan; Wang, Jianwei; Wu, Fan; Deng, Ying; Qin, Chuan; Jin, Qi

    2015-01-01

    The recently identified avian-originated influenza H7N9 virus causes severe pulmonary disease and may lead to death in humans. Currently, treatment options for the prevention and control of fatal H7N9 infections in humans remain limited. Here we characterize two human monoclonal antibodies (HuMAbs), HNIgGA6 and HNIgGB5, by screening a Fab antibody phage library derived from patients who recovered from H7N9 infection. Both antibodies exhibit high neutralizing activity against H7N9 virus in cells. Two amino acids in the receptor-binding site, 186V and 226L, are crucial for the binding of these two HuMAbs to viral haemagglutinin antigens. Prophylaxis with HNIgGA6 and HNIgGB5 confers significant immunity against H7N9 virus in a mouse model and significantly reduces the pulmonary virus titre. When administered post infection, therapeutic doses of the HuMAbs also provide robust protection against lethality. These antibodies might represent a potential alternative or adjunct to H7N9 pandemic interventions. PMID:25819694

  1. Evolution Rescues Folding of Human Immunodeficiency Virus-1 Envelope Glycoprotein GP120 Lacking a Conserved Disulfide Bond

    PubMed Central

    Hsu, Shang-Te D.; van Anken, Eelco; Liscaljet, I. Marije; Dankers, Martijn; Bontjer, Ilja; Land, Aafke; Braakman, Ineke; Bonvin, Alexandre M.J.J.; Berkhout, Ben

    2008-01-01

    The majority of eukaryotic secretory and membrane proteins contain disulfide bonds, which are strongly conserved within protein families because of their crucial role in folding or function. The exact role of these disulfide bonds during folding is unclear. Using virus-driven evolution we generated a viral glycoprotein variant, which is functional despite the lack of an absolutely conserved disulfide bond that links two antiparallel β-strands in a six-stranded β-barrel. Molecular dynamics simulations revealed that improved hydrogen bonding and side chain packing led to stabilization of the β-barrel fold, implying that β-sheet preference codirects glycoprotein folding in vivo. Our results show that the interactions between two β-strands that are important for the formation and/or integrity of the β-barrel can be supported by either a disulfide bond or β-sheet favoring residues. PMID:18753405

  2. An electrochemiluminescence assay for analysis of rabies virus glycoprotein content in rabies vaccines

    PubMed Central

    Smith, Todd G.; Ellison, James A.; Ma, Xiaoyue; Kuzmina, Natalia; Carson, William C.; Rupprecht, Charles E.

    2015-01-01

    Vaccine potency testing is necessary to evaluate the immunogenicity of inactivated rabies virus (RABV) vaccine preparations before human or veterinary application. Currently, the NIH test is recommended by the WHO expert committee to evaluate RABV vaccine potency. However, numerous disadvantages are inherent concerning cost, number of animals and biosafety requirements. As such, several in vitro methods have been proposed for the evaluation of vaccines based on RABV glycoprotein (G) quality and quantity, which is expected to correlate with vaccine potency. In this study an antigen-capture electrochemiluminescent (ECL) assay was developed utilizing anti-RABV G monoclonal antibodies (MAb) to quantify RABV G. One MAb 2-21-14 was specific for a conformational epitope so that only immunogenic, natively-folded G was captured in the assay. A second MAb (62-80-6) that binds a linear epitope or MAb 2-21-14 was used for detection of RABV G. Vaccine efficacy was also assessed in vivo using pre-exposure vaccination of mice. Purified native RABV G induced a RABV neutralizing antibody (rVNA) response with a geometric mean titer of 4.2 IU/ml and protected 100% of immunized mice against RABV challenge, while an experimental vaccine with a lower quality and quantity of G induced a rVNA titer <0.05 IU/ml and protected <50% of immunized mice. These preliminary results support the hypothesis that in vivo immunogenicity may be predicted from the in vitro measurement of RABV G using an ECL assay. Based upon these results, the ECL assay may have utility in replacement of the NIH test. PMID:23742991

  3. Architecture of the hepatitis C virus E1 glycoprotein transmembrane domain studied by NMR.

    PubMed

    Zazrin, Hadas; Shaked, Hadassa; Chill, Jordan H

    2014-03-01

    Oligomerization of hepatitis C viral envelope proteins E1 and E2 is essential to virus fusion and assembly. Although interactions within the transmembrane (TM) domains of these glycoproteins have proven contributions to the E1/E2 heterodimerization process and consequent infectivity, there is little structural information on this entry mechanism. Here, as a first step towards our long-term goal of understanding the interaction between E1 and E2 TM-domains, we have expressed, purified and characterized E1-TM using structural biomolecular NMR methods. An MBP-fusion expression system yielded sufficient quantities of pure E1-TM, which was solubilized in two membrane-mimicking environments, SDS- and LPPG-micelles, affording samples amenable to NMR studies. Triple resonance assignment experiments and relaxation measurements provided information on the secondary structure and global fold of E1-TM in these environments. In SDS micelles E1-TM adopts a helical conformation, with helical stretches at residues 354-363 and 371-379 separated by a more flexible segment of residues 364-370. In LPPG micelles a helical conformation was observed for residues 354-377 with greater flexibility in the 366-367 dyad, suggesting LPPG provides a more native environment for the peptide. Replacement of key positively charged residue K370 with an alanine did not affect the secondary structure of E1-TM but did change the relative positioning within the micelle of the two helices. These results lay the foundation for structure determination of E1-TM and a molecular understanding of how E1-TM flexibility enhances its interaction with E2-TM during heterodimerization and membrane fusion. PMID:24192053

  4. A Cysteine Zipper Stabilizes a Pre-Fusion F Glycoprotein Vaccine for Respiratory Syncytial Virus

    PubMed Central

    Stewart-Jones, Guillaume B. E.; Thomas, Paul V.; Chen, Lei; Chuang, Gwo-Yu; Georgiev, Ivelin S.; McLellan, Jason S.; Srivatsan, Sanjay; Zhou, Tongqing; Baxa, Ulrich; Mascola, John R.; Graham, Barney S.; Kwong, Peter D.

    2015-01-01

    Recombinant subunit vaccines should contain minimal non-pathogen motifs to reduce potential off-target reactivity. We recently developed a vaccine antigen against respiratory syncytial virus (RSV), which comprised the fusion (F) glycoprotein stabilized in its pre-fusion trimeric conformation by “DS-Cav1” mutations and by an appended C-terminal trimerization motif or “foldon” from T4-bacteriophage fibritin. Here we investigate the creation of a cysteine zipper to allow for the removal of the phage foldon, while maintaining the immunogenicity of the parent DS-Cav1+foldon antigen. Constructs without foldon yielded RSV F monomers, and enzymatic removal of the phage foldon from pre-fusion F trimers resulted in their dissociation into monomers. Because the native C terminus of the pre-fusion RSV F ectodomain encompasses a viral trimeric coiled-coil, we explored whether introduction of cysteine residues capable of forming inter-protomer disulfides might allow for stable trimers. Structural modeling indicated the introduced cysteines to form disulfide “rings”, with each ring comprising a different set of inward facing residues of the coiled-coil. Three sets of rings could be placed within the native RSV F coiled-coil, and additional rings could be added by duplicating portions of the coiled-coil. High levels of neutralizing activity in mice, equivalent to that of the parent DS-Cav1+foldon antigen, were elicited by a 4-ring stabilized RSV F trimer with no foldon. Structure-based alteration of a viral coiled-coil to create a cysteine zipper thus allows a phage trimerization motif to be removed from a candidate vaccine antigen. PMID:26098893

  5. A Cysteine Zipper Stabilizes a Pre-Fusion F Glycoprotein Vaccine for Respiratory Syncytial Virus.

    PubMed

    Stewart-Jones, Guillaume B E; Thomas, Paul V; Chen, Man; Druz, Aliaksandr; Joyce, M Gordon; Kong, Wing-Pui; Sastry, Mallika; Soto, Cinque; Yang, Yongping; Zhang, Baoshan; Chen, Lei; Chuang, Gwo-Yu; Georgiev, Ivelin S; McLellan, Jason S; Srivatsan, Sanjay; Zhou, Tongqing; Baxa, Ulrich; Mascola, John R; Graham, Barney S; Kwong, Peter D

    2015-01-01

    Recombinant subunit vaccines should contain minimal non-pathogen motifs to reduce potential off-target reactivity. We recently developed a vaccine antigen against respiratory syncytial virus (RSV), which comprised the fusion (F) glycoprotein stabilized in its pre-fusion trimeric conformation by "DS-Cav1" mutations and by an appended C-terminal trimerization motif or "foldon" from T4-bacteriophage fibritin. Here we investigate the creation of a cysteine zipper to allow for the removal of the phage foldon, while maintaining the immunogenicity of the parent DS-Cav1+foldon antigen. Constructs without foldon yielded RSV F monomers, and enzymatic removal of the phage foldon from pre-fusion F trimers resulted in their dissociation into monomers. Because the native C terminus of the pre-fusion RSV F ectodomain encompasses a viral trimeric coiled-coil, we explored whether introduction of cysteine residues capable of forming inter-protomer disulfides might allow for stable trimers. Structural modeling indicated the introduced cysteines to form disulfide "rings", with each ring comprising a different set of inward facing residues of the coiled-coil. Three sets of rings could be placed within the native RSV F coiled-coil, and additional rings could be added by duplicating portions of the coiled-coil. High levels of neutralizing activity in mice, equivalent to that of the parent DS-Cav1+foldon antigen, were elicited by a 4-ring stabilized RSV F trimer with no foldon. Structure-based alteration of a viral coiled-coil to create a cysteine zipper thus allows a phage trimerization motif to be removed from a candidate vaccine antigen. PMID:26098893

  6. Rabies Virus Vector Transgene Expression Level and Cytotoxicity Improvement Induced by Deletion of Glycoprotein Gene

    PubMed Central

    Ohara, Shinya; Sato, Sho; Oyama, Kei; Tsutsui, Ken-Ichiro; Iijima, Toshio

    2013-01-01

    The glycoprotein (G) of rabies virus (RV) is required for binding to neuronal receptors and for viral entry. G-deleted RV vector is a powerful tool for investigating the organization and function of the neural circuits. It gives the investigator the ability to genetically target initial infection to particular neurons and to control trans-synaptic propagation. In this study we have quantitatively evaluated the effect of G gene deletion on the cytotoxicity and transgene expression level of the RV vector. We compared the characteristics of the propagation-competent RV vector (rHEP5.0-CVSG-mRFP) and the G-deleted RV vector (rHEP5.0-ΔG-mRFP), both of which are based on the attenuated HEP-Flury strain and express monomeric red fluorescent protein (mRFP) as a transgene. rHEP5.0-ΔG-mRFP showed lower cytotoxicity than rHEP5.0-CVSG-mRFP, and within 16 days of infection we found no change in the basic electrophysiological properties of neurons infected with the rHEP5.0-ΔG-mRFP. The mRFP expression level of rHEP5.0-ΔG-mRFP was much higher than that of rHEP5.0-CVSG-mRFP, and 3 days after infection the retrogradely infected neurons were clearly visualized by the expressed fluorescent protein without any staining. This may be due to the low cytotoxicity and/or the presumed change in the polymerase gene (L) expression level of the G-deleted RV vector. Although the mechanisms remains to be clarified, the results of this study indicate that deletion of the G gene greatly improves the usability of the RV vector for studying the organization and function of the neural circuits by decreasing the cytotoxicity and increasing the transgene expression level. PMID:24244660

  7. Nucleotide sequence specifying the glycoprotein gene, gB, of herpes simplex virus type 1.

    PubMed

    Bzik, D J; Fox, B A; DeLuca, N A; Person, S

    1984-03-01

    The nucleotide sequence thought to specify the glycoprotein gene, gB, of the KOS strain of herpes simplex virus type 1 (HSV-1) has been determined. A 3.1-kilobase (kb), viral-specified RNA was mapped to the left half of the BamHI-G fragment (0.345 to 0.399 map units). TATA, CAT-box, and possible mRNA start sequences characteristic of HSV-1 genes are found near 0.368 map units. The first available ATG codon is at 0.366 and the first in-phase chain terminator at 0.348 map units. A polyA-addition signal (AATAAA) occurs 17 nucleotides past the chain terminator. Translation of these sequences would yield a 100.3-kilodalton (kDa) polypeptide characterized by a 5' signal sequence, nine N-linked saccharide addition sites, a strongly hydrophobic membrane-spanning sequence, and a highly charged 3' cytoplasmic anchor sequence. Two mutants of KOS, tsJ12 and tsJ20, that are temperature-sensitive for viral growth and for the production of gB, have been physically mapped to 0.357 to 0.360 and 0.360 to 0.364 map units, respectively (DeLuca et al., in preparation). The nucleotide sequence of the mutants was determined in these regions. In both cases a single amino acid replacement within the 100.3-kDa polypeptide is predicted from the sequence analysis. PMID:6324454

  8. An electrochemiluminescence assay for analysis of rabies virus glycoprotein content in rabies vaccines.

    PubMed

    Smith, Todd G; Ellison, James A; Ma, Xiaoyue; Kuzmina, Natalia; Carson, William C; Rupprecht, Charles E

    2013-07-18

    Vaccine potency testing is necessary to evaluate the immunogenicity of inactivated rabies virus (RABV) vaccine preparations before human or veterinary application. Currently, the NIH test is recommended by the WHO expert committee to evaluate RABV vaccine potency. However, numerous disadvantages are inherent concerning cost, number of animals and biosafety requirements. As such, several in vitro methods have been proposed for the evaluation of vaccines based on RABV glycoprotein (G) quality and quantity, which is expected to correlate with vaccine potency. In this study an antigen-capture electrochemiluminescent (ECL) assay was developed utilizing anti-RABV G monoclonal antibodies (MAb) to quantify RABV G. One MAb 2-21-14 was specific for a conformational epitope so that only immunogenic, natively folded G was captured in the assay. MAb 2-21-14 or a second MAb (62-80-6) that binds a linear epitope was used for detection of RABV G. Vaccine efficacy was also assessed in vivo using pre-exposure vaccination of mice. Purified native RABV G induced a RABV neutralizing antibody (rVNA) response with a geometric mean titer of 4.2IU/ml and protected 100% of immunized mice against RABV challenge, while an experimental vaccine with a lower quality and quantity of G induced a rVNA titer<0.05IU/ml and protected <50% of immunized mice. These preliminary results support the hypothesis that in vivo immunogenicity may be predicted from the in vitro measurement of RABV G using an ECL assay. Based upon these results, the ECL assay may have utility in replacement of the NIH test. PMID:23742991

  9. Basis for receptor specificity of nonecotropic murine leukemia virus surface glycoprotein gp70SU.

    PubMed Central

    Ott, D; Rein, A

    1992-01-01

    Murine leukemia viruses (MuLVs) initiate infection of NIH 3T3 cells by binding of the viral envelope (Env) protein to a cell surface receptor. Interference assays have shown that MuLVs can be divided into four groups, each using a distinct receptor: ecotropic, polytropic, amphotropic, and 10A1. In this study, we have attempted to map the determinants within viral Env proteins by constructing chimeric env genes. Chimeras were made in all six pairwise combinations between Moloney MCF (a polytropic MuLV), amphotropic MuLV, and 10A1, using a conserved EcoRI site in the middle of the Env coding region. The receptor specificity of each chimera was determined by using an interference assay. We found that amphotropic receptor specificity of each chimera was determined by using an interference assay. We found that amphotropic receptor specificity seems to map to the N-terminal portion of surface glycoprotein gp70SU. The difference between amphotropic and 10A1 receptor specificity can be attributed to one or more of only six amino acid differences in this region. Nearly all other cases showed evidence of interaction between Env domains in the generation of receptor specificity. Thus, a chimera composed exclusively of MCF and amphotropic sequences was found to exhibit 10A1 receptor specificity. None of the chimeras were able to infect cells by using the MCF receptor; however, two chimeras containing the C-terminal portion of MCF gp70SU could bind to this receptor, while they were able to infect cells via the amphotropic receptor. This result raises the possibility that receptor binding maps to the C-terminal portion of MCF gp70SU but requires MCF N-terminal sequences for a functional interaction with the MCF receptor. Images PMID:1321266

  10. Vaccination by cholera toxin conjugated to a herpes simplex virus type 2 glycoprotein D peptide.

    PubMed

    Drew, M D; Estrada-Correa, A; Underdown, B J; McDermott, M R

    1992-09-01

    Immunization of BALB/cJ mice with a peptide corresponding to residues 1 to 23 of glycoprotein D [gD(1-23)] from herpes simplex virus type 2 (HSV-2) elicits antibody responses which correlate with protection against lethal HSV-2 infection. In the present study, we examined the ability of cholera toxin (CTX) to act as an immunogenic carrier for gD(1-23). The number of gD(1-23) residues conjugated to CTX affected its binding to GM1 ganglioside and physiological toxicity, both of which are factors affecting oral immunogenicity. The antibody response elicited after intraperitoneal (i.p.) immunization with the CTX-gD(1-23) conjugate was protective against a lethal i.p. challenge with HSV-2. In other experiments, mice were immunized i.p. on day 0 and subsequent immunizations conducted on days 14 and 28 were administered either intragastrically or intravaginally (i.vag.). Intraperitoneal priming followed by either i.p or intragastric boosting resulted in anti-HSV-2 antibodies in vaginal washings and in protection against a lethal i.vag. challenge with HSV-2. Intraperitoneal priming followed by i.vag. boosting did not elicit anti-HSV-2 antibodies in vaginal washings and did not protect mice against a lethal i.vag. challenge with HSV-2. These results suggest that CTX can act as a systemic and an oral delivery molecule for the covalently linked gD(1-23) peptide and that such conjugates can elicit protective immune responses against systemic and genital HSV-2 infection. PMID:1383408

  11. Genomic and antigenic variations of porcine reproductive and respiratory syndrome virus major envelope GP5 glycoprotein.

    PubMed Central

    Pirzadeh, B; Gagnon, C A; Dea, S

    1998-01-01

    The objective of the present study was to evaluate the importance of genomic and antigenic variations which may have affected the major envelope glycoprotein GP5 of porcine reproductive and respiratory syndrome virus (PRRSV) isolates responsible for outbreaks in Quebec and Ontario, in comparison with the modified-live U.S. vaccine strain (MLV) and the European prototype strain from Lelystad (LV). Nucleotide sequence analyses of the open reading frame (ORF)5 genes showed that all of the isolates studied were heterogenous, amino acid (aa) identities varied from 88 to 99% with the MLV strain, and between 51 and 54% with the LV strain. The aa substitutions were randomly scattered across the protein, although one region between residues 26 and 39 was found to correspond to a hypervariable region which involved 0 to 3 potential N-glycosylation sites. The ORF5 encoded products of 5 of these isolates, including the MLV and LV strains, were expressed in E. coli as recombinant proteins fused to the glutathione S-transferase (GST) protein and used to raise hyperimmune anti-ORF5 sera in rabbits. The reactivity patterns of strain-specific hyperimmune anti-ORF5 sera and a panel of 4 monoclonal antibodies directed against the ORF5 gene product of the Quebec IAF-Klop strain of PRRSV, indicated that GP5 of field isolates also underwent antigenic variations. The data suggest that neutralizing epitopes, independent of conformation and glycosylation, are also associated with antigenic variability of the GP5 of PRRSV. PMID:9684045

  12. Designed protein mimics of the Ebola virus glycoprotein GP2 α-helical bundle: Stability and pH effects

    PubMed Central

    Harrison, Joseph S; Higgins, Chelsea D; Chandran, Kartik; Lai, Jonathan R

    2011-01-01

    Ebola virus (EboV) belongs to the Filoviridae family of viruses that causes severe and fatal hemhorragic fever. Infection by EboV involves fusion between the virus and host cell membranes mediated by the envelope glycoprotein GP2 of the virus. Similar to the envelope glycoproteins of other viruses, the central feature of the GP2 ectodomain postfusion structure is a six-helix bundle formed by the protein's N- and C-heptad repeat regions (NHR and CHR, respectively). Folding of this six-helix bundle provides the energetic driving force for membrane fusion; in other viruses, designed agents that disrupt formation of the six-helix bundle act as potent fusion inhibitors. To interrogate determinants of EboV GP2-mediated membrane fusion, we designed model proteins that consist of the NHR and CHR segments linked by short protein linkers. Circular dichroism and gel filtration studies indicate that these proteins adopt stable α-helical folds consistent with design. Thermal denaturation indicated that the GP2 six-helix bundle is highly stable at pH 5.3 (melting temperature, Tm, of 86.8 ± 2.0°C and van't Hoff enthalpy, ΔHvH, of −28.2 ± 1.0 kcal/mol) and comparable in stability to other viral membrane fusion six-helix bundles. We found that the stability of our designed α-helical bundle proteins was dependent on buffering conditions with increasing stability at lower pH. Small pH differences (5.3–6.1) had dramatic effects (ΔTm = 37°C) suggesting a mechanism for conformational control that is dependent on environmental pH. These results suggest a role for low pH in stabilizing six-helix bundle formation during the process of GP2-mediated viral membrane fusion. PMID:21739501

  13. Varicella-zoster virus glycoproteins B and E are major targets of CD4+ and CD8+ T cells reconstituting during zoster after allogeneic transplantation

    PubMed Central

    Kleemann, Patrick; Distler, Eva; Wagner, Eva M.; Thomas, Simone; Klobuch, Sebastian; Aue, Steffi; Schnürer, Elke; Schild, Hansjörg; Theobald, Matthias; Plachter, Bodo; Tenzer, Stefan; Meyer, Ralf G.; Herr, Wolfgang

    2012-01-01

    Background After allogeneic hematopoietic stem-cell transplantation patients are at increased risk for herpes zoster as long as varicella-zoster virus specific T-cell reconstitution is impaired. This study aimed to identify immunodominant varicella-zoster virus antigens that drive recovery of virus-specific T cells after transplantation. Design and Methods Antigens were purified from a varicella-zoster virus infected cell lysate by high-performance liquid chromatography and were identified by quantitative mass spectrometric analysis. To approximate in vivo immunogenicity for memory T cells, antigen preparations were consistently screened with ex vivo PBMC of varicella-zoster virus immune healthy individuals in sensitive interferon-γ ELISpot assays. Candidate virus antigens identified by the approach were genetically expressed in PBMC using electroporation of in vitro transcribed RNA encoding full-length proteins and were then analyzed for recognition by CD4+ and CD8+ memory T cells. Results Varicella-zoster virus encoded glycoproteins B and E, and immediate early protein 62 were identified in immunoreactive lysate material. Predominant CD4+ T-cell reactivity to these proteins was observed in healthy virus carriers. Furthermore, longitudinal screening in allogeneic stem-cell transplantation patients showed strong expansions of memory T cells recognizing glycoproteins B and E after onset of herpes zoster, while immediate early protein 62 reactivity remained moderate. Reactivity to viral glycoproteins boosted by acute zoster was mediated by both CD4+ and CD8+ T cells. Conclusions Our data demonstrate that glycoproteins B and E are major targets of varicella-zoster virus specific CD4+ and CD8+ T-cell reconstitution occurring during herpes zoster after allogeneic stem-cell transplantation. Varicella-zoster virus glycoproteins B and E might form the basis for novel non-hazardous zoster subunit vaccines suitable for immunocompromised transplant patients. PMID:22207687

  14. Immunocompetent truncated E2 glycoprotein of bovine viral diarrhea virus (BVDV) expressed in Nicotiana tabacum plants: a candidate antigen for new generation of veterinary vaccines.

    PubMed

    Nelson, Guillermo; Marconi, Patricia; Periolo, Osvaldo; La Torre, José; Alvarez, María Alejandra

    2012-06-22

    The bovine viral diarrhea virus (BVDV) is the etiological agent responsible for a wide spectrum of clinical diseases in cattle. The glycoprotein E2 is the major envelope protein of this virus and the strongest inductor of the immune response. There are several available commercial vaccines against bovine viral diarrhea (BVD), which show irregular performances. Here, we report the use of tobacco plants as an alternative productive platform for the expression of the truncated version of E2 glycoprotein (tE2) from the BVDV. The tE2 sequence, lacking the transmembrane domain, was cloned into the pK7WG2 Agrobacterium binary vector. The construct also carried the 2S2 Arabidopsis thaliana signal for directing the protein into the plant secretory pathway, the Kozak sequence, an hexa-histidine tag to facilitate protein purification and the KDEL endoplasmic reticulum retention signal. The resulting plasmid (pK-2S2-tE2-His-KDEL) was introduced into Agrobacterium tumefaciens strain EHA101 by electroporation. The transformed A. tumefaciens was then used to express tE2 in leaves of Nicotiana tabacum plants. Western blot and ELISA using specific monoclonal antibodies confirmed the presence of the recombinant tE2 protein in plant extracts. An estimated amount of 20 μg of tE2 per gram of fresh leaves was regularly obtained with this plant system. Injection of guinea pigs with plant extracts containing 20 μg of rtE2 induced the production of BVDV specific antibodies at equal or higher levels than those induced by whole virus vaccines. This is the first report of the production of an immunocompetent tE2 in N. tabacum plants, having the advantage to be free of any eventual animal contaminant. PMID:22554468

  15. A 45,000-M(r) glycoprotein in the Sendai virus envelope triggers virus-cell fusion.

    PubMed Central

    Kumar, M; Hassan, M Q; Tyagi, S K; Sarkar, D P

    1997-01-01

    Sendai virus envelopes devoid of hemagglutinin-neuraminidase but containing the fusion protein (F-virosomes) were prepared. F-virosomes exhibited discernible serine protease activity at neutral pH. Electrophoretic analysis of the protein profile of the F-virosomes under nonreducing conditions, by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing, led to the identification of a previously unknown glycoprotein with a relative molecular weight of 45,000 (45K protein) associated with the F protein. The identity of the 45K protein, as distinct from F protein, was established by Western blot analysis with F- and 45K-specific antibodies. This 45K protein forms a nexus with the F protein through noncovalent hydrophobic interactions, as proved by its sensitivity to urea treatment, and it is essential for the proteolytic activity of the F-virosomes as well as for the fusion of the viral envelope with host cell membrane. N-terminal sequence analysis (first 11 amino acids) of this protein showed strong homology (> 90%) to flavivirus NS3 serine proteases but no similarity to any of the Sendai viral proteins. On the basis of the N-terminal sequence, oligonucleotides were designed corresponding to the sense and antisense DNA sequences. Dot blot hybridization and primer extension with these oligonucleotides with the viral and the host genome confirmed the host origin of this protein. Further, the limited proteolytic digestion of the target membrane resulted in significant inhibition of viral fusion with it. On the basis of these results, we postulate a model for the molecular mechanism of F protein-induced membrane fusion, which may provide a rationale for other paramyxoviruses. PMID:9261357

  16. Disulfide-bonded discontinuous epitopes on the glycoprotein of vesicular stomatitis virus (New Jersey serotype).

    PubMed

    Grigera, P R; Keil, W; Wagner, R R

    1992-06-01

    Intrachain disulfide bonds between paired cysteines in the glycoprotein (G) of vesicular stomatitis virus (VSV) are required for the recognition of discontinuous epitopes by specific monoclonal antibodies (MAbs) (W. Keil and R. R. Wagner, Virology 170:392-407, 1989). Cleavage by Staphylococcus aureus V8 protease of the 517-amino-acid VSV-New Jersey G protein, limited to the glutamic acid at residue 110, resulted in a protein (designated GV8) with greatly retarded migration by polyacrylamide gel electrophoresis (PAGE) under nonreducing conditions. By Western blot (immunoblot) analysis, protein GV8 was found to lose discontinuous epitope IV, which maps within the first 193 NH2-terminal amino acids. These data, coupled with those obtained by PAGE migration of a vector-expressed, truncated protein (G1-193) under reducing and nonreducing conditions, lead us to postulate the existence of a major loop structure within the first 193 NH2-terminal amino acids of the G protein, possibly anchored by a disulfide bond between cysteine 108 and cysteine 169, encompassing epitope IV. Site-directed mutants in which 10 of the 12 cysteines were individually converted to serines in vaccinia virus-based vectors expressing these single-site mutant G proteins were also constructed, each of which was then tested by immunoprecipitation for its capacity to recognize epitope-specific MAbs. These results showed that mutations in NH2-terminal cysteines 130, 174, and, to a lesser extent, 193 all resulted in the loss of neutralization epitope VIII. A mutation at NH2-terminal cysteine 130 also resulted in the loss of neutralization epitope VII, as did a mutation at cysteine 108 to a lesser extent. Both epitopes VII and VIII disappeared when mutations were made in COOH-distal cysteine 235, 240, or 273, the general map locations of epitopes VII and VIII. These studies also reveal that distal, as well as proximal, cysteine residues markedly influence the disulfide-bond secondary structure, which

  17. Chikungunya Virus Glycoproteins Pseudotype with Lentiviral Vectors and Reveal a Broad Spectrum of Cellular Tropism

    PubMed Central

    Wang, Hua; Liu, Shuangchun; Yu, Lianhua; Sun, Lingfen; Qu, Ying

    2014-01-01

    Background Outbreaks of the Chikungunya virus (CHIKV) infection has been documented in over 40 countries, resulting in clinical symptoms characterized by fever and joint pain. Diagnosing CHIKV in a clinical lab setting is often omitted because of the high lab safety requirement. An infection system that mimics CHIKV infection will permit clinical evaluation of the production of neutralizing antibody for both disease diagnostics and treatment. Methodology/Principal Findings We generated a CHIKV construct expressing CHIKV structural proteins. This construct permits the production of CHIKV pseudo-viral particles with a luciferase reporter. The pseudo-virus was able to infect a wide range of cell lines. The pseudovirus could be neutralized by the addition of neutralizing antibodies from patients. Conclusions Taken together, we have developed a powerful system that can be handled at biosafety level 2 laboratories for evaluation of existence of CHIKV neutralizing antibodies. PMID:25333782

  18. The catalytic triad of the influenza C virus glycoprotein HEF esterase: characterization by site-directed mutagenesis and functional analysis.

    PubMed

    Pleschka, S; Klenk, H D; Herrler, G

    1995-10-01

    Influenza C virus is able to inactivate its own cellular receptors by virtue of a sialate 9-O-acetylesterase that releases the acetyl residue at position C-9 of N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2). The receptor-destroying enzyme activity is a function of the surface glycoprotein HEF and this esterase belongs to the class of serine hydrolases. In their active site, these enzymes contain a catalytic triad made up of a serine, a histidine and an aspartic acid residue. Sequence comparison with other serine esterases has indicated that, in addition to serine-71 (S71), the amino acids histidine-368 or -369 (H368/369) and aspartic acid 261 (D261) are the most likely candidates to form the catalytic triad of the influenza C virus glycoprotein. By site-directed mutagenesis, mutants were generated in which alanine substituted for either of these amino acids. Using a phagemid expression vector, pSP1D-HEF the HEF gene was expressed in both COS 7 and MDCK I cells. The glycoprotein was obtained in a functional form only in the latter cells, as indicated by its transport to the cell surface and measurable enzyme activity. The low level of expression could be increased by stimulating the NF-KB-binding activity of the cytomegalovirus immediate-early promoter/enhancer element of the vector. The esterase activity of the mutant proteins was compared with that of the wild-type glycoprotein. With Neu5,9Ac2 as the substrate, the esterase specific activities of the S71/A mutant and the H368,369/A mutant were reduced by more than 90%. In the case of the D261/A mutant the specific activity was reduced by 64%. From this data we conclude that S71, H368/369 and D261 are likely to represent the catalytic triad of the influenza C virus glycoprotein HEF. In addition, N280 is proposed to stabilize the oxyanion of the presumptive transition state intermediate formed by the enzyme-substrate complex. PMID:7595356

  19. The pseudorabies virus gII gene is closely related to the gB glycoprotein gene of herpes simplex virus.

    PubMed Central

    Robbins, A K; Dorney, D J; Wathen, M W; Whealy, M E; Gold, C; Watson, R J; Holland, L E; Weed, S D; Levine, M; Glorioso, J C

    1987-01-01

    We have looked for conserved DNA sequences between four herpes simplex virus type 1 (HSV-1) glycoprotein genes encoding gB, gC, gD, and gE and pseudorabies virus (PRV) DNA, HSV-1 DNA fragments representing these four glycoprotein-coding sequences were hybridized to restriction enzyme fragments of PRV DNA by the Southern blot procedure. Specific hybridization was observed only when HSV-1 gB DNA was used as probe. This region of hybridization was localized to a 5.2-kilobase (kb) region mapping at approximately 0.15 map units on the PRV genome. Northern blot (RNA blot) analysis, with a 1.2-kb probe derived from this segment, revealed a predominant hybridizing RNA species of approximately 3 kb in PRV-infected PK15 cells. DNA sequence analysis of the region corresponding to this RNA revealed a single large open reading frame with significant nucleotide homology with the gB gene of HSV-1 KOS 321. In addition, the beginning of the sequenced PRV region also contained the end of an open reading frame with amino acid homology to HSV-1 ICP 18.5, a protein that may be involved in viral glycoprotein transport. This sequence partially overlaps the PRV gB homolog coding sequence. We have shown that the PRV gene with homology to HSV-1 gB encoded the gII glycoprotein gene by expressing a 765-base-pair segment of the PRV open reading frame in Escherichia coli as a protein fused to beta-galactosidase. Antiserum, raised in rabbits, against this fusion protein immunoprecipitated a specific family of PRV glycoproteins of apparent molecular mass 110, 68, and 55 kilodaltons that have been identified as the gII family of glycoproteins. Analysis of the predicted amino acid sequence indicated that the PRV gII protein shares 50% amino acid homology with the aligned HSV-1 gB protein. All 10 cysteine residues located outside of the signal sequence, as well as 4 of 6 potential N-linked glycosylation sites, were conserved between the two proteins. The primary protein sequence for HSV-1 gB regions

  20. Antiviral activity of a single-domain antibody immunotoxin binding to glycoprotein D of herpes simplex virus 2.

    PubMed

    Geoghegan, Eileen M; Zhang, Hong; Desai, Prashant J; Biragyn, Arya; Markham, Richard B

    2015-01-01

    Despite years of research dedicated to preventing the sexual transmission of herpes simplex virus 2 (HSV-2), there is still no protective vaccine or microbicide against one of the most common sexually transmitted infections in the world. Using a phage display library constructed from a llama immunized with recombinant HSV-2 glycoprotein D, we identified a single-domain antibody VHH, R33, which binds to the viral surface glycoprotein D. Although R33 does not demonstrate any HSV-2 neutralization activity in vitro, when expressed with the cytotoxic domain of exotoxin A, the resulting immunotoxin (R33ExoA) specifically and potently kills HSV-2-infected cells, with a 50% neutralizing dilution (IC50) of 6.7 nM. We propose that R33ExoA could be used clinically to prevent transmission of HSV-2 through killing of virus-producing epithelial cells during virus reactivation. R33 could also potentially be used to deliver other cytotoxic effectors to HSV-2-infected cells. PMID:25385102

  1. Antiviral Activity of a Single-Domain Antibody Immunotoxin Binding to Glycoprotein D of Herpes Simplex Virus 2

    PubMed Central

    Geoghegan, Eileen M.; Zhang, Hong; Desai, Prashant J.; Biragyn, Arya

    2014-01-01

    Despite years of research dedicated to preventing the sexual transmission of herpes simplex virus 2 (HSV-2), there is still no protective vaccine or microbicide against one of the most common sexually transmitted infections in the world. Using a phage display library constructed from a llama immunized with recombinant HSV-2 glycoprotein D, we identified a single-domain antibody VHH, R33, which binds to the viral surface glycoprotein D. Although R33 does not demonstrate any HSV-2 neutralization activity in vitro, when expressed with the cytotoxic domain of exotoxin A, the resulting immunotoxin (R33ExoA) specifically and potently kills HSV-2-infected cells, with a 50% neutralizing dilution (IC50) of 6.7 nM. We propose that R33ExoA could be used clinically to prevent transmission of HSV-2 through killing of virus-producing epithelial cells during virus reactivation. R33 could also potentially be used to deliver other cytotoxic effectors to HSV-2-infected cells. PMID:25385102

  2. Mutagenesis of the La Crosse Virus glycoprotein supports a role for Gc (1066-1087) as the fusion peptide

    SciTech Connect

    Plassmeyer, Matthew L.; Soldan, Samantha S.; Stachelek, Karen M.; Roth, Susan M.; Martin-Garcia, Julio; Gonzalez-Scarano, Francisco . E-mail: scarano@mail.med.upenn.edu

    2007-02-20

    The La Crosse Virus (LACV) M segment encodes two glycoproteins (Gn and Gc), and plays a critical role in the neuropathogenesis of LACV infection as the primary determinant of neuroinvasion. A recent study from our group demonstrated that the region comprising the membrane proximal two-thirds of Gc, amino acids 860-1442, is critical in mediating LACV fusion and entry. Furthermore, computational analysis identified structural similarities between a portion of this region, amino acids 970-1350, and the E1 fusion protein of two alphaviruses: Sindbis virus and Semliki Forrest virus (SFV). Within the region 970-1350, a 22-amino-acid hydrophobic segment (1066-1087) is predicted to correlate structurally with the fusion peptides of class II fusion proteins. We performed site-directed mutagenesis of key amino acids in this 22-amino acid segment and determined the functional consequences of these mutations on fusion and entry. Several mutations within this hydrophobic domain affected glycoprotein expression to some extent, but all mutations either shifted the pH threshold of fusion below that of the wild-type protein, reduced fusion efficiency, or abrogated cell-to-cell fusion and pseudotype entry altogether. These results, coupled with the aforementioned computational modeling, suggest that the LACV Gc functions as a class II fusion protein and support a role for the region Gc 1066-1087 as a fusion peptide.

  3. The use of chimeric virus-like particles harbouring a segment of hantavirus Gc glycoprotein to generate a broadly-reactive hantavirus-specific monoclonal antibody.

    PubMed

    Zvirbliene, Aurelija; Kucinskaite-Kodze, Indre; Razanskiene, Ausra; Petraityte-Burneikiene, Rasa; Klempa, Boris; Ulrich, Rainer G; Gedvilaite, Alma

    2014-02-01

    Monoclonal antibodies (MAbs) against viral glycoproteins have important diagnostic and therapeutic applications. In most cases, the MAbs specific to viral glycoproteins are raised against intact virus particles. The biosynthesis of viral glycoproteins in heterologous expression systems such as bacteria, yeast, insect or mammalian cells is often problematic due to their low expression level, improper folding and limited stability. To generate MAbs against hantavirus glycoprotein Gc, we have used initially a recombinant yeast-expressed full-length Puumala virus (PUUV) Gc protein. However, this approach was unsuccessful. As an alternative recombinant antigen, chimeric virus-like particles (VLPs) harboring a segment of PUUV Gc glycoprotein were generated in yeast Saccharomyces cerevisiae. A 99 amino acid (aa)-long segment of Gc protein was inserted into the major capsid protein VP1 of hamster polyomavirus at previously defined positions: either site #1 (aa 80-89) or site #4 (aa 280-289). The chimeric proteins were found to self-assemble to VLPs as evidenced by electron microscopy. Chimeric VLPs induced an efficient insert-specific antibody response in immunized mice. Monoclonal antibody (clone #10B8) of IgG isotype specific to hantavirus Gc glycoprotein was generated. It recognized recombinant full-length PUUV Gc glycoprotein both in ELISA and Western blot assay and reacted specifically with hantavirus-infected cells in immunofluorescence assay. Epitope mapping studies revealed the N-terminally located epitope highly conserved among different hantavirus strains. In conclusion, our approach to use chimeric VLPs was proven useful for the generation of virus-reactive MAb against hantavirus Gc glycoprotein. The generated broadly-reactive MAb #10B8 might be useful for various diagnostic applications. PMID:24513568

  4. The Use of Chimeric Virus-like Particles Harbouring a Segment of Hantavirus Gc Glycoprotein to Generate a Broadly-Reactive Hantavirus-Specific Monoclonal Antibody

    PubMed Central

    Zvirbliene, Aurelija; Kucinskaite-Kodze, Indre; Razanskiene, Ausra; Petraityte-Burneikiene, Rasa; Klempa, Boris; Ulrich, Rainer G.; Gedvilaite, Alma

    2014-01-01

    Monoclonal antibodies (MAbs) against viral glycoproteins have important diagnostic and therapeutic applications. In most cases, the MAbs specific to viral glycoproteins are raised against intact virus particles. The biosynthesis of viral glycoproteins in heterologous expression systems such as bacteria, yeast, insect or mammalian cells is often problematic due to their low expression level, improper folding and limited stability. To generate MAbs against hantavirus glycoprotein Gc, we have used initially a recombinant yeast-expressed full-length Puumala virus (PUUV) Gc protein. However, this approach was unsuccessful. As an alternative recombinant antigen, chimeric virus-like particles (VLPs) harboring a segment of PUUV Gc glycoprotein were generated in yeast Saccharomyces cerevisiae. A 99 amino acid (aa)-long segment of Gc protein was inserted into the major capsid protein VP1 of hamster polyomavirus at previously defined positions: either site #1 (aa 80–89) or site #4 (aa 280–289). The chimeric proteins were found to self-assemble to VLPs as evidenced by electron microscopy. Chimeric VLPs induced an efficient insert-specific antibody response in immunized mice. Monoclonal antibody (clone #10B8) of IgG isotype specific to hantavirus Gc glycoprotein was generated. It recognized recombinant full-length PUUV Gc glycoprotein both in ELISA and Western blot assay and reacted specifically with hantavirus-infected cells in immunofluorescence assay. Epitope mapping studies revealed the N-terminally located epitope highly conserved among different hantavirus strains. In conclusion, our approach to use chimeric VLPs was proven useful for the generation of virus-reactive MAb against hantavirus Gc glycoprotein. The generated broadly-reactive MAb #10B8 might be useful for various diagnostic applications. PMID:24513568

  5. Recovery of Recombinant Crimean Congo Hemorrhagic Fever Virus Reveals a Function for Non-structural Glycoproteins Cleavage by Furin

    PubMed Central

    Bergeron, Éric; Zivcec, Marko; Chakrabarti, Ayan K.; Nichol, Stuart T.; Albariño, César G.; Spiropoulou, Christina F.

    2015-01-01

    Crimean Congo hemorrhagic fever virus (CCHFV) is a negative-strand RNA virus of the family Bunyaviridae (genus: Nairovirus). In humans, CCHFV causes fever, hemorrhage, severe thrombocytopenia, and high fatality. A major impediment in precisely determining the basis of CCHFV’s high pathogenicity has been the lack of methodology to produce recombinant CCHFV. We developed a reverse genetics system based on transfecting plasmids into BSR-T7/5 and Huh7 cells. In our system, bacteriophage T7 RNA polymerase produced complementary RNA copies of the viral S, M, and L segments that were encapsidated with the support, in trans, of CCHFV nucleoprotein and L polymerase. The system was optimized to systematically recover high yields of infectious CCHFV. Additionally, we tested the ability of the system to produce specifically designed CCHFV mutants. The M segment encodes a polyprotein that is processed by host proprotein convertases (PCs), including the site-1 protease (S1P) and furin-like PCs. S1P and furin cleavages are necessary for producing the non-structural glycoprotein GP38, while S1P cleavage yields structural Gn. We studied the role of furin cleavage by rescuing a recombinant CCHFV encoding a virus glycoprotein precursor lacking a functional furin cleavage motif (RSKR mutated to ASKA). The ASKA mutation blocked glycoprotein precursor’s maturation to GP38, and Gn precursor’s maturation to Gn was slightly diminished. Furin cleavage was not essential for replication, as blocking furin cleavage resulted only in transient reduction of CCHFV titers, suggesting that either GP38 and/or decreased Gn maturation accounted for the reduced virion production. Our data demonstrate that nairoviruses can be produced by reverse genetics, and the utility of our system uncovered a function for furin cleavage. This viral rescue system could be further used to study the CCHFV replication cycle and facilitate the development of efficacious vaccines to counter this biological and public

  6. LC–MS/MS Quantitation of Esophagus Disease Blood Serum Glycoproteins by Enrichment with Hydrazide Chemistry and Lectin Affinity Chromatography

    PubMed Central

    2015-01-01

    Changes in glycosylation have been shown to have a profound correlation with development/malignancy in many cancer types. Currently, two major enrichment techniques have been widely applied in glycoproteomics, namely, lectin affinity chromatography (LAC)-based and hydrazide chemistry (HC)-based enrichments. Here we report the LC–MS/MS quantitative analyses of human blood serum glycoproteins and glycopeptides associated with esophageal diseases by LAC- and HC-based enrichment. The separate and complementary qualitative and quantitative data analyses of protein glycosylation were performed using both enrichment techniques. Chemometric and statistical evaluations, PCA plots, or ANOVA test, respectively, were employed to determine and confirm candidate cancer-associated glycoprotein/glycopeptide biomarkers. Out of 139, 59 common glycoproteins (42% overlap) were observed in both enrichment techniques. This overlap is very similar to previously published studies. The quantitation and evaluation of significantly changed glycoproteins/glycopeptides are complementary between LAC and HC enrichments. LC–ESI–MS/MS analyses indicated that 7 glycoproteins enriched by LAC and 11 glycoproteins enriched by HC showed significantly different abundances between disease-free and disease cohorts. Multiple reaction monitoring quantitation resulted in 13 glycopeptides by LAC enrichment and 10 glycosylation sites by HC enrichment to be statistically different among disease cohorts. PMID:25134008

  7. Evidence that maturation of the N-linked glycans of the respiratory syncytial virus (RSV) glycoproteins is required for virus-mediated cell fusion: The effect of {alpha}-mannosidase inhibitors on RSV infectivity

    SciTech Connect

    McDonald, Terence P.; Jeffree, Chris E.; Li, Ping; Rixon, Helen W. McL.; Brown, Gaie; Aitken, James D.; MacLellan, Kirsty; Sugrue, Richard J. . E-mail: rjsugrue@ntu.edu.sg

    2006-07-05

    Glycan heterogeneity of the respiratory syncytial virus (RSV) fusion (F) protein was demonstrated by proteomics. The effect of maturation of the virus glycoproteins-associated glycans on virus infectivity was therefore examined using the {alpha}-mannosidase inhibitors deoxymannojirimycin (DMJ) and swainsonine (SW). In the presence of SW the N-linked glycans on the F protein appeared in a partially mature form, whereas in the presence of DMJ no maturation of the glycans was observed. Neither inhibitor had a significant effect on G protein processing or on the formation of progeny virus. Although the level of infectious virus and syncytia formation was not significantly affected by SW-treatment, DMJ-treatment correlated with a one hundred-fold reduction in virus infectivity. Our data suggest that glycan maturation of the RSV glycoproteins, in particular those on the F protein, is an important step in virus maturation and is required for virus infectivity.

  8. [Creation of DNA vaccine vector based on codon-optimized gene of rabies virus glycoprotein (G protein) with consensus amino acid sequence].

    PubMed

    Starodubova, E S; Kuzmenko, Y V; Latanova, A A; Preobrazhenskaya, O V; Karpov, V L

    2016-01-01

    An optimized design of the rabies virus glycoprotein (G protein) for use within DNA vaccines has been suggested. The design represents a territorially adapted antigen constructed taking into account glycoprotein amino acid sequences of the rabies viruses registered in the Russian Federation and the vaccine Vnukovo-32 strain. Based on the created consensus amino acid sequence, the nucleotide codon-optimized sequence of this modified glycoprotein was obtained and cloned into the pVAX1 plasmid (a vector of the last generation used in the creation of DNA vaccines). A twofold increase in this gene expression compared to the expression of the Vnukovo-32 strain viral glycoprotein gene in a similar vector was registered in the transfected cell culture. It has been demonstrated that the accumulation of modified G protein exceeds the number of the control protein synthesized using the plasmid with the Vnukovo-32 strain viral glycoprotein gene by 20 times. Thus, the obtained modified rabies virus glycoprotein can be considered to be a promising DNA vaccine antigen. PMID:27239860

  9. Expression and immunogenicity of recombinant glycoprotein D of herpes simplex virus 1 in Drosophila S2 cells.

    PubMed

    Mao, Hongyan; Zhao, Xiaofei; Zhu, Hongjuan; Guo, Jingxia; Ma, Zhenghai

    2016-05-18

    Herpes simplex virus type 1 (HSV-1) is responsible for cold sores in the general population, but also contributes to the development of other more serious diseases in some circumstances. The viral glycoprotein D (gD) is essential for virus entry into host cells. In the present study, the Drosophila melanogaster Schneider 2 (S2) expression system (DES) was evaluated for the expression of recombinant gD1. The DNA sequences encoding the full-length gD1 (369aa, FLgD1) and a truncated gD1 form corresponding to the ectodomain (314aa, EgD1) were cloned into S2 expression vector pMT/BiP/V5-HisA to generate pMT-EgD1 and pMT-FLgD1, respectively. Two forms of gD1 gene were fitted with a hexahistidine tag to facilitate their purification. Cell populations expressing the highest gD1 levels were selected by using a limiting dilution assay. Western blot, flow cytometry (FACS), and confocal immunofluoresence assay demonstrated that the full-length form is restrained in the lipid membranes of the cell and the ectodomain form is secreted into the medium. Recombinant ectodomain gD1 was scaled up and purified from the culture medium using nickel nitrilotriacetic acid affinity chromatography, and a maximum production level of 56.8 mg/L of recombinant gD1 was obtained in a shake-flask culture of S2 cells after induction with 5 µM CdCl2 for 4 days. Mice were then immunized with recombinant purified gD1 and produced high titers of antibody measured by enzyme-linked immunosorbent assay (ELISA; 1:5,120,000) as well as high plaque neutralization titer (1:320). Overall, the data indicated that stable expression in S2 cells is a practical way of synthesizing gD1 for use in structural and functional studies in the further study. PMID:26835587

  10. Structural Flexibility of a Conserved Antigenic Region in Hepatitis C Virus Glycoprotein E2 Recognized by Broadly Neutralizing Antibodies

    PubMed Central

    Meola, Annalisa; Tarr, Alexander W.; England, Patrick; Meredith, Luke W.; McClure, C. Patrick; Foung, Steven K. H.; McKeating, Jane A.; Ball, Jonathan K.; Rey, Felix A.

    2014-01-01

    ABSTRACT Neutralizing antibodies (NAbs) targeting glycoprotein E2 are important for the control of hepatitis C virus (HCV) infection. One conserved antigenic site (amino acids 412 to 423) is disordered in the reported E2 structure, but a synthetic peptide mimicking this site forms a β-hairpin in complex with three independent NAbs. Our structure of the same peptide in complex with NAb 3/11 demonstrates a strikingly different extended conformation. We also show that residues 412 to 423 are essential for virus entry but not for E2 folding. Together with the neutralizing capacity of the 3/11 Fab fragment, this indicates an unexpected structural flexibility within this epitope. NAbs 3/11 and AP33 (recognizing the extended and β-hairpin conformations, respectively) display similar neutralizing activities despite converse binding kinetics. Our results suggest that HCV utilizes conformational flexibility as an immune evasion strategy, contributing to the limited immunogenicity of this epitope in patients, similar to the conformational flexibility described for other enveloped and nonenveloped viruses. IMPORTANCE Approximately 180 million people worldwide are infected with hepatitis C virus (HCV), and neutralizing antibodies play an important role in controlling the replication of this major human pathogen. We show here that one of the most conserved antigenic sites within the major glycoprotein E2 (amino acids 412 to 423), which is disordered in the recently reported crystal structure of an E2 core fragment, can adopt different conformations in the context of the infectious virus particle. Recombinant Fab fragments recognizing different conformations of this antigenic site have similar neutralization activities in spite of converse kinetic binding parameters. Of note, an antibody response targeting this antigenic region is less frequent than those targeting other more immunogenic regions in E2. Our results suggest that the observed conformational flexibility in this

  11. Control of virus diseases in soybeans.

    PubMed

    Hill, John H; Whitham, Steven A

    2014-01-01

    Soybean, one of the world's most important sources of animal feed and vegetable oil, can be infected by numerous viruses. However, only a small number of the viruses that can potentially infect soybean are considered as major economic problems to soybean production. Therefore, we consider management options available to control diseases caused by eight viruses that cause, or have the potential to cause, significant economic loss to producers. We summarize management tactics in use and suggest direction for the future. Clearly, the most important tactic is disease resistance. Several resistance genes are available for three of the eight viruses discussed. Other options include use of virus-free seed and avoidance of alternative virus hosts when planting. Attempts at arthropod vector control have generally not provided consistent disease management. In the future, disease management will be considerably enhanced by knowledge of the interaction between soybean and viral proteins. Identification of genes required for soybean defense may represent key regulatory hubs that will enhance or broaden the spectrum of basal resistance to viruses. It may be possible to create new recessive or dominant negative alleles of host proteins that do not support viral functions but perform normal cellular function. The future approach to virus control based on gene editing or exploiting allelic diversity points to necessary research into soybean-virus interactions. This will help to generate the knowledge needed for rational design of durable resistance that will maximize global production. PMID:25410106

  12. Immune response and resistance to Rous sarcoma virus challenge of chickens immunized with cell-associated glycoproteins provided with a recombinant avian leukosis virus.

    PubMed Central

    Chebloune, Y; Rulka, J; Cosset, F L; Valsesia, S; Ronfort, C; Legras, C; Drynda, A; Kuzmak, J; Nigon, V M; Verdier, G

    1991-01-01

    The Rous-associated virus 1 env gene, which encodes the envelope gp85 and gp37 glycoproteins, was isolated and inserted in place of the v-erbB oncogene into an avian erythroblastosis virus-based vector, carrying the neo resistance gene substituted for the v-erbA oncogene, to generate the pNEA recombinant vector. A helper-free virus stock of the pNEA vector was produced on an avian transcomplementing cell line and used to infect primary chicken embryo fibroblasts (CEFs) or quail QT6 cells. These infected cells, selected with G418 (CEF/NEA and QT6/NEA, respectively) were found to be resistant to superinfections with subgroup A retroviruses. The CEF/NEA preparations were used as a cell-associated antigen to inoculate adult chickens by the intravenous route compared with direct inoculations of NEA recombinant helper-free virus used as a cell-free antigen. Chickens injected with the cell-associated antigen (CEF/NEA) exhibited an immune response demonstrated by induction of high titers of neutralizing antibodies and were found to be protected against tumor production after Rous sarcoma virus A challenge. Conversely, no immune response and no protection against Rous sarcoma virus A challenge were observed in chickens directly inoculated with cell-free NEA recombinant virus or in sham-inoculated chickens. PMID:1654445

  13. The Insulin Degrading Enzyme Binding Domain of Varicella-Zoster Virus (VZV) Glycoprotein E is Important for Cell-to-Cell Spread and VZV Infectivity, while a Glycoprotein I Binding Domain is Essential for Infection

    PubMed Central

    Ali, Mir A.; Li, Qingxue; Fischer, Elizabeth R.; Cohen, Jeffrey I.

    2009-01-01

    Varicella-zoster virus (VZV) glycoprotein E (gE) interacts with glycoprotein I and with insulin degrading enzyme (IDE), which is a receptor for the virus. We found that a VZV gE deletion mutant could only be grown in cells expressing gE. Expression of VZV gE on the surface of cells did not interfere with VZV infection. HSV deleted for gE is impaired for cell-to-cell spread; VZV gE could not complement this activity in an HSV gE null mutant. VZV lacking the IDE binding domain of gE grew to peak titers nearly equivalent to parental virus; however, it was impaired for cell-to-cell spread and for infectivity with cell-free virus. VZV deleted for a region of gE that binds glycoprotein I could not replicate in cell culture unless grown in cells expressing gE. We conclude that the IDE binding domain is important for efficient cell-to-cell spread and infectivity of cell-free virus. PMID:19233447

  14. Accessory human cytomegalovirus glycoprotein US9 in the unique short component of the viral genome promotes cell-to-cell transmission of virus in polarized epithelial cells.

    PubMed Central

    Maidji, E; Tugizov, S; Jones, T; Zheng, Z; Pereira, L

    1996-01-01

    Human cytomegalovirus (CMV) encodes accessory glycoproteins that are dispensable for virus growth in nonpolarized cells in culture. We report that CMV deletion mutants lacking the gene for accessory glycoprotein US9 in the unique short component of the viral genome are impaired in plaque formation in polarized human retinal pigment epithelial (ARPE-19) cells. Comparison of CMV deletion mutants in US9 with herpes simplex virus type 1 deletion mutants lacking glycoproteins gE and gI showed that both of these mutants are impaired in altering junctional complexes and increasing paracellular permeability in polarized ARPE-19 cells cultured on permeable filter supports. Results of functional studies indicate that CMV US9 and homologs of gE have analogous roles in promoting virus spread across lateral membranes of polarized epithelial cells. PMID:8970961

  15. Secreted Herpes Simplex Virus-2 Glycoprotein G Modifies NGF-TrkA Signaling to Attract Free Nerve Endings to the Site of Infection

    PubMed Central

    Cabrera, Jorge Rubén; Viejo-Borbolla, Abel; Martinez-Martín, Nadia; Blanco, Soledad; Wandosell, Francisco; Alcamí, Antonio

    2015-01-01

    Herpes simplex virus type 1 (HSV-1) and HSV-2 are highly prevalent viruses that cause a variety of diseases, from cold sores to encephalitis. Both viruses establish latency in peripheral neurons but the molecular mechanisms facilitating the infection of neurons are not fully understood. Using surface plasmon resonance and crosslinking assays, we show that glycoprotein G (gG) from HSV-2, known to modulate immune mediators (chemokines), also interacts with neurotrophic factors, with high affinity. In our experimental model, HSV-2 secreted gG (SgG2) increases nerve growth factor (NGF)-dependent axonal growth of sympathetic neurons ex vivo, and modifies tropomyosin related kinase (Trk)A-mediated signaling. SgG2 alters TrkA recruitment to lipid rafts and decreases TrkA internalization. We could show, with microfluidic devices, that SgG2 reduced NGF-induced TrkA retrograde transport. In vivo, both HSV-2 infection and SgG2 expression in mouse hindpaw epidermis enhance axonal growth modifying the termination zone of the NGF-dependent peptidergic free nerve endings. This constitutes, to our knowledge, the discovery of the first viral protein that modulates neurotrophins, an activity that may facilitate HSV-2 infection of neurons. This dual function of the chemokine-binding protein SgG2 uncovers a novel strategy developed by HSV-2 to modulate factors from both the immune and nervous systems. PMID:25611061

  16. Production of pseudorabies virus recombinant glycoprotein B and its use in an agar gel immunodiffusion (AGID) test for detection of antibodies with sensitivity and specificity equal to the virus neutralization assay.

    PubMed

    Serena, María Soledad; Geisler, Christoph; Metz, Germán Ernesto; Mórtola, Eduardo Carlos; Echeverría, María Gabriela

    2016-04-01

    Pseudorabies virus (PrV) causes Aujeszky's disease (AD), which affects mainly swine, but also cattle, sheep, and wild animals, resulting in substantial economic losses due to animal mortality and lost productivity worldwide. To combat PrV, eradication programs using PrV strains lacking the gene encoding glycoprotein E (gE) are ongoing in several countries. These eradication programs have generated a currently unmet demand for affordable, easy-to-use, and sensitive tests that can detect PrV infection in pigs infected with either wild-type virus or vaccine strain (gE-deleted) virus. To meet this demand, we used the baculovirus-insect cell system to produce recombinant glycoprotein B (gB) as antigen for an immune assay. The high GC-content (70% average) of the gB gene from the Argentinian PrV CL15 strain necessitated the use of betaine as a PCR enhancer to amplify the extracellular domain. Recombinant gB was expressed at high levels and reacted strongly with sera from PrV infected pigs. We used the recombinant gB to develop an agar gel immunodiffusion (AGID) test for detection of PrV antibodies. Compared to the gold standard virus neutralization (VN) assay, the AGID sensitivity and specificity were 95% and 96.6% respectively. Thus, recombinant gB produced in the baculovirus-insect cell system is a viable source of antigen for the detection of PrV antibodies in AGID tests. Considering its relatively lower cost, simplicity of use and result interpretation, our AGID is a valuable alternative tool to the VN assay. PMID:26800775

  17. Preparation and diagnostic use of a novel recombinant single-chain antibody against rabies virus glycoprotein.

    PubMed

    Yuan, Ruosen; Chen, Xiaoxu; Chen, Yan; Gu, Tiejun; Xi, Hualong; Duan, Ye; Sun, Bo; Yu, Xianghui; Jiang, Chunlai; Liu, Xintao; Wu, Chunlai; Kong, Wei; Wu, Yongge

    2014-02-01

    Rabies virus (RABV) causes a fatal infectious disease, but effective protection may be achieved with the use of rabies immunoglobulin and a rabies vaccine. Virus-neutralizing antibodies (VNA), which play an important role in the prevention of rabies, are commonly evaluated by the RABV neutralizing test. For determining serum VNA levels or virus titers during the RABV vaccine manufacturing process, reliability of the assay method is highly important and mainly dependent on the diagnostic antibody. Most diagnostic antibodies are monoclonal antibodies (mAbs) made from hybridoma cell lines and are costly and time consuming to prepare. Thus, production of a cost-effective mAb for determining rabies VNA levels or RABV titers is needed. In this report, we describe the prokaryotic production of a RABV-specific single-chain variable fragment (scFv) protein with a His-tag (scFv98H) from a previously constructed plasmid in a bioreactor, including the purification and refolding process as well as the functional testing of the protein. The antigen-specific binding characteristics, affinity, and relative affinity of the purified protein were tested. The scFv98H antibody was compared with a commercial RABV nucleoprotein mAb for assaying the VNA level of anti-rabies serum samples from different sources or testing the growth kinetics of RABV strains for vaccine manufactured in China. The results indicated that scFv98H may be used as a novel diagnostic tool to assay VNA levels or virus titers and may be used as an alternative for the diagnostic antibody presently employed for these purposes. PMID:24241896

  18. Feline immunodeficiency virus envelope glycoprotein mediates apoptosis in activated PBMC by a mechanism dependent on gp41 function

    SciTech Connect

    Garg, Himanshu; Joshi, Anjali; Tompkins, Wayne A. . E-mail: Wayne_Tompkins@ncsu.edu

    2004-12-20

    Feline Immunodeficiency Virus (FIV) is a lentivirus that causes immunodeficiency in cats, which parallels HIV-1-induced immunodeficiency in humans. It has been established that HIV envelope (Env) glycoprotein mediates T cell loss via a mechanism that requires CXCR4 binding. The Env glycoprotein of FIV, similar to HIV, requires CXCR4 binding for viral entry, as well as inducing membrane fusion leading to syncytia formation. However, the role of FIV Env in T cell loss and the molecular mechanisms governing this process have not been elucidated. We studied the role of Env glycoprotein in FIV-mediated T cell apoptosis in an in vitro model. Our studies demonstrate that membrane-expressed FIV Env induces apoptosis in activated feline peripheral blood mononuclear cells (PBMC) by a mechanism that requires CXCR4 binding, as the process was inhibited by CXCR4 antagonist AMD3100 in a dose-dependent manner. Interestingly, studies regarding the role of CD134, the recently identified primary receptor of FIV, suggest that binding to CD134 may not be important for induction of apoptosis in PBMC. However, inhibiting Env-mediated fusion post CXCR4 binding by FIV gp41-specific fusion inhibitor also inhibited apoptosis. Under similar conditions, a fusion-defective gp41 mutant was unable to induce apoptosis in activated PBMC. Our findings are the first report suggesting the potential of FIV Env to mediate apoptosis in bystander cells by a process that is dependent on gp41 function.

  19. Rainbow Trout Sleeping Disease Virus Is an Atypical Alphavirus

    PubMed Central

    Villoing, Stéphane; Béarzotti, Monique; Chilmonczyk, Stefan; Castric, Jeannette; Brémont, Michel

    2000-01-01

    Sleeping disease (SD) is currently a matter of concern for salmonid fish farmers in most parts of the world. A viral etiology of SD has recently been suspected, since virus-like particles have been observed in infected rainbow trout cells. In salmonid-derived cell lines, the maximal rate of virus production was observed at 10°C, while little virus was produced at 14°C. Through biochemical, physicochemical, and morphological studies, SD virus (SDV) was shown to be an enveloped virus of roughly 60 nm in diameter. The genome consists of 12 kb of RNA, with the appearance of a 26S subgenomic RNA during the time course of SDV replication. The screening of a random-primed cDNA library constructed from the genomic RNA of semipurified virions facilitated the identification of a specific SDV cDNA clone having an open reading frame related to the alphavirus E2 glycoproteins. To extend the comparison between SDV structural proteins and the alphavirus protein counterparts, the nucleotide sequence of the total 4.1-kb subgenomic RNA has been determined. The 26S RNA encodes a 1,324-amino-acid polyprotein exhibiting typical alphavirus structural protein organization. SDV structural proteins showed several remarkable features compared to other alphaviruses: (i) unusually large individual proteins, (ii) very low homology (ranging from 30 to 34%) (iii) an unglycosylated E3 protein, and (iv) and E1 fusion domain sharing mutations implicated in the pH threshold. Although phylogenetically related to the Semliki Forest virus group of alphaviruses, SDV should be considered an atypical member, able to naturally replicate in lower vertebrates. PMID:10590104

  20. Virus neutralizing antibody response in mice and dogs with a bicistronic DNA vaccine encoding rabies virus glycoprotein and canine parvovirus VP2.

    PubMed

    Patial, Sonika; Chaturvedi, V K; Rai, A; Saini, M; Chandra, Rajesh; Saini, Y; Gupta, Praveen K

    2007-05-16

    A bicistronic DNA vaccine against rabies and parvovirus infection of dogs was developed by subcloning rabies glycoprotein and canine parvovirus (CPV) VP2 genes into a bicistronic vector. After characterizing the expression of both the proteins in vitro, the bicistronic DNA vaccine was injected in mice and induced immune response was compared with monocistronic DNA vaccines. There was no significant difference in ELISA and virus neutralizing (VN) antibody responses against rabies and CPV in mice immunized with either bicistronic or monocistronic DNA vaccine. Further, there was significantly similar protection in mice immunized with either bicistronic or monocistronic rabies DNA vaccine on rabies virus challenge. Similarly, dogs immunized with monocistronic and bicistronic DNA vaccines developed comparable VN antibodies against rabies and CPV. This study indicated that bicistronic DNA vaccine can be used in dogs to induce virus neutralizing immune responses against both rabies and CPV. PMID:17391817

  1. The anti-inflammatory glycoprotein, CD200, restores neurogenesis and enhances amyloid phagocytosis in a mouse model of Alzheimer's disease.

    PubMed

    Varnum, Megan M; Kiyota, Tomomi; Ingraham, Kaitlin L; Ikezu, Seiko; Ikezu, Tsuneya

    2015-11-01

    Cluster of Differentiation-200 (CD200) is an anti-inflammatory glycoprotein expressed in neurons, T cells, and B cells, and its receptor is expressed on glia. Both Alzheimer's disease patients and mouse models display age-related or amyloid-β peptide (Aβ)-induced reductions in CD200. The goal of this study was to determine if neuronal CD200 expression restores hippocampal neurogenesis and reduces Aβ in the amyloid precursor protein mouse model. Amyloid precursor protein and wild-type mice were injected at 6 months of age with an adeno-associated virus expressing CD200 into the hippocampus and sacrificed at 12 months. CD200 expression restored neural progenitor cell proliferation and differentiation in the subgranular and granular cell layers of the dentate gyrus and reduced diffuse but not thioflavin-S(+) plaques in the hippocampus. In vitro studies demonstrated that CD200-stimulated microglia increased neural differentiation of neural stem cells and enhanced axon elongation and dendrite number. CD200 also enhanced Aβ uptake by microglia. These data indicate that CD200 is capable of enhancing microglia-mediated Aβ clearance and neural differentiation and has potential as a therapeutic for Alzheimer's disease. PMID:26315370

  2. A novel human T-leukemia virus type 1 cell-to-cell transmission assay permits definition of SU glycoprotein amino acids important for infectivity.

    PubMed Central

    Delamarre, L; Rosenberg, A R; Pique, C; Pham, D; Dokhélar, M C

    1997-01-01

    Human T-leukemia virus type 1 (HTLV-1) envelope glycoproteins play a major role in viral transmission, which in the case of this virus occurs almost exclusively via cell-to-cell contact. Until very recently, the lack of an HTLV-1 infectivity assay precluded the determination of the HTLV-1 protein domains required for infectivity. Here, we describe an assay which allows the quantitative evaluation of HTLV-1 cell-to-cell transmission in a single round of infection. Using this assay, we demonstrate that in this system, cell-to-cell transmission is at least 100 times more efficient than transmission with free viral particles. We have examined 46 surface (SU) glycoprotein mutants in order to define the amino acids of the HTLV-1 SU glycoprotein required for full infectivity. We demonstrate that these amino acids are distributed along the entire length of the SU glycoprotein, including the N-terminus and C-terminus regions, which have not been previously defined as being important for HTLV-1 glycoprotein function. For most of the mutated glycoproteins, the capacity to mediate cell-to-cell transmission is correlated with the ability to induce formation of syncytia. This result indicates that the fusion capacity is the main factor responsible for infectivity mediated by the HTLV-1 SU envelope glycoprotein, as is the case for other retroviral glycoproteins. However, other factors must also intervene, since two of the mutated glycoproteins were correctly fusogenic but could not mediate cell-to-cell transmission. Existence of this phenotype shows that capacity for fusion is not sufficient to confer infectivity, even in cell-to-cell transmission, and could suggest that postfusion events involve the SU. PMID:8985345

  3. Interaction between the Hemagglutinin-Neuraminidase and Fusion Glycoproteins of Human Parainfluenza Virus Type III Regulates Viral Growth In Vivo

    PubMed Central

    Xu, Rui; Palmer, Samantha G.; Porotto, Matteo; Palermo, Laura M.; Niewiesk, Stefan; Wilson, Ian A.; Moscona, Anne

    2013-01-01

    ABSTRACT Paramyxoviruses, enveloped RNA viruses that include human parainfluenza virus type 3 (HPIV3), cause the majority of childhood viral pneumonia. HPIV3 infection starts when the viral receptor-binding protein engages sialic acid receptors in the lung and the viral envelope fuses with the target cell membrane. Fusion/entry requires interaction between two viral surface glycoproteins: tetrameric hemagglutinin-neuraminidase (HN) and fusion protein (F). In this report, we define structural correlates of the HN features that permit infection in vivo. We have shown that viruses with an HN-F that promotes growth in cultured immortalized cells are impaired in differentiated human airway epithelial cell cultures (HAE) and in vivo and evolve in HAE into viable viruses with less fusogenic HN-F. In this report, we identify specific structural features of the HN dimer interface that modulate HN-F interaction and fusion triggering and directly impact infection. Crystal structures of HN, which promotes viral growth in vivo, show a diminished interface in the HN dimer compared to the reference strain’s HN, consistent with biochemical and biological data indicating decreased dimerization and decreased interaction with F protein. The crystallographic data suggest a structural explanation for the HN’s altered ability to activate F and reveal properties that are critical for infection in vivo. IMPORTANCE Human parainfluenza viruses cause the majority of childhood cases of croup, bronchiolitis, and pneumonia worldwide. Enveloped viruses must fuse their membranes with the target cell membranes in order to initiate infection. Parainfluenza fusion proceeds via a multistep reaction orchestrated by the two glycoproteins that make up its fusion machine. In vivo, viruses adapt for survival by evolving to acquire a set of fusion machinery features that provide key clues about requirements for infection in human beings. Infection of the lung by parainfluenzavirus is determined by

  4. Respiratory syncytial virus glycoproteins uptake occurs through clathrin-mediated endocytosis in a human epithelial cell line

    PubMed Central

    Gutiérrez-Ortega, Abel; Sánchez-Hernández, Carla; Gómez-García, Beatriz

    2008-01-01

    Cell-surface viral proteins most frequently enter the cell through clathrin or caveolae endocytosis. Respiratory syncytial virus antigen internalization by immune cells is via caveolin, however, uptake of paramyxovirus cell membrane proteins by non-immune cells is done through clathrin-coated pits. In this work, the uptake of respiratory syncytial virus cell surface glycoproteins by non-immune human epithelial cells was investigated through indirect immunofluorescence with polyclonal anti-RSV antibody and confocal lasser-scanner microscopy. Clathrin and caveolae internalization pathways were monitored through specific inhibitors monodansylcadaverine (MDC) and methyl-beta-cyclodextrin (MBCD), respectively. Internalization of RSV antigens was inhibited by MDC but not by MBCD, implying that clathrin-mediated endocytosis is the major uptake route of RSV antigens by an epithelial human cell line. PMID:18950517

  5. Evolutionary dynamics of Newcastle disease virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A comprehensive dataset of Newcastle disease viruses (NDV) genome sequences was evaluated using bioinformatics to characterize the evolutionary forces affecting NDV genomes. Despite evidence of recombination in most genes, only one event in the fusion gene of genotype V viruses produced evolutionar...

  6. Structure-Based Mutational Analysis of the Highly Conserved Domain IV of Glycoprotein H of Pseudorabies Virus

    PubMed Central

    Fuchs, Walter; Backovic, Marija; Klupp, Barbara G.; Rey, Felix A.

    2012-01-01

    Glycoprotein H (gH) is an envelope protein conserved in the Herpesviridae. Together with glycoprotein B (gB), the heterodimeric complex of gH and glycoprotein L (gL) mediates penetration and direct viral cell-to-cell spread. In herpes simplex and pseudorabies virus (PrV), coexpression of gH/gL, gB, and gD induces membrane fusion to form polykaryocytes. The recently determined crystal structure of a core fragment of PrV gH revealed marked structural similarity to other gH proteins (M. Backovic et al., Proc. Natl. Acad. Sci. U. S. A. 107:22635–22640, 2010). Within the membrane-proximal part (domain IV), a conserved negatively charged surface loop (flap) is flanked by intramolecular disulfide bonds. Together with an N-linked carbohydrate moiety, this flap covers an underlying patch of hydrophobic residues. To investigate the functional relevance of these structures, nonconservative amino acid substitutions were introduced by site-directed mutagenesis. The mutated proteins were tested for correct expression, fusion activity, and functional complementation of gH-deleted PrV. Several single amino acid changes within the flap and the hydrophobic patch were tolerated, and deletion of the glycosylation site had only minor effects. However, multiple alanine substitutions within the flap or the hydrophobic patch led to significant defects. gH function was also severely affected by disruption of the disulfide bond at the C terminus of the flap and after introduction of cysteine pairs designed to bridge the central part of the flap with the hydrophobic patch. Interestingly, all mutated gH proteins were able to complement gH-deleted PrV, but fusion-deficient gH mutants resulted in a pronounced delay in virus entry. PMID:22623768

  7. N-Glycans on the Nipah Virus Attachment Glycoprotein Modulate Fusion and Viral Entry as They Protect against Antibody Neutralization

    PubMed Central

    Biering, Scott B.; Huang, Andrew; Vu, Andy T.; Robinson, Lindsey R.; Bradel-Tretheway, Birgit; Choi, Eric

    2012-01-01

    Nipah virus (NiV) is the deadliest known paramyxovirus. Membrane fusion is essential for NiV entry into host cells and for the virus' pathological induction of cell-cell fusion (syncytia). The mechanism by which the attachment glycoprotein (G), upon binding to the cell receptors ephrinB2 or ephrinB3, triggers the fusion glycoprotein (F) to execute membrane fusion is largely unknown. N-glycans on paramyxovirus glycoproteins are generally required for proper protein conformational integrity, transport, and sometimes biological functions. We made conservative mutations (Asn to Gln) at the seven potential N-glycosylation sites in the NiV G ectodomain (G1 to G7) individually or in combination. Six of the seven N-glycosylation sites were found to be glycosylated. Moreover, pseudotyped virions carrying these N-glycan mutants had increased antibody neutralization sensitivities. Interestingly, our results revealed hyperfusogenic and hypofusogenic phenotypes for mutants that bound ephrinB2 at wild-type levels, and the mutant's cell-cell fusion phenotypes generally correlated to viral entry levels. In addition, when removing multiple N-glycans simultaneously, we observed synergistic or dominant-negative membrane fusion phenotypes. Interestingly, our data indicated that 4- to 6-fold increases in fusogenicity resulted from multiple mechanisms, including but not restricted to the increase of F triggering. Altogether, our results suggest that NiV-G N-glycans play a role in shielding virions against antibody neutralization, while modulating cell-cell fusion and viral entry via multiple mechanisms. PMID:22915812

  8. Membrane anchoring domain of herpes simplex virus glycoprotein gB is sufficient for nuclear envelope localization.

    PubMed Central

    Gilbert, R; Ghosh, K; Rasile, L; Ghosh, H P

    1994-01-01

    We have used the glycoprotein gB of herpes simplex virus type 1 (gB-1), which buds from the inner nuclear membrane, as a model protein to study localization of membrane proteins in the nuclear envelope. To determine whether specific domains of gB-1 glycoprotein are involved in localization in the nuclear envelope, we have used deletion mutants of gB-1 protein as well as chimeric proteins constructed by replacing the domains of the cell surface glycoprotein G of vesicular stomatitis virus with the corresponding domains of gB. Mutant and chimeric proteins expressed in COS cells were localized by immunoelectron microscopy. A chimeric protein (gB-G) containing the ectodomain of gB and the transmembrane and cytoplasmic domains of G did not localize in the nuclear envelope. When the ectodomain of G was fused to the transmembrane and cytoplasmic domains of gB, however, the resulting chimeric protein (G-gB) was localized in the nuclear envelope. Substitution of the transmembrane domain of G with the 69 hydrophobic amino acids containing the membrane anchoring domain of gB allowed the hybrid protein (G-tmgB) to be localized in the nuclear envelope, suggesting that residues 721 to 795 of gB can promote retention of proteins in the nuclear envelope. Deletion mutations in the hydrophobic region further showed that a transmembrane segment of 21 hydrophobic amino acids, residues 774 to 795 of gB, was sufficient for localization in the nuclear envelope. Since wild-type gB and the mutant and chimeric proteins that were localized in the nuclear envelope were also retained in the endoplasmic reticulum, the membrane spanning segment of gB could also influence retention in the endoplasmic reticulum. Images PMID:8139012

  9. Mutational Analysis of the Candidate Internal Fusion Peptide of the Avian Leukosis and Sarcoma Virus Subgroup A Envelope Glycoprotein

    PubMed Central

    Hernandez, Lorraine D.; White, Judith M.

    1998-01-01

    The transmembrane subunit (TM) of the avian leukosis and sarcoma virus (ALSV) envelope glycoprotein (Env) contains a stretch of conserved hydrophobic amino acids internal to its amino terminus (residues 21 to 42). By analogy with similar sequences in other viral envelope glycoproteins, this region has been proposed to be a fusion peptide. We investigated the role of this region by changing each of three hydrophobic residues (Ile-21, Val-30, and Ile-39) to glutamatic acid and lysine in the ALSV subgroup A Env. Like wild-type (wt) Env, all six mutant Env proteins were proteolytically processed, oligomerized, and expressed at the cell surface in a form that bound Tva, the ALSV subgroup A receptor. Like wt Env, Ile21Glu, Ile21Lys, Val30Glu, and Val30Lys changed conformation upon binding Tva, as assayed by sensitivity to thermolysin. Ile39Glu and Ile39Lys were cleaved by thermolysin in both the absence and presence of Tva. Although incorporated into virus particles at approximately equal levels, all mutant Envs were compromised in their ability to support infection. The mutants at residues 21 and 30 showed levels of infection 2 to 3 orders of magnitude lower than that of wt Env. The mutants at residue 39 were noninfectious. Furthermore, none of the mutants displayed activity in a cell-cell fusion assay. Our results support the contention that residues 21 to 42 of ALSV subgroup A Env constitute its fusion peptide. PMID:9525653

  10. The role of eukaryotic subtilisin-like endoproteases for the activation of human immunodeficiency virus glycoproteins in natural host cells.

    PubMed Central

    Hallenberger, S; Moulard, M; Sordel, M; Klenk, H D; Garten, W

    1997-01-01

    Proteolytic activation of the precursor envelope glycoproteins gp160 of human immunodeficiency virus type 1 (HIV-1) and gp140 of HIV-2, a prerequisite for viral infection, results in the formation of gp120/gp41 and gp125/gp36, respectively. Cleavage is mediated by cellular proteases. Furin, a member of the eukaryotic subtilisin family, has been shown to be an activating protease for HIV. Here, we compared the presence of furin and other mammalian subtilisins in lymphatic cells and tissues. Northern blot analyses revealed that furin and the recently discovered protease LPC/PC7 were the only subtilisin-like enzymes transcribed in such cells. Furin was identified as an enzymatically active endoprotease present in different lymphocytic, as well as monocytic, cell lines. When expressed from vaccinia virus vectors, the proprotein convertases were correctly processed, transported, and secreted into the media and enzymatically active. Coexpression of different subtilisins with the HIV envelope precursors revealed that furin and LPC/PC7 are able to cleave HIV-1 gp160. Moreover, both enzymes proteolytically processed the envelope precursor of HIV-2. gp140 was also cleaved to some extent by PC1, which is not, however, present in lymphatic cells. Furin- and LPC/PC7-catalyzed cleavage of HIV-1 gp160 resulted in biologically active envelope protein. In conclusion, among the known members of the subtilisin family, only furin and LPC/PC7 fulfill the requirements of a protease responsible for in vivo activation of HIV envelope glycoproteins. PMID:8995623

  11. Identification of an immunodominant epitope in the C terminus of glycoprotein 5 of porcine reproductive and respiratory syndrome virus.

    PubMed

    Rodriguez, M J; Sarraseca, J; Fominaya, J; Cortés, E; Sanz, A; Casal, J I

    2001-05-01

    Glycoprotein 5 (GP(5)) is the major glycoprotein of porcine reproductive and respiratory syndrome virus (PRRSV). Expression of GP(5) has been improved by removing the transmembrane regions. Vectors were constructed encoding complete GP(5) plus three mutants: GP(5) Ns (residues 28--201), GP(5)[30--67] (residues 30--67) and GP(5)[30--201] (residues 30--67/130--201). The three deletion mutants were expressed at levels 20--30 times higher than complete GP(5). GP(5)[30--201] was well recognized in ELISA or immunoblotting by a collection of pig sera. All the fragments were tested for the generation of MAbs, but only the polyhistidine-tagged fragment GP(5)[30--201]H elicited an antibody response sufficient to produce MABS: The two MAbs were positive for PRRSV in ELISA and immunoblotting, but negative for virus neutralization. MAb 4BE12 reacted with residues 130--170 and MAb 3AH9 recognized residues 170--201. This region was recognized strongly in immunoblotting by a collection of infected-pig sera. These results indicate diagnostic potential for this epitope. PMID:11297674

  12. Structure of Hepatitis C Virus Envelope Glycoprotein E1 Antigenic Site 314-324 in Complex with Antibody IGH526.

    PubMed

    Kong, Leopold; Kadam, Rameshwar U; Giang, Erick; Ruwona, Tinashe B; Nieusma, Travis; Culhane, Jeffrey C; Stanfield, Robyn L; Dawson, Philip E; Wilson, Ian A; Law, Mansun

    2015-08-14

    Hepatitis C virus (HCV) is a positive-strand RNA virus within the Flaviviridae family. The viral "spike" of HCV is formed by two envelope glycoproteins, E1 and E2, which together mediate viral entry by engaging host receptors and undergoing conformational changes to facilitate membrane fusion. While E2 can be readily produced in the absence of E1, E1 cannot be expressed without E2 and few reagents, including monoclonal antibodies (mAbs), are available for study of this essential HCV glycoprotein. A human mAb to E1, IGH526, was previously reported to cross-neutralize different HCV isolates, and therefore, we sought to further characterize the IGH526 neutralizing epitope to obtain information for vaccine design. We found that mAb IGH526 bound to a discontinuous epitope, but with a major component corresponding to E1 residues 314-324. The crystal structure of IGH526 Fab with this E1 glycopeptide at 1.75Å resolution revealed that the antibody binds to one face of an α-helical peptide. Single mutations on the helix substantially lowered IGH526 binding but did not affect neutralization, indicating either that multiple mutations are required or that additional regions are recognized by the antibody in the context of the membrane-associated envelope oligomer. Molecular dynamics simulations indicate that the free peptide is flexible in solution, suggesting that it requires stabilization for use as a candidate vaccine immunogen. PMID:26135247

  13. Myelin-associated glycoprotein gene mutation causes Pelizaeus-Merzbacher disease-like disorder.

    PubMed

    Lossos, Alexander; Elazar, Nimrod; Lerer, Israela; Schueler-Furman, Ora; Fellig, Yakov; Glick, Benjamin; Zimmerman, Bat-El; Azulay, Haim; Dotan, Shlomo; Goldberg, Sharon; Gomori, John M; Ponger, Penina; Newman, J P; Marreed, Hodaifah; Steck, Andreas J; Schaeren-Wiemers, Nicole; Mor, Nofar; Harel, Michal; Geiger, Tamar; Eshed-Eisenbach, Yael; Meiner, Vardiella; Peles, Elior

    2015-09-01

    Pelizaeus-Merzbacher disease is an X-linked hypomyelinating leukodystrophy caused by mutations or rearrangements in PLP1. It presents in infancy with nystagmus, jerky head movements, hypotonia and developmental delay evolving into spastic tetraplegia with optic atrophy and variable movement disorders. A clinically similar phenotype caused by recessive mutations in GJC2 is known as Pelizaeus-Merzbacher-like disease. Both genes encode proteins associated with myelin. We describe three siblings of a consanguineous family manifesting the typical infantile-onset Pelizaeus-Merzbacher disease-like phenotype slowly evolving into a form of complicated hereditary spastic paraplegia with mental retardation, dysarthria, optic atrophy and peripheral neuropathy in adulthood. Magnetic resonance imaging and spectroscopy were consistent with a demyelinating leukodystrophy. Using genetic linkage and exome sequencing, we identified a homozygous missense c.399C>G; p.S133R mutation in MAG. This gene, previously associated with hereditary spastic paraplegia, encodes myelin-associated glycoprotein, which is involved in myelin maintenance and glia-axon interaction. This mutation is predicted to destabilize the protein and affect its tertiary structure. Examination of the sural nerve biopsy sample obtained in childhood in the oldest sibling revealed complete absence of myelin-associated glycoprotein accompanied by ill-formed onion-bulb structures and a relatively thin myelin sheath of the affected axons. Immunofluorescence, cell surface labelling, biochemical analysis and mass spectrometry-based proteomics studies in a variety of cell types demonstrated a devastating effect of the mutation on post-translational processing, steady state expression and subcellular localization of myelin-associated glycoprotein. In contrast to the wild-type protein, the p.S133R mutant was retained in the endoplasmic reticulum and was subjected to endoplasmic reticulum-associated protein degradation by the

  14. Cassava virus diseases: biology, epidemiology, and management.

    PubMed

    Legg, James P; Lava Kumar, P; Makeshkumar, T; Tripathi, Leena; Ferguson, Morag; Kanju, Edward; Ntawuruhunga, Pheneas; Cuellar, Wilmer

    2015-01-01

    Cassava (Manihot esculenta Crantz.) is the most important vegetatively propagated food staple in Africa and a prominent industrial crop in Latin America and Asia. Its vegetative propagation through stem cuttings has many advantages, but deleteriously it means that pathogens are passed from one generation to the next and can easily accumulate, threatening cassava production. Cassava-growing continents are characterized by specific suites of viruses that affect cassava and pose particular threats. Of major concern, causing large and increasing economic impact in Africa and Asia are the cassava mosaic geminiviruses that cause cassava mosaic disease in Africa and Asia and cassava brown streak viruses causing cassava brown streak disease in Africa. Latin America, the center of origin and domestication of the crop, hosts a diverse set of virus species, of which the most economically important give rise to cassava frog skin disease syndrome. Here, we review current knowledge on the biology, epidemiology, and control of the most economically important groups of viruses in relation to both farming and cultural practices. Components of virus control strategies examined include: diagnostics and surveillance, prevention and control of infection using phytosanitation, and control of disease through the breeding and promotion of varieties that inhibit virus replication and/or movement. We highlight areas that need further research attention and conclude by examining the likely future global outlook for virus disease management in cassava. PMID:25591878

  15. Ebola (Ebola Virus Disease): Signs and Symptoms

    MedlinePlus

    ... Virus Disease) About Ebola Questions & Answers 2014 West Africa Outbreak What's New Timeline Case Counts Previous Case ... U.S. Q&A: 2014 Ebola Outbreak 2014 West Africa Ebola Outbreak Communication Resources Guinea Guinea-Bissau Liberia ...

  16. The UL24 protein of herpes simplex virus 1 affects the sub-cellular distribution of viral glycoproteins involved in fusion

    SciTech Connect

    Ben Abdeljelil, Nawel; Rochette, Pierre-Alexandre; Pearson, Angela

    2013-09-15

    Mutations in UL24 of herpes simplex virus type 1 can lead to a syncytial phenotype. We hypothesized that UL24 affects the sub-cellular distribution of viral glycoproteins involved in fusion. In non-immortalized human foreskin fibroblasts (HFFs) we detected viral glycoproteins B (gB), gD, gH and gL present in extended blotches throughout the cytoplasm with limited nuclear membrane staining; however, in HFFs infected with a UL24-deficient virus (UL24X), staining for the viral glycoproteins appeared as long, thin streaks running across the cell. Interestingly, there was a decrease in co-localized staining of gB and gD with F-actin at late times in UL24X-infected HFFs. Treatment with chemical agents that perturbed the actin cytoskeleton hindered the formation of UL24X-induced syncytia in these cells. These data support a model whereby the UL24 syncytial phenotype results from a mislocalization of viral glycoproteins late in infection. - Highlights: • UL24 affects the sub-cellular distribution of viral glycoproteins required for fusion. • Sub-cellular distribution of viral glycoproteins varies in cell-type dependent manner. • Drugs targeting actin microfilaments affect formation of UL24-related syncytia in HFFs.

  17. Venezuelan Equine Encephalitis Virus Replicon Particles Encoding Respiratory Syncytial Virus Surface Glycoproteins Induce Protective Mucosal Responses in Mice and Cotton Rats▿

    PubMed Central

    Mok, Hoyin; Lee, Sujin; Utley, Thomas J.; Shepherd, Bryan E.; Polosukhin, Vasiliy V.; Collier, Martha L.; Davis, Nancy L.; Johnston, Robert E.; Crowe, James E.

    2007-01-01

    Respiratory syncytial virus (RSV) is an important viral pathogen that causes severe lower respiratory tract infection in infants, the elderly, and immunocompromised individuals. There are no licensed RSV vaccines to date. To prevent RSV infection, immune responses in both the upper and lower respiratory tracts are required. Previously, immunization with Venezuelan equine encephalitis virus replicon particles (VRPs) demonstrated effectiveness in inducing mucosal protection against various pathogens. In this study, we developed VRPs encoding RSV fusion (F) or attachment (G) glycoproteins and evaluated the immunogenicity and efficacy of these vaccine candidates in mice and cotton rats. VRPs, when administered intranasally, induced surface glycoprotein-specific virus neutralizing antibodies in serum and immunoglobulin A (IgA) antibodies in secretions at the respiratory mucosa. In addition, fusion protein-encoding VRPs induced gamma interferon (IFN-γ)-secreting T cells in the lungs and spleen, as measured by reaction with an H-2Kd-restricted CD8+ T-cell epitope. In animals vaccinated with F protein VRPs, challenge virus replication was reduced below the level of detection in both the upper and lower respiratory tracts following intranasal RSV challenge, while in those vaccinated with G protein VRPs, challenge virus was detected in the upper but not the lower respiratory tract. Close examination of histopathology of the lungs of vaccinated animals following RSV challenge revealed no enhanced inflammation. Immunization with VRPs induced balanced Th1/Th2 immune responses, as measured by the cytokine profile in the lungs and antibody isotype of the humoral immune response. These results represent an important first step toward the use of VRPs encoding RSV proteins as a prophylactic vaccine for RSV. PMID:17928349

  18. A recombinant Rift Valley fever virus glycoprotein subunit vaccine confers full protection against Rift Valley fever challenge in sheep

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Rift Valley fever virus (RVFV) is a mosquito-borne zoonotic pathogen causing disease outbreaks in Africa and the Arabian Peninsula. The virus has great potential for transboundary spread due to the presence of competent vectors in non-endemic areas. There is currently no fully licensed vaccine suita...

  19. Human Respiratory Syncytial Virus Memphis 37 Grown in HEp-2 Cells Causes more Severe Disease in Lambs than Virus Grown in Vero Cells

    PubMed Central

    Derscheid, Rachel J.; van Geelen, Albert; McGill, Jodi L.; Gallup, Jack M.; Cihlar, Tomas; Sacco, Randy E.; Ackermann, Mark R.

    2013-01-01

    Respiratory syncytial virus (RSV) is the most common cause of bronchiolitis in infants and young children. A small percentage of these individuals develop severe and even fatal disease. To better understand the pathogenesis of severe disease and develop therapies unique to the less-developed infant immune system, a model of infant disease is needed. The neonatal lamb pulmonary development and physiology is similar to that of infants, and sheep are susceptible to ovine, bovine, or human strains of RSV. RSV grown in Vero (African green monkey) cells has a truncated attachment G glycoprotein as compared to that grown in HEp-2 cells. We hypothesized that the virus grown in HEp-2 cells would cause more severe clinical symptoms and cause more severe pathology. To confirm the hypothesis, lambs were inoculated simultaneously by two different delivery methods (intranasal and nebulized inoculation) with either Vero-grown or HEp-2-grown RSV Memphis 37 (M37) strain of virus to compare viral infection and disease symptoms. Lambs infected with HEp-2 cell-derived virus by either intranasal or nebulization inoculation had significantly higher levels of viral RNA in lungs as well as greater clinical disease including both gross and histopathologic lesions compared to lambs similarly inoculated with Vero-grown virus. Thus, our results provide convincing in vivo evidence for differences in viral infectivity that corroborate previous in vitro mechanistic studies demonstrating differences in the G glycoprotein expression by RSV grown in Vero cells. PMID:24284879

  20. Further characterization of the immune response in mice to inactivated and live rabies vaccines expressing Ebola virus glycoprotein.

    PubMed

    Papaneri, Amy B; Wirblich, Christoph; Cooper, Kurt; Jahrling, Peter B; Schnell, Matthias J; Blaney, Joseph E

    2012-09-21

    We have previously developed (a) replication-competent, (b) replication-deficient, and (c) chemically inactivated rabies virus (RABV) vaccines expressing Ebola virus (EBOV) glycoprotein (GP) that induce humoral immunity against each virus and confer protection from both lethal RABV and mouse-adapted EBOV challenge in mice. Here, we expand our investigation of the immunogenic properties of these bivalent vaccines in mice. Both live and killed vaccines induced primary EBOV GP-specific T-cells and a robust recall response as measured by interferon-γ ELISPOT assay. In addition to cellular immunity, an effective filovirus vaccine will likely require a multivalent humoral immune response against multiple virus species. As a proof-of-principle experiment, we demonstrated that inactivated RV-GP could be formulated with another inactivated RABV vaccine expressing the nontoxic fragment of botulinum neurotoxin A heavy chain (HC50) without a reduction in immunity to each component. Finally, we demonstrated that humoral immunity to GP could be induced by immunization of mice with inactivated RV-GP in the presence of pre-existing immunity to RABV. The ability of these novel vaccines to induce strong humoral and cellular immunity indicates that they should be further evaluated in additional animal models of infection. PMID:22884661

  1. Candidate topical microbicides bind herpes simplex virus glycoprotein B and prevent viral entry and cell-to-cell spread.

    PubMed

    Cheshenko, Natalia; Keller, Marla J; MasCasullo, Veronica; Jarvis, Gary A; Cheng, Hui; John, Minnie; Li, Jin-Hua; Hogarty, Kathleen; Anderson, Robert A; Waller, Donald P; Zaneveld, Lourens J D; Profy, Albert T; Klotman, Mary E; Herold, Betsy C

    2004-06-01

    Topical microbicides designed to prevent acquisition of sexually transmitted infections are urgently needed. Nonoxynol-9, the only commercially available spermicide, damages epithelium and may enhance human immunodeficiency virus transmission. The observation that herpes simplex virus (HSV) and human immunodeficiency virus bind heparan sulfate provided the rationale for the development of sulfated or sulfonated polymers as topical agents. Although several of the polymers have advanced to clinical trials, the spectrum and mechanism of anti-HSV activity and the effects on soluble mediators of inflammation have not been evaluated. The present studies address these gaps. The results indicate that PRO 2000, polystyrene sulfonate, cellulose sulfate, and polymethylenehydroquinone sulfonate inhibit HSV infection 10,000-fold and are active against clinical isolates, including an acyclovir-resistant variant. The compounds formed stable complexes with glycoprotein B and inhibit viral binding, entry, and cell-to-cell spread. The effects may be long lasting due to the high affinity and stability of the sulfated compound-virus complex, as evidenced by surface plasmon resonance studies. The candidate microbicides retained their antiviral activities in the presence of cervical secretions and over a broad pH range. There was little reduction in cell viability following repeated exposure of human endocervical cells to these compounds, although a reduction in secretory leukocyte protease inhibitor levels was observed. These studies support further development and rigorous evaluation of these candidate microbicides. PMID:15155195

  2. Envelope Glycoprotein Internalization Protects Human and Simian Immunodeficiency Virus-Infected Cells from Antibody-Dependent Cell-Mediated Cytotoxicity

    PubMed Central

    von Bredow, Benjamin; Arias, Juan F.; Heyer, Lisa N.; Gardner, Matthew R.; Farzan, Michael; Rakasz, Eva G.

    2015-01-01

    ABSTRACT The cytoplasmic tails of human and simian immunodeficiency virus (HIV and SIV, respectively) envelope glycoproteins contain a highly conserved, membrane-proximal endocytosis motif that prevents the accumulation of Env on the surface of infected cells prior to virus assembly. Using an assay designed to measure the killing of virus-infected cells by antibody-dependent cell-mediated cytotoxicity (ADCC), we show that substitutions in this motif increase the susceptibility of HIV-1- and SIV-infected cells to ADCC in a manner that directly correlates with elevated Env levels on the surface of virus-infected cells. In the case of HIV-1, this effect is additive with a deletion in vpu recently shown to enhance the susceptibility of HIV-1-infected cells to ADCC as a result of tetherin-mediated retention of budding virions on the cell surface. These results reveal a previously unappreciated role for the membrane-proximal endocytosis motif of gp41 in protecting HIV-1- and SIV-infected cells from antibody responses by regulating the amount of Env present on the cell surface. IMPORTANCE This study reveals an unappreciated role for the membrane-proximal endocytosis motif of gp41 in protecting HIV-1- and SIV-infected cells from elimination by Env-specific antibodies. Thus, strategies designed to interfere with this mechanism of Env internalization may improve the efficacy of antibody-based vaccines and antiretroviral therapies designed to enhance the immunological control of HIV-1 replication in chronically infected individuals. PMID:26269175

  3. Generation and Efficacy Evaluation of Recombinant Classical Swine Fever Virus E2 Glycoprotein Expressed in Stable Transgenic Mammalian Cell Line

    PubMed Central

    Hua, Rong-Hong; Huo, Hong; Li, Ye-Nan; Xue, Yao; Wang, Xiao-Lei; Guo, Li-Ping; Zhou, Bin; Song, Yong; Bu, Zhi-Gao

    2014-01-01

    Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), which is a highly contagious swine disease that causes significant economic loses to the pig industry worldwide. The envelope E2 glycoprotein of CSFV is the most important viral antigen in inducing protective immune response against CSF. In this study, we generated a mammalian cell clone (BCSFV-E2) that could stably produce a secreted form of CSFV E2 protein (mE2). The mE2 protein was shown to be N-linked glycosylated and formed a homodimer. The vaccine efficacy of mE2 was evaluated by immunizing pigs. Twenty-five 6-week-old Landrace piglets were randomly divided into five groups. Four groups were intramuscularly immunized with mE2 emulsified in different adjuvants twice at four-week intervals. One group was used as the control group. All mE2-vaccinated pigs developed CSFV-neutralizing antibodies two weeks after the first vaccination with neutralizing antibody titers ranging from 1∶40 to 1∶320. Two weeks after the booster vaccination, the neutralizing antibody titers increased greatly and ranged from 1∶10,240 to 1∶81,920. At 28 weeks after the booster vaccine was administered, the neutralizing antibody titers ranged from 1∶80 to 1∶10240. At 32 weeks after the first vaccination, pigs in all the groups were challenged with a virulent CSFV strain at a dose of 1×105 TCID50. At two weeks after the challenge, all the mE2-immunized pigs survived and exhibited no obvious symptoms of CSF. The neutralizing antibody titer at this time was 20,480. Unvaccinated pigs in the control group exhibited symptoms of CSF 3–4 days after challenge and were euthanized from 7–9 days after challenge when the pigs became moribund. These results indicate that the mE2 is a good candidate for the development of a safe and effective CSFV subunit vaccine. PMID:25198669

  4. Variable regions A and B in the envelope glycoproteins of feline leukemia virus subgroup B and amphotropic murine leukemia virus interact with discrete receptor domains.

    PubMed Central

    Tailor, C S; Kabat, D

    1997-01-01

    The surface (SU) envelope glycoproteins of feline leukemia virus subgroup B (FeLV-B) and amphotropic murine leukemia virus (A-MLV) are highly related, even in the variable regions VRA and VRB that have been shown to be required for receptor recognition. However, FeLV-B and A-MLV use different sodium-dependent phosphate symporters, Pit1 and Pit2, respectively, as receptors for infection. Pit1 and Pit2 are predicted to have 10 membrane-spanning domains and five extracellular loops. The close relationship of the retroviral envelopes enabled us to generate pseudotype virions carrying chimeric FeLV-B/A-MLV envelope glycoproteins. We found that some of the pseudotype viruses could not use Pit1 or Pit2 proteins but could efficiently utilize specific chimeric Pit1/Pit2 proteins as receptors. By studying Mus dunni tail fibroblasts expressing chimeric Pit1/Pit2 proteins and pseudotype virions carrying chimeric FeLV-B/A-MLV envelopes, we show that FeLV-B and A-MLV VRA and VRB interact in a modular manner with specific receptor domains. Our results suggest that FeLV-B VRA interacts with Pit1 extracellular loops 4 and 5 and that residues Phe-60 and Pro-61 of FeLV-B VRA are essential for receptor choice. However, this interaction is insufficient for infection, and an additional interaction between FeLV-B VRB and Pit1 loop 2 is essential. Similarly, A-MLV infection requires interaction of A-MLV VRA with Pit2 loops 4 and 5 and VRB with Pit2 loop 2, with residues Tyr-60 and Val-61 of A-MLV VRA being critical for receptor recognition. Together, our results suggest that FeLV-B and A-MLV infections require two major discrete interactions between the viral SU envelope glycoproteins and their respective receptors. We propose a common two-step mechanism for interaction between retroviral envelope glycoproteins and cell surface receptors. PMID:9371598

  5. Control of sweet potato virus diseases.

    PubMed

    Loebenstein, Gad

    2015-01-01

    Sweet potato (Ipomoea batatas) is ranked seventh in global food crop production and is the third most important root crop after potato and cassava. Sweet potatoes are vegetative propagated from vines, root slips (sprouts), or tubers. Therefore, virus diseases can be a major constrain, reducing yields markedly, often more than 50%. The main viruses worldwide are Sweet potato feathery mottle virus (SPFMV) and Sweet potato chlorotic stunt virus (SPCSV). Effects on yields by SPFMV or SPCSV alone are minor, or but in complex infection by the two or other viruses yield losses of 50%. The orthodox way of controlling viruses in vegetative propagated crops is by supplying the growers with virus-tested planting material. High-yielding plants are tested for freedom of viruses by PCR, serology, and grafting to sweet potato virus indicator plants. After this, meristem tips are taken from those plants that reacted negative. The meristems were grown into plants which were kept under insect-proof conditions and away from other sweet potato material for distribution to farmers after another cycle of reproduction. PMID:25591876

  6. Chikungunya virus-like particles are more immunogenic in a lethal AG129 mouse model compared to glycoprotein E1 or E2 subunits.

    PubMed

    Metz, Stefan W; Martina, Byron E; van den Doel, Petra; Geertsema, Corinne; Osterhaus, Albert D; Vlak, Just M; Pijlman, Gorben P

    2013-12-01

    Chikungunya virus (CHIKV) causes acute illness characterized by fever and long-lasting arthritic symptoms. The need for a safe and effective vaccine against CHIKV infections is on the rise due to on-going vector spread and increasing severity of clinical complications. Here we report the results of a comparative vaccination-challenge experiment in mice using three different vaccine candidates produced in insect cells by recombinant baculoviruses: (i) secreted (s)E1 and (ii) sE2 CHIKV glycoprotein subunits (2 μg/immunization), and (iii) CHIKV virus-like particles (VLPs) (1 μg E2 equivalent/immunization). These experiments show that vaccination with two subsequent administrations of 1 μg of Matrix M adjuvanted CHIKV VLPs completely protected AG129 mice from lethal CHIKV challenge. Vaccination with E1 and E2 subunits provided partial protection, with half of the mice surviving but with significantly lower neutralizing antibody titres as compared to the VLP vaccinated mice. This study provides evidence that even a modest neutralizing antibody response is sufficient to protect mice from CHIKV infections. Neutralization was the prominent correlate of protection. In addition, CHIKV VLPs provide a superior immune response and protection against CHIKV-induced disease in mice as compared to individual CHIKV-sE1 and -sE2 subunits. PMID:24099875

  7. Immune responses of chickens inoculated with a recombinant fowlpox vaccine coexpressing glycoprotein B of infectious laryngotracheitis virus and chicken IL-18.

    PubMed

    Chen, Hong-Ying; Cui, Pei; Cui, Bao-An; Li, He-Ping; Jiao, Xian-Qin; Zheng, Lan-Lan; Cheng, Guo; Chao, An-Jun

    2011-11-01

    Infectious laryngotracheitis virus (ILTV) is an alphaherpesvirus that causes severe and economically significant respiratory disease in poultry worldwide. Herein, the immunogenicity of two recombinant fowlpox viruses (rFPV-gB and rFPV-gB/IL18) containing ILTV glycoprotein B (gB) and chicken interleukin-18 (IL-18) were investigated in a challenge model. One-day-old specific-pathogen-free chickens were vaccinated by wing-web puncture with the two rFPVs and challenged with the virulent ILTV CG strain. There were differences in antibody levels elicited by either rFPV-gB/IL18 or rFPV-gB as determined using ELISA. The ratios of CD4(+) to CD8(+) in chickens immunized with rFPV-gB/IL18 were higher (P < 0.05) than in those immunized with rFPV-gB, and the level of proliferative response of the T cells in the rFPV-gB/IL18-vaccinated group was higher (P < 0.05) than that in the rFPV-gB group. All chickens immunized with rFPV-gB/IL18 were protected (10/10), whereas only eight of 10 of the chickens immunized with the rFPV-gB were protected. The results showed that the protective efficacy of the rFPV-gB vaccine could be enhanced by simultaneous expression of chicken IL-18. PMID:22077232

  8. Anticardiolipin, anti-ß2-glycoprotein I and antiprothrombin antibodies in black South African patients with infectious disease

    PubMed Central

    Loizou, S; Singh, S; Wypkema, E; Asherson, R

    2003-01-01

    Objectives: To investigate IgG, IgM, and IgA, antiphospholipid antibodies (aPL), against cardiolipin (aCL), ß2-glycoprotein I (anti-ß2GPI), and prothrombin (anti-PT), in black South African patients with infectious disease. Unlike patients with systemic lupus erythematosus (SLE) and the antiphospholipid syndrome (APS), raised levels of aPL in infectious diseases are not usually associated with thrombotic complications. Patients and methods: Serum samples from 272 patients with a variety of infectious diseases (100 HIV positive, 112 leprosy, 25 syphilis, 25 malaria, and 10 HCV patients) were studied and compared with autoantibody levels in 100 normal controls. All three aPL were measured using commercial enzyme linked immunosorbent assay (ELISA) kits. Results: Raised levels of all thee aPL were found in all patient groups studied: aCL in 7%, anti-ß2GPI in 6%, and aPT in 43% of 100 HIV patients, in 29%, 89%, and 21% of 112 patients with leprosy, in 8%, 8%, and 28% of 25 patients with syphilis, in 12%, 8%, and 28% of 25 patients with malaria, and in 20%, 30%, and 30% of 10 HCV patients studied, respectively. Conclusions: The prevalence of aCL and anti-ß2GPI in black South African HIV positive patients, or those with syphilis, malaria, or hepatitis C virus is lower than reported for mixed race or white populations. aPT were the most prevalent aPL detected in these patient groups, except in patients with leprosy, for whom anti-ß2GPI was the most prevalent, and where the spectrum of aPL was similar to that seen in patients with SLE and APS. PMID:14583576

  9. 9 CFR 113.212 - Bursal Disease Vaccine, Killed Virus.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false Bursal Disease Vaccine, Killed Virus..., DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Killed Virus Vaccines § 113.212 Bursal Disease Vaccine, Killed Virus. Bursal Disease...

  10. 9 CFR 113.212 - Bursal Disease Vaccine, Killed Virus.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Bursal Disease Vaccine, Killed Virus..., DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Killed Virus Vaccines § 113.212 Bursal Disease Vaccine, Killed Virus. Bursal Disease...

  11. 9 CFR 113.212 - Bursal Disease Vaccine, Killed Virus.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Bursal Disease Vaccine, Killed Virus..., DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Killed Virus Vaccines § 113.212 Bursal Disease Vaccine, Killed Virus. Bursal Disease...

  12. 9 CFR 113.212 - Bursal Disease Vaccine, Killed Virus.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false Bursal Disease Vaccine, Killed Virus..., DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Killed Virus Vaccines § 113.212 Bursal Disease Vaccine, Killed Virus. Bursal Disease...

  13. 9 CFR 113.212 - Bursal Disease Vaccine, Killed Virus.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false Bursal Disease Vaccine, Killed Virus..., DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Killed Virus Vaccines § 113.212 Bursal Disease Vaccine, Killed Virus. Bursal Disease...

  14. Viral glycoproteins: biological role and application in diagnosis.

    PubMed

    Banerjee, Nilotpal; Mukhopadhyay, Sumi

    2016-03-01

    The viruses that infect humans cause a huge global disease burden and produce immense challenge towards healthcare system. Glycoproteins are one of the major components of human pathogenic viruses. They have been demonstrated to have important role(s) in infection and immunity. Concomitantly high titres of antibodies against these antigenic viral glycoproteins have paved the way for development of novel diagnostics. Availability of appropriate biomarkers is necessary for advance diagnosis of infectious diseases especially in case of outbreaks. As human mobilization has increased manifold nowadays, dissemination of infectious agents became quicker that paves the need of rapid diagnostic system. In case of viral infection it is an emergency as virus spreads and mutates very fast. This review encircles the vast arena of viral glycoproteins, their importance in health and disease and their diagnostic applications. PMID:26925438

  15. Molecular characterization of duck enteritis virus CHv strain UL49.5 protein and its colocalization with glycoprotein M

    PubMed Central

    Lin, Meng; Wang, Mingshu; Gao, Xinghong; Zhu, Dekang; Chen, Shun; Liu, Mafeng; Yin, Zhongqiong; Wang, Yin; Chen, Xiaoyue

    2014-01-01

    The UL49.5 gene of most herpesviruses is conserved and encodes glycoprotein N. However, the UL49.5 protein of duck enteritis virus (DEV) (pUL49.5) has not been reported. In the current study, the DEV pUL49.5 gene was first subjected to molecular characterization. To verify the predicted intracellular localization of gene expression, the recombinant plasmid pEGFP-C1/pUL49.5 was constructed and used to transfect duck embryo fibroblasts. Next, the recombinant plasmid pDsRed1-N1/glycoprotein M (gM) was produced and used for co-transfection with the pEGFP-C1/pUL49.5 plasmid to determine whether DEV pUL49.5 and gM (a conserved protein in herpesviruses) colocalize. DEV pUL49.5 was thought to be an envelope glycoprotein with a signal peptide and two transmembrane domains. This protein was also predicted to localize in the cytoplasm and endoplasmic reticulum with a probability of 66.7%. Images taken by a fluorescence microscope at different time points revealed that the DEV pUL49.5 and gM proteins were both expressed in the cytoplasm. Overlap of the two different fluorescence signals appeared 12 h after transfection and continued to persist until the end of the experiment. These data indicate a possible interaction between DEV pUL49.5 and gM. PMID:24690604

  16. Genome locations of temperature-sensitive mutants in glycoprotein gB of herpes simplex virus type 1.

    PubMed

    DeLuca, N; Person, S; Bzik, D J; Snipes, W

    1984-09-01

    A plasmid containing a herpes simplex virus type 1 (HSV-1) insert from strain KOS, prototypic coordinates 0.345 to 0.368 (3.45 kilobases) was mutagenized in vitro, and potential mutations were introduced into intact viral DNA by cotransfection. Functions normally associated with the glycoprotein gB are in the 1-9 complementation group, and the above coordinates include those that specify the gB glycoprotein gene. Following cotransfection, individual plaques were screened for temperature sensitivity (ts) of viral growth. A total of seven ts mutants was obtained, of which four were spurious mutations due to alterations outside the cloned sequences, presumably mediated by some aspect of the Ca-precipitation-cotransfection method. The remaining three did not complement known mutants of the 1-9 complementation group. These three mutants, along with tsJ12 (P.A. Schaffer, G.M. Aron, N. Biswal, and M. Benyesh-Melnick, 1973, Virology 52, 57-71) and tsJ33 (C.-T. Chu, D.S. Parris, R.A.F. Dixon, F.E. Farber, and P.A. Schaffer, 1979, Virology 98, 168-181), were physically located by marker-rescue experiments to three different restriction fragments between 0.345 to 0.368 map units. Sodium dodecyl sulfate-gel electrophoresis was used to analyze the glycoproteins synthesized during continuous or pulse-chase labeling protocols. All five mutants were found to synthesize a precursor of gB but did not accumulate mature gB during a pulse, a chase, or continuous labeling at the nonpermissive temperature. PMID:6091335

  17. Human immunodeficiency virus type 1 glycoprotein precursor retains a CD4-p56lck complex in the endoplasmic reticulum.

    PubMed Central

    Crise, B; Rose, J K

    1992-01-01

    The cell surface glycoprotein, CD4, is the receptor for human immunodeficiency virus (HIV) in T lymphocytes. Following HIV infection, there is reduced expression of CD4 on the cell surface, and this downregulation probably results, at least in part, from the formation of complexes containing the HIV type 1 (HIV-1) glycoprotein precursor (gp160) and CD4 that are not transported from the endoplasmic reticulum (ER). At the plasma membrane of T cells, CD4 is tightly associated with a cytoplasmic tyrosine kinase (p56lck) that is involved in T-cell activation. Using a transient expression system with HeLa cells, we show by pulse-labeling and immunoprecipitation that newly synthesized CD4 can associate with p56lck before CD4 is transported from the ER. In the presence of HIV-1 gp160, a ternary complex of gp160-CD4 and p56lck forms in the ER. Using confocal immunofluorescence microscopy, we observed complete retention of p56lck in the ER. Such mislocation of a tyrosine kinase to the cytoplasmic face of the ER could play a role in lymphocyte killing caused by HIV infection or expression of gp160 alone. Images PMID:1548763

  18. Structural basis for immunization with postfusion respiratory syncytial virus fusion F glycoprotein (RSV F) to elicit high neutralizing antibody titers

    SciTech Connect

    Swanson, Kurt A.; Settembre, Ethan C.; Shaw, Christine A.; Dey, Antu K.; Rappuoli, Rino; Mandl, Christian W.; Dormitzer, Philip R.; Carfi, Andrea

    2012-02-07

    Respiratory syncytial virus (RSV), the main cause of infant bronchiolitis, remains a major unmet vaccine need despite more than 40 years of vaccine research. Vaccine candidates based on a chief RSV neutralization antigen, the fusion (F) glycoprotein, have foundered due to problems with stability, purity, reproducibility, and potency. Crystal structures of related parainfluenza F glycoproteins have revealed a large conformational change between the prefusion and postfusion states, suggesting that postfusion F antigens might not efficiently elicit neutralizing antibodies. We have generated a homogeneous, stable, and reproducible postfusion RSV F immunogen that elicits high titers of neutralizing antibodies in immunized animals. The 3.2-{angstrom} X-ray crystal structure of this substantially complete RSV F reveals important differences from homology-based structural models. Specifically, the RSV F crystal structure demonstrates the exposure of key neutralizing antibody binding sites on the surface of the postfusion RSV F trimer. This unanticipated structural feature explains the engineered RSV F antigen's efficiency as an immunogen. This work illustrates how structural-based antigen design can guide the rational optimization of candidate vaccine antigens.

  19. Antibody Treatment of Ebola and Sudan Virus Infection via a Uniquely Exposed Epitope within the Glycoprotein Receptor-Binding Site.

    PubMed

    Howell, Katie A; Qiu, Xiangguo; Brannan, Jennifer M; Bryan, Christopher; Davidson, Edgar; Holtsberg, Frederick W; Wec, Anna Z; Shulenin, Sergey; Biggins, Julia E; Douglas, Robin; Enterlein, Sven G; Turner, Hannah L; Pallesen, Jesper; Murin, Charles D; He, Shihua; Kroeker, Andrea; Vu, Hong; Herbert, Andrew S; Fusco, Marnie L; Nyakatura, Elisabeth K; Lai, Jonathan R; Keck, Zhen-Yong; Foung, Steven K H; Saphire, Erica Ollmann; Zeitlin, Larry; Ward, Andrew B; Chandran, Kartik; Doranz, Benjamin J; Kobinger, Gary P; Dye, John M; Aman, M Javad

    2016-05-17

    Previous efforts to identify cross-neutralizing antibodies to the receptor-binding site (RBS) of ebolavirus glycoproteins have been unsuccessful, largely because the RBS is occluded on the viral surface. We report a monoclonal antibody (FVM04) that targets a uniquely exposed epitope within the RBS; cross-neutralizes Ebola (EBOV), Sudan (SUDV), and, to a lesser extent, Bundibugyo viruses; and shows protection against EBOV and SUDV in mice and guinea pigs. The antibody cocktail ZMapp™ is remarkably effective against EBOV (Zaire) but does not cross-neutralize other ebolaviruses. By replacing one of the ZMapp™ components with FVM04, we retained the anti-EBOV efficacy while extending the breadth of protection to SUDV, thereby generating a cross-protective antibody cocktail. In addition, we report several mutations at the base of the ebolavirus glycoprotein that enhance the binding of FVM04 and other cross-reactive antibodies. These findings have important implications for pan-ebolavirus vaccine development and defining broadly protective antibody cocktails. PMID:27160900

  20. Palmitoylation of the feline immunodeficiency virus envelope glycoprotein and its effect on fusion activity and envelope incorporation into virions

    SciTech Connect

    Gonzalez, Silvia A.; Paladino, Monica G.; Affranchino, Jose L.

    2012-06-20

    The feline immunodeficiency virus (FIV) envelope glycoprotein (Env) possesses a short cytoplasmic domain of 53 amino acids containing four highly conserved cysteines at Env positions 804, 811, 815 and 848. Since palmitoylation of transmembrane proteins occurs at or near the membrane anchor, we investigated whether cysteines 804, 811 and 815 are acylated and analyzed the relevance of these residues for Env functions. Replacement of cysteines 804, 811 and 815 individually or in combination by serine residues resulted in Env glycoproteins that were efficiently expressed and processed. However, mutations C804S and C811S reduced Env fusogenicity by 93% and 84%, respectively, compared with wild-type Env. By contrast, mutant C815S exhibited a fusogenic capacity representing 50% of the wild-type value. Remarkably, the double mutation C804S/C811S abrogated both Env fusion activity and Env incorporation into virions. Finally, by means of Click chemistry assays we demonstrated that the four FIV Env cytoplasmic cysteines are palmitoylated.

  1. Can mutational GC-pressure create new linear B-cell epitopes in herpes simplex virus type 1 glycoprotein B?

    PubMed

    Khrustalev, Vladislav Victorovich

    2009-01-01

    We showed that GC-content of nucleotide sequences coding for linear B-cell epitopes of herpes simplex virus type 1 (HSV1) glycoprotein B (gB) is higher than GC-content of sequences coding for epitope-free regions of this glycoprotein (G + C = 73 and 64%, respectively). Linear B-cell epitopes have been predicted in HSV1 gB by BepiPred algorithm ( www.cbs.dtu.dk/services/BepiPred ). Proline is an acrophilic amino acid residue (it is usually situated on the surface of protein globules, and so included in linear B-cell epitopes). Indeed, the level of proline is much higher in predicted epitopes of gB than in epitope-free regions (17.8% versus 1.8%). This amino acid is coded by GC-rich codons (CCX) that can be produced due to nucleotide substitutions caused by mutational GC-pressure. GC-pressure will also lead to disappearance of acrophobic phenylalanine, isoleucine, methionine and tyrosine coded by GC-poor codons. Results of our "in-silico directed mutagenesis" showed that single nonsynonymous substitutions in AT to GC direction in two long epitope-free regions of gB will cause formation of new linear epitopes or elongation of previously existing epitopes flanking these regions in 25% of 539 possible cases. The calculations of GC-content and amino acid content have been performed by CodonChanges algorithm ( www.barkovsky.hotmail.ru ). PMID:19811425

  2. Crystal structure of the Hendra virus attachment G glycoprotein bound to a potent cross-reactive neutralizing human monoclonal antibody.

    PubMed

    Xu, Kai; Rockx, Barry; Xie, Yihu; DeBuysscher, Blair L; Fusco, Deborah L; Zhu, Zhongyu; Chan, Yee-Peng; Xu, Yan; Luu, Truong; Cer, Regina Z; Feldmann, Heinz; Mokashi, Vishwesh; Dimitrov, Dimiter S; Bishop-Lilly, Kimberly A; Broder, Christopher C; Nikolov, Dimitar B

    2013-01-01

    The henipaviruses, represented by Hendra (HeV) and Nipah (NiV) viruses are highly pathogenic zoonotic paramyxoviruses with uniquely broad host tropisms responsible for repeated outbreaks in Australia, Southeast Asia, India and Bangladesh. The high morbidity and mortality rates associated with infection and lack of licensed antiviral therapies make the henipaviruses a potential biological threat to humans and livestock. Henipavirus entry is initiated by the attachment of the G envelope glycoprotein to host cell membrane receptors. Previously, henipavirus-neutralizing human monoclonal antibodies (hmAb) have been isolated using the HeV-G glycoprotein and a human naïve antibody library. One cross-reactive and receptor-blocking hmAb (m102.4) was recently demonstrated to be an effective post-exposure therapy in two animal models of NiV and HeV infection, has been used in several people on a compassionate use basis, and is currently in development for use in humans. Here, we report the crystal structure of the complex of HeV-G with m102.3, an m102.4 derivative, and describe NiV and HeV escape mutants. This structure provides detailed insight into the mechanism of HeV and NiV neutralization by m102.4, and serves as a blueprint for further optimization of m102.4 as a therapeutic agent and for the development of entry inhibitors and vaccines. PMID:24130486

  3. Specific pseudorabies virus infection of the rat visual system requires both gI and gp63 glycoproteins.

    PubMed Central

    Whealy, M E; Card, J P; Robbins, A K; Dubin, J R; Rziha, H J; Enquist, L W

    1993-01-01

    Transneuronal transport of pseudorabies virus (PRV) from the retina to visual centers that mediate visual discrimination and reflexes requires specific genes in the unique short region of the PRV genome. In contrast, these same viral genes are not required to infect retinorecipient areas of the brain involved in circadian rhythm regulation. In this report, we demonstrate that viral mutants carrying defined deletions of the genes encoding glycoprotein gI or gp63, or both, result in the same dramatic transport defect. Efficient export of either gI or gp63 from the endoplasmic reticulum to the Golgi apparatus in a fibroblast cell line requires the presence of both proteins. We also show that gI and gp63 physically interact, as demonstrated by pulse-chase and sucrose gradient sedimentation experiments. Complex formation is rapid compared with homodimerization of PRV glycoprotein gII. We suggest that gI and gp63 function in concert to affect neurotropism in the rat visual circuitry and that a heterodimer is likely to be the unit of function. Images PMID:8389905

  4. Further characterization of the immune response in mice to inactivated and live rabies vaccines expressing Ebola virus glycoprotein

    PubMed Central

    Papaneri, Amy B.; Wirblich, Christoph; Cooper, Kurt; Jahrling, Peter B.; Schnell, Matthias J.; Blaney, Joseph E.

    2012-01-01

    We have previously developed (a) replication-competent, (b) replication-deficient, and (c) chemically inactivated rabies virus (RABV) vaccines expressing ebolavirus (EBOV) glycoprotein (GP) that induce humoral immunity against each virus and confer protection from both lethal RABV and mouse-adapted EBOV challenge in mice. Here, we expand our investigation of the immunogenic properties of these bivalent vaccines in mice. Both live and killed vaccines induced primary EBOV GP-specific T-cells and a robust recall response as measured by interferon-γ ELISPOT assay. In addition to cellular immunity, an effective filovirus vaccine will likely require a multivalent humoral immune response against multiple virus species. As a proof-of-principle experiment, we demonstrated that inactivated RV-GP could be formulated with another inactivated RABV vaccine expressing the nontoxic fragment of botulinum neurotoxin A heavy chain (HC50) without a reduction in immunity to each component. Finally, we demonstrated that humoral immunity to GP could be induced by immunization of mice with inactivated RV-GP in the presence of pre-existing immunity to RABV. The ability of these novel vaccines to induce strong humoral and cellular immunity indicates that they should be further evaluated in additional animal models of infection. PMID:22884661

  5. Liposome-Mediated Herpes Simplex Virus Uptake Is Glycoprotein-D Receptor-Independent but Requires Heparan Sulfate.

    PubMed

    Burnham, Lorrie A; Jaishankar, Dinesh; Thompson, Jeffrey M; Jones, Kevin S; Shukla, Deepak; Tiwari, Vaibhav

    2016-01-01

    Cationic liposomes are widely used to facilitate introduction of genetic material into target cells during transfection. This study describes a non-receptor mediated herpes simplex virus type-1 (HSV-1) entry into the Chinese hamster ovary (CHO-K1) cells that naturally lack glycoprotein D (gD)-receptors using a commercially available cationic liposome: lipofectamine. Presence of cell surface heparan sulfate (HS) increased the levels of viral entry indicating a potential role of HS in this mode of entry. Loss of viral entry in the presence of actin de-polymerizing or lysosomotropic agents suggests that this mode of entry results in the endocytosis of the lipofectamine-virus mixture. Enhancement of HSV-1 entry by liposomes was also demonstrated in vivo using a zebrafish embryo model that showed stronger infection in the eyes and other tissues. Our study provides novel insights into gD receptor independent viral entry pathways and can guide new strategies to enhance the delivery of viral gene therapy vectors or oncolytic viruses. PMID:27446014

  6. Liposome-Mediated Herpes Simplex Virus Uptake Is Glycoprotein-D Receptor-Independent but Requires Heparan Sulfate

    PubMed Central

    Burnham, Lorrie A.; Jaishankar, Dinesh; Thompson, Jeffrey M.; Jones, Kevin S.; Shukla, Deepak; Tiwari, Vaibhav

    2016-01-01

    Cationic liposomes are widely used to facilitate introduction of genetic material into target cells during transfection. This study describes a non-receptor mediated herpes simplex virus type-1 (HSV-1) entry into the Chinese hamster ovary (CHO-K1) cells that naturally lack glycoprotein D (gD)-receptors using a commercially available cationic liposome: lipofectamine. Presence of cell surface heparan sulfate (HS) increased the levels of viral entry indicating a potential role of HS in this mode of entry. Loss of viral entry in the presence of actin de-polymerizing or lysosomotropic agents suggests that this mode of entry results in the endocytosis of the lipofectamine-virus mixture. Enhancement of HSV-1 entry by liposomes was also demonstrated in vivo using a zebrafish embryo model that showed stronger infection in the eyes and other tissues. Our study provides novel insights into gD receptor independent viral entry pathways and can guide new strategies to enhance the delivery of viral gene therapy vectors or oncolytic viruses. PMID:27446014

  7. An alternative conformation of the gp41 heptad repeat 1 region coiled coil exists in the human immunodeficiency virus (HIV-1) envelope glycoprotein precursor

    SciTech Connect

    Mische, Claudia C.; Yuan Wen; Strack, Bettina; Craig, Stewart; Farzan, Michael; Sodroski, Joseph . E-mail: joseph_sodroski@dfci.harvard.edu

    2005-07-20

    The human immunodeficiency virus (HIV-1) transmembrane envelope glycoprotein, gp41, which mediates virus-cell fusion, exists in at least three different conformations within the trimeric envelope glycoprotein complex. The structures of the prefusogenic and intermediate states are unknown; structures representing the postfusion state have been solved. In the postfusion conformation, three helical heptad repeat 2 (HR2) regions pack in an antiparallel fashion into the hydrophobic grooves on the surface of a triple-helical coiled coil formed by the heptad repeat 1 (HR1) regions. We studied the prefusogenic conformation of gp41 by mutagenic alteration of membrane-anchored and soluble forms of the HIV-1 envelope glycoproteins. Our results indicate that, in the HIV-1 envelope glycoprotein precursor, the gp41 HR1 region is in a conformation distinct from that of a trimeric coiled coil. Thus, the central gp41 coiled coil is formed during the transition of the HIV-1 envelope glycoproteins from the precursor state to the receptor-bound intermediate.

  8. Fusion protein of the paramyxovirus simian virus 5: nucleotide sequence of mRNA predicts a highly hydrophobic glycoprotein.

    PubMed Central

    Paterson, R G; Harris, T J; Lamb, R A

    1984-01-01

    The nucleotide sequence of the mRNA coding for the fusion glycoprotein (F) of the paramyxovirus, simian virus 5, has been obtained. There is a single large open reading frame on the mRNA that encodes a protein of 529 amino acids with a molecular weight of 56,531. The proteolytic cleavage/activation site of F, to yield F2 and F1, contains five arginine residues. Six potential glycosylation sites were identified in the protein, two on F2 and four on F1. The deduced amino acid sequence indicates that F is extensively hydrophobic over the length of the polypeptide chain. Three regions are very hydrophobic and could interact directly with membranes: these are the NH2-terminal putative signal peptide, the COOH-terminal putative membrane anchorage domain, and the NH2-terminal region of F1. Images PMID:6093114

  9. A sindbis virus replicon-based DNA vaccine encoding the rabies virus glycoprotein elicits immune responses and complete protection in mice from lethal challenge.

    PubMed

    Saxena, Sonal; Dahiya, Shyam S; Sonwane, Arvind A; Patel, Chhabi Lal; Saini, Mohini; Rai, A; Gupta, Praveen K

    2008-12-01

    A sindbis virus replicon-based DNA vaccine encoding rabies virus glycoprotein (G) was developed by subcloning rabies G gene into a sindbis virus replicon-based vaccine vector (pAlpha). The self-amplification of RNA transcripts and translation efficiency of rabies G was analyzed in pAlpha-Rab-G-transfected mammalian cells using RT-PCR, SDS-PAGE and Western blot analysis. The transfected cells also showed induction of apoptosis which is an important event in the enhancement of immune responses. Further, immune responses induced with replicon-based rabies DNA vaccine (pAlpha-Rab-G) was compared with conventional rabies DNA vaccine and commercial cell culture vaccine (Rabipur) in intramuscularly injected mice. The mice immunized with replicon-based rabies DNA vaccine induced humoral and cell mediated immune responses better than conventional rabies DNA vaccine however, comparable to Rabipur vaccine. On challenge with rabies virus CVS strain, replicon-based rabies DNA vaccine conferred complete protection similar to Rabipur. These results demonstrate that replicon-based rabies DNA vaccine is effective in inducing both humoral and cellular immune responses and can be considered as effective vaccine against rabies. PMID:18848857

  10. Foot-and-mouth disease virus L peptidase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Foot-and-mouth disease virus (FMDV), equine rhinitis A virus (ERAV) and bovine rhinitis B virus (BRBV) comprise the genus Aphthovirus of the Picornaviridae family. Seven genera within this family, Aphthoviruses, Cardioviruses, Erboviruses (ERBV), Kobuviruses, Senecaviruses, Sapeloviruses, and Tescho...

  11. Identification of amino acids controlling the low-pH-induced conformational change of rabies virus glycoprotein.

    PubMed Central

    Gaudin, Y; Raux, H; Flamand, A; Ruigrok, R W

    1996-01-01

    The glycoprotein (G) of rabies virus assumes at least three different conformations: the native state detected at the viral surface above pH 7, the activated state involved in the first step of the fusion process, and the fusion-inactive conformation (I). A new category of monoclonal antibodies (MAbs) which recognized specifically the I conformation at the viral surface has recently been described. These MAbs (17A4 and 29EC2) became neutralizing when the virus was preincubated at acidic pH to induce the conformational change toward the I state of G. Mutants escaping neutralization were then selected. In this study, we have investigated the fusion and the low-pH-induced fusion inactivation properties of these mutants. All of these mutants have fusion properties similar to those of the CVS parental strain, but five mutants (E282K, M44I, M44V, V392G, and M396T) were considerably slowed in their conformational change leading to the I state. These mutants allow us to define regions that control this conformational change. These results also reinforce the idea that structural transition toward the I state is irrelevant to the fusion process. Other mutations in amino acids 10, 13, and 15 are probably located in the epitopes of selecting MAbs. Furthermore, in electron microscopy, we observed a hexagonal lattice of glycoproteins at the viral surface of mutants M44I and V392G as well as strong cooperativity in the conformational change toward the I state. This finding demonstrates the existence of lateral interactions between the spikes of a rhabdovirus. PMID:8892855

  12. Deletion of a Predicted β-Sheet Domain within the Amino Terminus of Herpes Simplex Virus Glycoprotein K Conserved among Alphaherpesviruses Prevents Virus Entry into Neuronal Axons

    PubMed Central

    Jambunathan, Nithya; Charles, Anu-Susan; Subramanian, Ramesh; Saied, Ahmad A.; Naderi, Misagh; Rider, Paul; Brylinski, Michal; Chouljenko, Vladimir N.

    2015-01-01

    ABSTRACT We have shown previously that herpes simplex virus 1 (HSV-1) lacking expression of the entire glycoprotein K (gK) or expressing gK with a 38-amino-acid deletion (gKΔ31–68 mutation) failed to infect ganglionic neurons after ocular infection of mice. We constructed a new model for the predicted three-dimensional structure of gK, revealing that the gKΔ31–68 mutation spans a well-defined β-sheet structure within the amino terminus of gK, which is conserved among alphaherpesviruses. The HSV-1(McKrae) gKΔ31–68 virus was tested for the ability to enter into ganglionic neuronal axons in cell culture of explanted rat ganglia using a novel virus entry proximity ligation assay (VEPLA). In this assay, cell surface-bound virions were detected by the colocalization of gD and its cognate receptor nectin-1 on infected neuronal surfaces. Capsids that have entered into the cytoplasm were detected by the colocalization of the virion tegument protein UL37, with dynein required for loading of virion capsids onto microtubules for retrograde transport to the nucleus. HSV-1(McKrae) gKΔ31–68 attached to cell surfaces of Vero cells and ganglionic axons in cell culture as efficiently as wild-type HSV-1(McKrae). However, unlike the wild-type virus, the mutant virus failed to enter into the axoplasm of ganglionic neurons. This work suggests that the amino terminus of gK is a critical determinant for entry into neuronal axons and may serve similar conserved functions for other alphaherpesviruses. IMPORTANCE Alphaherpesviruses, unlike beta- and gammaherpesviruses, have the unique ability to infect and establish latency in neurons. Glycoprotein K (gK) and the membrane protein UL20 are conserved among all alphaherpesviruses. We show here that a predicted β-sheet domain, which is conserved among alphaherpesviruses, functions in HSV-1 entry into neuronal axons, suggesting that it may serve similar functions for other herpesviruses. These results are in agreement with our

  13. An mRNA Vaccine Encoding Rabies Virus Glycoprotein Induces Protection against Lethal Infection in Mice and Correlates of Protection in Adult and Newborn Pigs

    PubMed Central

    Voss, Daniel; Petsch, Benjamin; Baumhof, Patrick; Kramps, Thomas; Stitz, Lothar

    2016-01-01

    Rabies is a zoonotic infectious disease of the central nervous system (CNS). In unvaccinated or untreated subjects, rabies virus infection causes severe neurological symptoms and is invariably fatal. Despite the long-standing existence of effective vaccines, vaccine availability remains insufficient, with high numbers of fatal infections mostly in developing countries. Nucleic acid based vaccines have proven convincingly as a new technology for the fast development of vaccines against newly emerging pathogens, diseases where no vaccine exists or for replacing already existing vaccines. We used an optimized non-replicating rabies virus glycoprotein (RABV-G) encoding messenger RNA (mRNA) to induce potent neutralizing antibodies (VN titers) in mice and domestic pigs. Functional antibody titers were followed in mice for up to one year and titers remained stable for the entire observation period in all dose groups. T cell analysis revealed the induction of both, specific CD4+ as well as CD8+ T cells by RABV-G mRNA, with the induced CD4+ T cells being higher than those induced by a licensed vaccine. Notably, RABV-G mRNA vaccinated mice were protected against lethal intracerebral challenge infection. Inhibition of viral replication by vaccination was verified by qRT-PCR. Furthermore, we demonstrate that CD4+ T cells are crucial for the generation of neutralizing antibodies. In domestic pigs we were able to induce VN titers that correlate with protection in adult and newborn pigs. This study demonstrates the feasibility of a non-replicating mRNA rabies vaccine in small and large animals and highlights the promises of mRNA vaccines for the prevention of infectious diseases. PMID:27336830

  14. An mRNA Vaccine Encoding Rabies Virus Glycoprotein Induces Protection against Lethal Infection in Mice and Correlates of Protection in Adult and Newborn Pigs.

    PubMed

    Schnee, Margit; Vogel, Annette B; Voss, Daniel; Petsch, Benjamin; Baumhof, Patrick; Kramps, Thomas; Stitz, Lothar

    2016-06-01

    Rabies is a zoonotic infectious disease of the central nervous system (CNS). In unvaccinated or untreated subjects, rabies virus infection causes severe neurological symptoms and is invariably fatal. Despite the long-standing existence of effective vaccines, vaccine availability remains insufficient, with high numbers of fatal infections mostly in developing countries. Nucleic acid based vaccines have proven convincingly as a new technology for the fast development of vaccines against newly emerging pathogens, diseases where no vaccine exists or for replacing already existing vaccines. We used an optimized non-replicating rabies virus glycoprotein (RABV-G) encoding messenger RNA (mRNA) to induce potent neutralizing antibodies (VN titers) in mice and domestic pigs. Functional antibody titers were followed in mice for up to one year and titers remained stable for the entire observation period in all dose groups. T cell analysis revealed the induction of both, specific CD4+ as well as CD8+ T cells by RABV-G mRNA, with the induced CD4+ T cells being higher than those induced by a licensed vaccine. Notably, RABV-G mRNA vaccinated mice were protected against lethal intracerebral challenge infection. Inhibition of viral replication by vaccination was verified by qRT-PCR. Furthermore, we demonstrate that CD4+ T cells are crucial for the generation of neutralizing antibodies. In domestic pigs we were able to induce VN titers that correlate with protection in adult and newborn pigs. This study demonstrates the feasibility of a non-replicating mRNA rabies vaccine in small and large animals and highlights the promises of mRNA vaccines for the prevention of infectious diseases. PMID:27336830

  15. Induction of immune responses and protection in mice against rabies using a self-replicating RNA vaccine encoding rabies virus glycoprotein.

    PubMed

    Saxena, Sonal; Sonwane, Arvind A; Dahiya, Shyam S; Patel, Chhabi Lal; Saini, Mohini; Rai, A; Gupta, Praveen K

    2009-04-14

    A self-replicating RNA vaccine encoding rabies virus glycoprotein gene was developed utilizing sindbis virus RNA replicon. The in vitro transcribed RNA (Sin-Rab-G RNA) was transfected in mammalian cells and analysed for self-replication and expression of rabies glycoprotein. To generate immune responses against rabies, mice were immunized with 10microg of Sin-Rab-G RNA and immune responses developed were compared with mice immunized with rabies DNA vaccine and commercial cell culture vaccine (Rabipur). The self-replicating rabies RNA vaccine generated cellular and humoral IgG responses similar to rabies DNA vaccine. On challenge with rabies virus CVS strain, rabies RNA vaccine conferred protection similar to rabies DNA vaccine. These results demonstrated that replicon-based self-replicating rabies RNA vaccine with 10microg dose was effective in inducing immune responses and protection similar to rabies DNA vaccine. PMID:19081687

  16. Nucleotide sequences of Herpes Simplex Virus type 1 (HSV-1) affecting virus entry, cell fusion, and production of glycoprotein gB (VP7)

    SciTech Connect

    DeLuca, N.; Bzik, D.J.; Bond, V.C.; Person, S.; Snipes, W.

    1982-10-30

    The tsB5 strain of Herpes Simplex Virus type 1 (HSV-1) contains at least two mutations; one mutation specifies the syncytial phenotype and the other confers temperature sensitivity for virus growth. These functions are known to be located between the prototypic map coordinates 0.30 and 0.42. In this study it was demonstrated that tsB5 enters human embryonic lung (HEL) cells more rapidly than KOS, another strain of HSV-1. The EcoRI restriction fragment F from the KOS strain (map coordinates 0.315 to 0.421) was mapped with eight restriction endonucleases, and 16 recombinant plasmids were constructed which contained varying portions of the KOS genome. Recombinant viruses were generated by marker-rescue and marker-transfer cotransfection procedures, using intact DNA from one strain and a recombinant plasmid containing DNA from the other strain. The region of the crossover between the two nonisogenic strains was inferred by the identification of restriction sites in the recombinants that were characteristic of the parental strains. The recombinants were subjected to phenotypic analysis. Syncytium formation, rate of virus entry, and the production of gB were all separable by the crossovers that produced the recombinants. The KOS sequences which rescue the syncytial phenotype of tsB5 were localized to 1.5 kb (map coordinates 0.345 to 0.355), and the temperature-sensitive mutation was localized to 1.2 kb (0.360 to 0.368), giving an average separation between the mutations of 2.5 kb on the 150-kb genome. DNA sequences that specify a functional domain for virus entry were localized to the nucleotide sequences between the two mutations. All three functions could be encoded by the virus gene specifying the gB glycoprotein.

  17. Identification of N-glycans from Ebola virus glycoproteins by matrix-assisted laser desorption/ionisation time-of-flight and negative ion electrospray tandem mass spectrometry

    PubMed Central

    Ritchie, Gayle; Harvey, David J.; Stroeher, Ute; Feldmann, Friederike; Feldmann, Heinz; Wahl-Jensen, Victoria; Royle, Louise; Dwek, Raymond A.; Rudd, Pauline M.

    2012-01-01

    The larger fragment of the transmembrane glycoprotein (GP1) and the soluble glycoprotein (sGP) of Ebola virus were expressed in human embryonic kidney cells and the secreted products were purified from the supernatant for carbohydrate analysis. The N-glycans were released with PNGase F from within sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS-PAGE) gels. Identification of the glycans was made with normal-phase high-performance liquid chromatography (HPLC), matrix-assisted laser desorption/ionisation mass spectrometry, negative ion electrospray ionisation fragmentation mass spectrometry and exoglycosidase digestion. Most glycans were complex bi-, tri-and tetra-antennary compounds with reduced amounts of galactose. No bisected compounds were detected. Triantennary glycans were branched on the 6-antenna; fucose was attached to the core GlcNAc residue. Sialylated glycans were present on sGP but were largely absent from GP1, the larger fragment of the transmembrane glycoprotein. Consistent with this was the generally higher level of processing of carbohydrates found on sGP as evidenced by a higher percentage of galactose and lower levels of high-mannose glycans than were found on GP1. These results confirm and expand previous findings on partial characterisation of the Ebola virus transmembrane glycoprotein. They represent the first detailed data on carbohydrate structures of the Ebola virus sGP. PMID:20131323

  18. Mutants of the Rous sarcoma virus envelope glycoprotein that lack the transmembrane anchor and cytoplasmic domains: analysis of intracellular transport and assembly into virions.

    PubMed Central

    Perez, L G; Davis, G L; Hunter, E

    1987-01-01

    The envelope glycoprotein complex of Rous sarcoma virus consists of a knoblike, receptor-binding gp85 polypeptide that is linked through disulfide bonds to a membrane-spanning gp37 spike. We used oligonucleotide-directed mutagenesis to assess the role of the hydrophobic transmembrane region and hydrophilic cytoplasmic domain of gp37 in intracellular transport and assembly into virions. Early termination codons were introduced on either side of the hydrophobic transmembrane region, and the mutated env genes were expressed from the late promoter of simian virus 40. This resulted in the synthesis of glycoprotein complexes composed of a normal gp85 and a truncated gp37 molecule that lacked the cytoplasmic domain alone or both the cytoplasmic and transmembrane domains. The biosynthesis and intracellular transport of the truncated proteins were not significantly different from those of the wild-type glycoproteins, suggesting that any protein signals for biosynthesis and intracellular transport of this viral glycoprotein complex must reside in its extracellular domain. The glycoprotein complex lacking the cytoplasmic domain of gp37 is stably expressed on the cell surface in a manner similar to that of the wild type. In contrast, the complex lacking both the transmembrane and cytoplasmic domains is secreted as a soluble molecule into the media. It can be concluded, therefore, that the transmembrane domain alone is essential for anchoring the RSV env complex in the cell membrane and that the cytoplasmic domain is not required for anchor function. Insertion of the mutated genes into an infectious proviral genome allowed us to assess the ability of the truncated gene products to be assembled into virions and to determine whether such virions were infectious. Viral genomes encoding the secreted glycoprotein were noninfectious, whereas those encoding a glycoprotein complex lacking only the cytoplasmic domain of gp37 were infectious. Virions produced from these mutant

  19. N-Glycans on the Rift Valley Fever Virus Envelope Glycoproteins Gn and Gc Redundantly Support Viral Infection via DC-SIGN

    PubMed Central

    Phoenix, Inaia; Nishiyama, Shoko; Lokugamage, Nandadeva; Hill, Terence E.; Huante, Matthew B.; Slack, Olga A.L.; Carpio, Victor H.; Freiberg, Alexander N.; Ikegami, Tetsuro

    2016-01-01

    Rift Valley fever is a mosquito-transmitted, zoonotic disease that infects humans and ruminants. Dendritic cell specific intercellular adhesion molecule 3 (ICAM-3) grabbing non-integrin (DC-SIGN) acts as a receptor for members of the phlebovirus genus. The Rift Valley fever virus (RVFV) glycoproteins (Gn/Gc) encode five putative N-glycan sequons (asparagine (N)–any amino acid (X)–serine (S)/threonine (T)) at positions: N438 (Gn), and N794, N829, N1035, and N1077 (Gc). The N-glycosylation profile and significance in viral infection via DC-SIGN have not been elucidated. Gc N-glycosylation was first evaluated by using Gc asparagine (N) to glutamine (Q) mutants. Subsequently, we generated a series of recombinant RVFV MP-12 strain mutants, which encode N-to-Q mutations, and the infectivity of each mutant in Jurkat cells stably expressing DC-SIGN was evaluated. Results showed that Gc N794, N1035, and N1077 were N-glycosylated but N829 was not. Gc N1077 was heterogeneously N-glycosylated. RVFV Gc made two distinct N-glycoforms: “Gc-large” and “Gc-small”, and N1077 was responsible for “Gc-large” band. RVFV showed increased infection of cells expressing DC-SIGN compared to cells lacking DC-SIGN. Infection via DC-SIGN was increased in the presence of either Gn N438 or Gc N1077. Our study showed that N-glycans on the Gc and Gn surface glycoproteins redundantly support RVFV infection via DC-SIGN. PMID:27223297

  20. N-Glycans on the Rift Valley Fever Virus Envelope Glycoproteins Gn and Gc Redundantly Support Viral Infection via DC-SIGN.

    PubMed

    Phoenix, Inaia; Nishiyama, Shoko; Lokugamage, Nandadeva; Hill, Terence E; Huante, Matthew B; Slack, Olga A L; Carpio, Victor H; Freiberg, Alexander N; Ikegami, Tetsuro

    2016-01-01

    Rift Valley fever is a mosquito-transmitted, zoonotic disease that infects humans and ruminants. Dendritic cell specific intercellular adhesion molecule 3 (ICAM-3) grabbing non-integrin (DC-SIGN) acts as a receptor for members of the phlebovirus genus. The Rift Valley fever virus (RVFV) glycoproteins (Gn/Gc) encode five putative N-glycan sequons (asparagine (N)-any amino acid (X)-serine (S)/threonine (T)) at positions: N438 (Gn), and N794, N829, N1035, and N1077 (Gc). The N-glycosylation profile and significance in viral infection via DC-SIGN have not been elucidated. Gc N-glycosylation was first evaluated by using Gc asparagine (N) to glutamine (Q) mutants. Subsequently, we generated a series of recombinant RVFV MP-12 strain mutants, which encode N-to-Q mutations, and the infectivity of each mutant in Jurkat cells stably expressing DC-SIGN was evaluated. Results showed that Gc N794, N1035, and N1077 were N-glycosylated but N829 was not. Gc N1077 was heterogeneously N-glycosylated. RVFV Gc made two distinct N-glycoforms: "Gc-large" and "Gc-small", and N1077 was responsible for "Gc-large" band. RVFV showed increased infection of cells expressing DC-SIGN compared to cells lacking DC-SIGN. Infection via DC-SIGN was increased in the presence of either Gn N438 or Gc N1077. Our study showed that N-glycans on the Gc and Gn surface glycoproteins redundantly support RVFV infection via DC-SIGN. PMID:27223297

  1. Generation of virus like particles for epizootic hemorrhagic disease virus.

    PubMed

    Forzan, Mario; Maan, Sushila; Mazzei, Maurizio; Belaganahalli, Manjunatha N; Bonuccelli, Lucia; Calamari, Monica; Carrozza, Maria Luisa; Cappello, Valentina; Di Luca, Mariagrazia; Bandecchi, Patrizia; Mertens, Peter P C; Tolari, Francesco

    2016-08-01

    Epizootic hemorrhagic disease virus (EHDV) is a distinct species within the genus Orbivirus, within the family Reoviridae. The epizootic hemorrhagic disease virus genome comprises ten segments of linear, double stranded (ds) RNA, which are packaged within each virus particle. The EHDV virion has a three layered capsid-structure, generated by four major viral proteins: VP2 and VP5 (outer capsid layer); VP7 (intermediate, core-surface layer) and VP3 (innermost, sub-core layer). Although EHDV infects cattle sporadically, several outbreaks have recently occurred in this species in five Mediterranean countries, indicating a potential threat to the European cattle industry. EHDV is transmitted by biting midges of the genus Culicoides, which can travel long distances through wind-born movements (particularly over water), increasing the potential for viral spread in new areas/countries. Expression systems to generate self-assembled virus like particles (VLPs) by simultaneous expression of the major capsid-proteins, have been established for several viruses (including bluetongue virus). This study has developed expression systems for production of EHDV VLPs, for use as non-infectious antigens in both vaccinology and serology studies, avoiding the risk of genetic reassortment between vaccine and field strains and facilitating large scale antigen production. Genes encoding the four major-capsid proteins of a field strain of EHDV-6, were isolated and cloned into transfer vectors, to generate two recombinant baculoviruses. The expression of these viral genes was assessed in insect cells by monitoring the presence of specific viral mRNAs and by western blotting. Electron microscopy studies confirmed the formation and purification of assembled VLPs. PMID:27473984

  2. Generation and evaluation of recombinant Newcastle disease viruses (NDV) expressing the F and G proteins of avian metapneumovirus subtype C (aMPV-C) as bivalent vaccine against NDV and aMPV challenges in turkeys

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Previously we generated a Newcastle disease virus (NDV) LaSota strain-based recombinant virus expressing the glycoprotein (G) of avian metapneumovirus subgroup C (aMPV-C) as a bivalent vaccine, which provided a partial protection against aMPV-C challenge in turkeys. To improve the vaccine efficacy,...

  3. A Glycoprotein Subunit Vaccine Elicits a Strong Rift Valley Fever Virus Neutralizing Antibody Response in Sheep

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Rift Valley fever virus (RVFV), a member of the Bunyaviridae family, is a mosquito-borne zoonotic pathogen that causes serious morbidity and mortality in livestock and humans. The recent spread of the virus beyond its traditional endemic boundaries in Africa to the Arabian Peninsula coupled with the...

  4. Single amino acid changes in the viral glycoprotein M affect induction of alpha interferon by the coronavirus transmissible gastroenteritis virus.

    PubMed Central

    Laude, H; Gelfi, J; Lavenant, L; Charley, B

    1992-01-01

    Transmissible gastroenteritis virus, an enteropathogenic coronavirus of swine, is a potent inducer of alpha interferon (IFN-alpha) both in vitro and in vivo. Previous studies have shown that virus-infected fixed cells or viral suspensions were able to induce an early and strong IFN-alpha synthesis by naive lymphocytes. Two monoclonal antibodies directed against the viral membrane glycoprotein M (29,000; formerly E1) were found to markedly inhibit virus-induced IFN production, thus assigning to M protein a potential effector role in this phenomenon (B. Charley and H. Laude, J. Virol. 62:8-11, 1988). The present report describes the selection and characterization of a collection of 125 mutant viruses which escaped complement-mediated neutralization by two IFN induction-blocking anti-M protein monoclonal antibodies. Two of these mutants, designated H92 and dm49-4, were found to exhibit a markedly reduced interferogenic activity. IFN synthesis by lymphocytes incubated with purified suspensions of these mutants was 30- to 300-fold lower than that of the parental virus. The transcription of IFN-alpha genes following induction by each mutant was decreased proportionally, as evidenced by Northern (RNA) blot analysis. The sequence of the M gene of 20 complement-mediated neutralization-resistant mutants, including the 2 defective mutants, was determined by direct sequencing of genome RNA. Thirteen distinct amino acid changes were predicted, all located at positions 6 to 22 from the N terminus of the mature M protein and within the putative ectodomain of the molecule. Two substitutions, Thr-17 to Ile and Ser-19 to Pro, were assumed to generate the defective phenotypes of mutants dm49-4 and H92, respectively. The alteration of an Asn-Ser-Thr sequence in dm49-4 virus led to the synthesis of an M protein devoid of a glycan side chain, which suggests a possible involvement of this structure in IFN induction. Overall, these data supported the view that an interferogenic

  5. 9 CFR 113.205 - Newcastle Disease Vaccine, Killed Virus.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... Virus. 113.205 Section 113.205 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Killed Virus Vaccines § 113.205 Newcastle Disease Vaccine, Killed Virus. Newcastle Disease...

  6. 9 CFR 113.205 - Newcastle Disease Vaccine, Killed Virus.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... Virus. 113.205 Section 113.205 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Killed Virus Vaccines § 113.205 Newcastle Disease Vaccine, Killed Virus. Newcastle Disease...

  7. 9 CFR 113.205 - Newcastle Disease Vaccine, Killed Virus.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... Virus. 113.205 Section 113.205 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Killed Virus Vaccines § 113.205 Newcastle Disease Vaccine, Killed Virus. Newcastle Disease...

  8. 9 CFR 113.205 - Newcastle Disease Vaccine, Killed Virus.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... Virus. 113.205 Section 113.205 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Killed Virus Vaccines § 113.205 Newcastle Disease Vaccine, Killed Virus. Newcastle Disease...

  9. 9 CFR 113.205 - Newcastle Disease Vaccine, Killed Virus.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... Virus. 113.205 Section 113.205 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Killed Virus Vaccines § 113.205 Newcastle Disease Vaccine, Killed Virus. Newcastle Disease...

  10. High-efficiency gene transfer into CD34+ cells with a human immunodeficiency virus type 1-based retroviral vector pseudotyped with vesicular stomatitis virus envelope glycoprotein G.

    PubMed Central

    Akkina, R K; Walton, R M; Chen, M L; Li, Q X; Planelles, V; Chen, I S

    1996-01-01

    Currently, amphotropic retroviral vectors are widely used for gene transfer into CD34+ hematopoietic progenitor cells. The relatively low levels of transduction efficiency associated with these vectors in human cells is due to low viral titers and limitations in concentrating the virus because of the inherent fragility of retroviral envelopes. Here we show that a human immunodeficiency virus type 1 (HIV-1)-based retroviral vector containing the firefly luciferase reporter gene can be pseudotyped with a broad-host-range vesicular stomatitis virus envelope glycoprotein G (VSV-G). Higher-efficiency gene transfer into CD34+ cells was achieved with a VSV-G-pseudotyped HIV-1 vector than with a vector packaged in an amphotropic envelope. Concentration of virus without loss of viral infectivity permitted a higher multiplicity of infection, with a consequent higher efficiency of gene transfer, reaching 2.8 copies per cell. These vectors also showed remarkable stability during storage at 4 degrees C for a week. In addition, there was no significant loss of titer after freezing and thawing of the stock virus. The ability of VSV-G-pseudotyped retroviral vectors to achieve a severalfold increase in levels of transduction into CD34+ cells will allow high-efficiency gene transfer into hematopoietic progenitor cells for gene therapy purposes. Furthermore, since it has now become possible to infect CD34+ cells with pseudotyped HIV-1 with a high level of efficiency in vitro, many important questions regarding the effect of HIV-1 on lineage-specific differentiation of hematopoietic progenitors can now be addressed. PMID:8642689

  11. The herpes simplex virus receptor nectin-1 is down-regulated after trans-interaction with glycoprotein D

    SciTech Connect

    Stiles, Katie M.; Milne, Richard S.B.; Cohen, Gary H.; Eisenberg, Roselyn J.; Krummenacher, Claude

    2008-03-30

    During herpes simplex virus (HSV) entry, membrane fusion occurs either on the cell surface or after virus endocytosis. In both cases, binding of glycoprotein D (gD) to a receptor such as nectin-1 or HVEM is required. In this study, we co-cultured cells expressing gD with nectin-1 expressing cells to investigate the effects of gD on nectin-1 at cell contacts. After overnight co-cultures with gD expressing cells, there was a down-regulation of nectin-1 in B78H1-C10, SY5Y, A431 and HeLa cells, which HSV enters by endocytosis. In contrast, on Vero cells, which HSV enters at the plasma membrane, nectin-1 was not down-regulated. Further analysis of B78H1-derived cells showed that nectin-1 down-regulation corresponds to the ability of gD to bind nectin-1 and is achieved by internalization and low-pH-dependent degradation of nectin-1. Moreover, gD is necessary for virion internalization in B78H1 cells expressing nectin-1. These data suggest that the determinants of gD-mediated internalization of nectin-1 may direct HSV to an endocytic pathway during entry.

  12. Mapping of the structural gene of pseudorabies virus glycoprotein A and identification of two non-glycosylated precursor polypeptides.

    PubMed Central

    Mettenleiter, T C; Lukacs, N; Rziha, H J

    1985-01-01

    Cell-free translation of pseudorabies virus RNA isolated during the late phase of the infectious cycle yielded a variety of polypeptides. A monoclonal antibody directed against one of the major viral glycoproteins, gA, immunoprecipitated two polypeptides ranging in molecular weight from 78K to 83K. To localize the structural gene for gA, we used cloned BamHI fragments of the viral DNA to select specific mRNA species and immunoprecipitated their in vitro translation products with the anti-gA monoclonal antibody. This allowed us to map the genomic region encoding the mRNA for the gA within the short unique region of the viral genome on BamHI fragments 7 and 12. Additional polypeptides encoded by this region were characterized by their electrophoretic mobility. In three virus strains tested a similar, but strain-specific, pattern of the two gA precursors was found which was not dependent on the host cell or the state of infection after reaching the late phase. Images PMID:2981362

  13. The erythropoietin receptor transmembrane region is necessary for activation by the Friend spleen focus-forming virus gp55 glycoprotein.

    PubMed Central

    Zon, L I; Moreau, J F; Koo, J W; Mathey-Prevot, B; D'Andrea, A D

    1992-01-01

    The erythropoietin receptor (EPO-R), a member of the cytokine receptor superfamily, can be activated by binding either erythropoietin (EPO) or gp55, the Friend spleen focus-forming virus glycoprotein. The highly specific interaction between gp55 and EPO-R triggers cell proliferation and thereby causes the first stage of Friend virus-induced erythroleukemia. We have generated functional chimeric receptors containing regions of the EPO-R and the interleukin-3 receptor (AIC2A polypeptide), a related cytokine receptor which does not interact with gp55. All chimeric receptors were expressed at similar levels, had similar binding affinities for EPO, and conferred EPO-dependent cell growth. Only those chimeric receptors which contained the EPO-R transmembrane region were activated by gp55. These results demonstrate that the transmembrane region of the EPO-R is critical for activation by gp55. In addition, analysis of a soluble, secreted EPO-R and cysteine point mutants of the EPO-R show that the extracytoplasmic region of the EPO-R specifically interacts with gp55. Images PMID:1320192

  14. Effects of altered cytoplasmic domains on transport of the vesicular stomatitis virus glycoprotein are transferable to other proteins.

    PubMed Central

    Guan, J L; Ruusala, A; Cao, H; Rose, J K

    1988-01-01

    Alterations of the cytoplasmic domain of the vesicular stomatitis virus glycoprotein (G protein) were shown previously to affect transport of the protein from the endoplasmic reticulum, and recent studies have shown that this occurs without detectable effects on G protein folding and trimerization (R. W. Doms et al., J. Cell Biol., in press). Deletions within this domain slowed exit of the mutant proteins from the endoplasmic reticulum, and replacement of this domain with a foreign 12-amino-acid sequence blocked all transport out of the endoplasmic reticulum. To extend these studies, we determined whether such effects of cytoplasmic domain changes were transferable to other proteins. Three different assays showed that the effects of the mutations on transport of two membrane-anchored secretory proteins were the same as those observed with vesicular stomatitis virus G protein. In addition, possible effects on oligomerization were examined for both transported and nontransported forms of membrane-anchored human chorionic gonadotropin-alpha. These membrane-anchored forms, like the nonanchored human chorionic gonadotropin-alpha, had sedimentation coefficients consistent with a monomeric structure. Taken together, our results provide strong evidence that these cytoplasmic mutations affect transport by affecting interactions at or near the cytoplasmic side of the membrane. Images PMID:2841589

  15. Marek's disease virus and skin interactions.

    PubMed

    Couteaudier, Mathilde; Denesvre, Caroline

    2014-01-01

    Marek's disease virus (MDV) is a highly contagious herpesvirus which induces T-cell lymphoma in the chicken. This virus is still spreading in flocks despite forty years of vaccination, with important economical losses worldwide. The feather follicles, which anchor feathers into the skin and allow their morphogenesis, are considered as the unique source of MDV excretion, causing environmental contamination and disease transmission. Epithelial cells from the feather follicles are the only known cells in which high levels of infectious mature virions have been observed by transmission electron microscopy and from which cell-free infectious virions have been purified. Finally, feathers harvested on animals and dust are today considered excellent materials to monitor vaccination, spread of pathogenic viruses, and environmental contamination. This article reviews the current knowledge on MDV-skin interactions and discusses new approaches that could solve important issues in the future. PMID:24694064

  16. Monoclonal Antibodies to Distinct Sites on Herpes Simplex Virus (HSV) Glycoprotein D Block HSV Binding to HVEM

    PubMed Central

    Nicola, Anthony V.; Ponce de Leon, Manuel; Xu, Ruliang; Hou, Wangfang; Whitbeck, J. Charles; Krummenacher, Claude; Montgomery, Rebecca I.; Spear, Patricia G.; Eisenberg, Roselyn J.; Cohen, Gary H.

    1998-01-01

    HVEM (for herpesvirus entry mediator) is a member of the tumor necrosis factor receptor superfamily and mediates entry of many strains of herpes simplex virus (HSV) into normally nonpermissive Chinese hamster ovary (CHO) cells. We used sucrose density centrifugation to demonstrate that purified HSV-1 KOS virions bind directly to a soluble, truncated form of HVEM (HVEMt) in the absence of any other cell-associated components. Therefore, HVEM mediates HSV entry by serving as a receptor for the virus. We previously showed that soluble, truncated forms of HSV glycoprotein D (gDt) bind to HVEMt in vitro. Here we show that antibodies specific for gD, but not the other entry glycoproteins gB, gC, or the gH/gL complex, completely block HSV binding to HVEM. Thus, virion gD is the principal mediator of HSV binding to HVEM. To map sites on virion gD which are necessary for its interaction with HVEM, we preincubated virions with gD-specific monoclonal antibodies (MAbs). MAbs that recognize antigenic sites Ib and VII of gD were the only MAbs which blocked the HSV-HVEM interaction. MAbs from these two groups failed to coprecipitate HVEMt in the presence of soluble gDt, whereas the other anti-gD MAbs coprecipitated HVEMt and gDt. Previous mapping data indicated that site VII includes amino acids 11 to 19 and site Ib includes 222 to 252. The current experiments indicate that these sites contain residues important for HSV binding to HVEM. Group Ib and VII MAbs also blocked HSV entry into HVEM-expressing CHO cells. These results suggest that the mechanism of neutralization by these MAbs is via interference with the interaction between gD in the virus and HVEM on the cell. Group Ia and II MAbs failed to block HSV binding to HVEM yet still neutralized HVEM-mediated entry, suggesting that these MAbs block entry at a step other than HVEM binding. PMID:9557640

  17. Monoclonal Antibodies Directed against Conserved Epitopes on the Nucleocapsid Protein and the Major Envelope Glycoprotein of Equine Arteritis Virus

    PubMed Central

    Weiland, Emilie; Bolz, Sylvia; Weiland, Frank; Herbst, Werner; Raamsman, Martin J. B.; Rottier, Peter J. M.; De Vries, Antoine A. F.

    2000-01-01

    We recently developed a highly effective immunization procedure for the generation of monoclonal antibodies (MAbs) directed against the porcine reproductive and respiratory syndrome virus (E. Weiland, M. Wieczorek-Krohmer, D. Kohl, K. K. Conzelmann, and F. Weiland, Vet. Microbiol. 66:171–186, 1999). The same method was used to produce a panel of 16 MAbs specific for the equine arteritis virus (EAV). Ten MAbs were directed against the EAV nucleocapsid (N) protein, and five MAbs recognized the major viral envelope glycoprotein (GL). Two of the EAV GL-specific MAbs and one antibody of unknown specificity neutralized virus infectivity. A comparison of the reactivities of the MAbs with 1 U.S. and 22 newly obtained European field isolates of EAV demonstrated that all N-specific MAbs, the three nonneutralizing anti-GL MAbs, and the weakest neutralizing MAb (MAb E7/d15-c9) recognized conserved epitopes. In contrast, the two MAbs with the highest neutralization titers bound to 17 of 23 (MAb E6/A3) and 10 of 23 (MAb E7/d15-c1) of the field isolates. Ten of the virus isolates reacted with only one of these two MAbs, indicating that they recognized different epitopes. The GL-specific MAbs and the strongly neutralizing MAb of unknown specificity (MAb E6/A3) were used for the selection of neutralization-resistant (NR) virus variants. The observation that the E6/A3-specific NR virus variants were neutralized by MAb E7/d15-c1 and that MAb E6/A3 blocked the infectivity of the E7/d15-c1-specific NR escape mutant confirmed that these antibodies reacted with distinct antigenic sites. Immunoelectron microscopy revealed for the first time that the antigenic determinants recognized by the anti-GL MAbs were localized on the virion surface. Surprisingly, although the immunofluorescence signal obtained with the neutralizing antibodies was relatively weak, they mediated binding of about three times as much gold granules to the viral envelope than the nonneutralizing anti-GL MAbs. PMID

  18. Identification of the V1 region as a linear neutralizing epitope of the simian immunodeficiency virus SIVmac envelope glycoprotein.

    PubMed Central

    Jurkiewicz, E; Hunsmann, G; Schäffner, J; Nisslein, T; Lüke, W; Petry, H

    1997-01-01

    The sequence variability of viral structure polypeptides has been associated with immune escape mechanisms. The V1 region of simian immunodeficiency virus (SIV) is a highly variable region of the SIVmac env gene. Here, we describe the V1 region as a linear neutralizing epitope. V1 region-specific neutralizing antibodies (NAb) were first demonstrated in a rabbit infected with a recombinant vaccinia virus carrying the env gene of human immunodeficiency virus type 2 strain ben (HIV-2ben). Since we detected in this animal V1 region-specific NAb that were able to neutralize not only human immunodeficiency virus type 2 but also SIVmac32H, we investigated whether a similar immune response is evoked in macaques (Macaca mulatta) either infected with SIVmac or immunized with the external glycoprotein (gp130) of the same virus. Distinctly lower NAb titers were found in the SIVmac-infected animals than in the gp130-immunized macaques. Since the NAb titers in both groups were high enough for competition experiments, we used five overlapping peptides encompassing the whole V1 region for a detailed identification of the epitope. In each of the 12 macaques investigated, we detected a high level of NAb reacting with at least one peptide located in the central part of the V1 region. The relatively high degree of divergence, especially within the central part of the V1 region, which characterized the evolution of the retroviral sequences from the original inoculum in the infected macaques suggests the development of escape mutants. Furthermore, 3 of 12 animals developed NAb directed against the amino-terminal end of the V1 region epitope. Sequence analysis, however, revealed relatively low levels of genetic drift and genetic variability within this part of the V1 region. The induction of V1 env-specific NAb not only in gp130-immunized macaques but also in SIVmac-infected animals in combination with the increased genetic variability of this region in vivo indicates a marked biological

  19. Evaluation of lumpy skin disease virus, a capripoxvirus, as a replication-deficient vaccine vector.

    PubMed

    Aspden, Kate; Passmore, Jo-Ann; Tiedt, Friedrich; Williamson, Anna-Lise

    2003-08-01

    Lumpy skin disease virus (LSDV), a capripoxvirus with a host range limited to ruminants, was evaluated as a replication-deficient vaccine vector for use in non-ruminant hosts. By using the rabies virus glycoprotein (RG) as a model antigen, it was demonstrated that recombinant LSDV encoding the rabies glycoprotein (rLSDV-RG) was able to express RG in both permissive (ruminant) and non-permissive (non-ruminant) cells. The recombinant LSDV, however, replicated to maturity only in permissive but not in non-permissive cells. Recombinant LSDV-RG was assessed for its ability to generate immunity against RG in non-ruminant hosts (rabbits and mice). Rabbits inoculated with rLSDV-RG produced rabies virus (RV) neutralizing antibodies at levels twofold higher than those reported by the WHO to be protective. BALB/c mice immunized with rLSDV-RG elicited levels of RV-specific cellular immunity (T-cell proliferation) comparable with those of mice immunized with a commercial inactivated rabies vaccine (Verorab; Pasteur Merieux). Most importantly, mice immunized with rLSDV-RG were protected from an aggressive intracranial rabies virus challenge. PMID:12867628

  20. Ebola virus disease in nonendemic countries.

    PubMed

    Wong, Samson Sai-Yin; Wong, Sally Cheuk-Ying

    2015-05-01

    The 2014 West African outbreak of Ebola virus disease was unprecedented in its scale and has resulted in transmissions outside endemic countries. Clinicians in nonendemic countries will most likely face the disease in returning travelers, either among healthcare workers, expatriates, or visiting friends and relatives. Clinical suspicion for the disease must be heightened for travelers or contacts presenting with compatible clinical syndromes, and strict infection control measures must be promptly implemented to minimize the risk of secondary transmission within healthcare settings or in the community. We present a concise review on human filoviral disease with an emphasis on issues that are pertinent to clinicians practicing in nonendemic countries. PMID:25882189

  1. Newcastle disease virus vaccine potency determination

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Potency of inactivated Newcastle disease virus (NDV) vaccines is determined using vaccination and challenge. If the minimum killed viral antigen necessary for clinical protection can be determined, vaccines meeting or exceeding this dose might be considered of adequate potency. In these studies, c...

  2. Newcastle disease virus past, present and future

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Newcastle disease viruses (NDV) are endemic in many countries around the world and have caused outbreaks in most countries since it was identified in 1926. Many countries vaccinate poultry to prevent economic losses from sickness and death. The majority of vaccines administered are formulated from N...

  3. Repertoire of Epitopes Recognized by Serum IgG from Humans Vaccinated with Herpes Simplex Virus 2 Glycoprotein D

    PubMed Central

    Huang, Zhen-Yu; Cairns, Tina M.; Gallagher, John R.; Lou, Huan; Ponce-de-Leon, Manuel; Belshe, Robert B.; Eisenberg, Roselyn J.; Cohen, Gary H.

    2014-01-01

    ABSTRACT The results of a clinical trial of a subunit vaccine against genital herpes were recently reported (R. B. Belshe, P. A. Leone, D. I. Bernstein, A. Wald, M. J. Levin, J. T. Stapleton, I. Gorfinkel, R. L. Morrow, M. G. Ewell, A. Stokes-Riner, G. Dubin, T. C. Heineman, J. M. Schulte, C. D. Deal, N. Engl. J. Med. 366:34–43, 2012, doi:10.1056/NEJMoa1103151). The vaccine consisted of a soluble form of herpes simplex virus 2 (HSV-2) glycoprotein D (gD2) with adjuvant. The goal of the current study was to examine the composition of the humoral response to gD2 within a selected subset of vaccinated individuals. Serum samples from 30 vaccine recipients were selected based upon relative enzyme-linked immunosorbent assay (ELISA) titers against gD2; 10 samples had high titers, 10 had medium titers, and the remaining 10 had low ELISA titers. We employed a novel, biosensor-based monoclonal antibody (MAb)-blocking assay to determine whether gD2 vaccination elicited IgG responses against epitopes overlapping those of well-characterized MAbs. Importantly, IgGs from the majority of gD2-immunized subjects competed for gD binding with four antigenically distinct virus-neutralizing MAbs (MC2, MC5, MC23, and DL11). Screening of patient IgGs against overlapping peptides spanning the gD2 ectodomain revealed that about half of the samples contained antibodies against linear epitopes within the N and C termini of gD2. We found that the virus-neutralizing abilities of the 10 most potent samples correlated with overall gD-binding activity and to an even greater extent with the combined content of IgGs against the epitopes of MAbs MC2, MC5, MC23, and DL11. This suggests that optimal virus-neutralizing activity is achieved by strong and balanced responses to the four major discontinuous neutralizing epitopes of gD2. IMPORTANCE Several herpes simplex virus 2 (HSV-2) subunit vaccine studies have been conducted in human subjects using a recombinant form of HSV-2 glycoprotein D (gD2

  4. An Open Receptor-Binding Cavity of Hemagglutinin-Esterase-Fusion Glycoprotein from Newly-Identified Influenza D Virus: Basis for Its Broad Cell Tropism

    PubMed Central

    Song, Hao; Qi, Jianxun; Khedri, Zahra; Diaz, Sandra; Yu, Hai; Chen, Xi; Varki, Ajit; Shi, Yi; Gao, George F.

    2016-01-01

    Influenza viruses cause seasonal flu each year and pandemics or epidemic sporadically, posing a major threat to public health. Recently, a new influenza D virus (IDV) was isolated from pigs and cattle. Here, we reveal that the IDV utilizes 9-O-acetylated sialic acids as its receptor for virus entry. Then, we determined the crystal structures of hemagglutinin-esterase-fusion glycoprotein (HEF) of IDV both in its free form and in complex with the receptor and enzymatic substrate analogs. The IDV HEF shows an extremely similar structural fold as the human-infecting influenza C virus (ICV) HEF. However, IDV HEF has an open receptor-binding cavity to accommodate diverse extended glycan moieties. This structural difference provides an explanation for the phenomenon that the IDV has a broad cell tropism. As IDV HEF is structurally and functionally similar to ICV HEF, our findings highlight the potential threat of the virus to public health. PMID:26816272

  5. An Open Receptor-Binding Cavity of Hemagglutinin-Esterase-Fusion Glycoprotein from Newly-Identified Influenza D Virus: Basis for Its Broad Cell Tropism.

    PubMed

    Song, Hao; Qi, Jianxun; Khedri, Zahra; Diaz, Sandra; Yu, Hai; Chen, Xi; Varki, Ajit; Shi, Yi; Gao, George F

    2016-01-01

    Influenza viruses cause seasonal flu each year and pandemics or epidemic sporadically, posing a major threat to public health. Recently, a new influenza D virus (IDV) was isolated from pigs and cattle. Here, we reveal that the IDV utilizes 9-O-acetylated sialic acids as its receptor for virus entry. Then, we determined the crystal structures of hemagglutinin-esterase-fusion glycoprotein (HEF) of IDV both in its free form and in complex with the receptor and enzymatic substrate analogs. The IDV HEF shows an extremely similar structural fold as the human-infecting influenza C virus (ICV) HEF. However, IDV HEF has an open receptor-binding cavity to accommodate diverse extended glycan moieties. This structural difference provides an explanation for the phenomenon that the IDV has a broad cell tropism. As IDV HEF is structurally and functionally similar to ICV HEF, our findings highlight the potential threat of the virus to public health. PMID:26816272

  6. Chimeric antigen receptor (CAR)-engineered T cells redirected against hepatitis C virus (HCV) E2 glycoprotein

    PubMed Central

    Sautto, Giuseppe A; Wisskirchen, Karin; Clementi, Nicola; Castelli, Matteo; Diotti, Roberta A; Graf, Julia; Clementi, Massimo; Burioni, Roberto; Protzer, Ulrike; Mancini, Nicasio

    2016-01-01

    Objective The recent availability of novel antiviral drugs has raised new hope for a more effective treatment of hepatitis C virus (HCV) infection and its severe sequelae. However, in the case of non-responding or relapsing patients, alternative strategies are needed. To this end we have used chimeric antigen receptors (CARs), a very promising approach recently used in several clinical trials to redirect primary human T cells against different tumours. In particular, we designed the first CARs against HCV targeting the HCV/E2 glycoprotein (HCV/E2). Design Anti-HCV/E2 CARs were composed of single-chain variable fragments (scFvs) obtained from a broadly cross-reactive and cross-neutralising human monoclonal antibody (mAb), e137, fused to the intracellular signalling motif of the costimulatory CD28 molecule and the CD3ζ domain. Activity of CAR-grafted T cells was evaluated in vitro against HCV/E2-transfected cells as well as hepatocytes infected with cell culture-derived HCV (HCVcc). Results In this proof-of-concept study, retrovirus-transduced human T cells expressing anti-HCV/E2 CARs were endowed with specific antigen recognition accompanied by degranulation and secretion of proinflammatory and antiviral cytokines, such as interferon γ, interleukin 2 and tumour necrosis factor α. Moreover, CAR-grafted T cells were capable of lysing target cells of both hepatic and non-hepatic origin expressing on their surface the HCV/E2 glycoproteins of the most clinically relevant genotypes, including 1a, 1b, 2a, 3a, 4 and 5. Finally, and more importantly, they were capable of lysing HCVcc-infected hepatocytes. Conclusions Clearance of HCV-infected cells is a major therapeutic goal in chronic HCV infection, and adoptive transfer of anti-HCV/E2 CARs-grafted T cells represents a promising new therapeutic tool. PMID:25661083

  7. Self-assembled or mixed peptide amphiphile micelles from Herpes simplex virus glycoproteins as potential immunomodulatory treatment.

    PubMed

    Accardo, Antonella; Vitiello, Mariateresa; Tesauro, Diego; Galdiero, Marilena; Finamore, Emiliana; Martora, Francesca; Mansi, Rosalba; Ringhieri, Paola; Morelli, Giancarlo

    2014-01-01

    The use of micelle aggregates formed from peptide amphiphiles (PAs) as potential synthetic self-adjuvant vaccines to treat Herpes simplex virus (HSV) infection are reported here. The PAs were based on epitopes gB409-505 and gD301-309, selected from HSV envelope glycoprotein B (gB) and glycoprotein D (gD), that had their N-terminus modified with hydrophobic moieties containing two C18 hydrocarbon chains. Pure and mixed micelles of gB and/or gD peptide epitopes were easily prepared after starting with the synthesis of corresponding PAs by solid phase methods. Structural characterization of the aggregates confirmed that they were sufficiently stable and compatible with in vivo use: critical micelle concentration values around 4.0 ⋅ 10(-7) mol ⋅ Kg(-1); hydrodynamic radii (RH) between 50-80 nm, and a zeta potential (ζ) around - 40 mV were found for all aggregates. The in vitro results indicate that both peptide epitopes and micelles, at 10 μM, triggered U937 and RAW 264.7 cells to release appreciable levels of cytokines. In particular, interleukin (IL)-23-, IL-6-, IL-8- or macrophage inflammatory protein (MIP)-2-, and tumor necrosis factor (TNF)-α-release increased considerably when cells were treated with the gB-micelles or gD-micelles compared with the production of the same cytokines when the stimulus was the single gB or gD peptide. PMID:24855352

  8. Complete nucleotide sequence of the gene for the specific glycoprotein (gp55) of Friend spleen focus-forming virus.

    PubMed Central

    Amanuma, H; Katori, A; Obata, M; Sagata, N; Ikawa, Y

    1983-01-01

    The complete nucleotide sequence of the gene for the specific glycoprotein (gp55) of the polycythemic strain of Friend spleen focus-forming virus (SFFV) was derived from the cloned SFFV DNA intermediate. The gp55 gene is present within 1.4 kilobases of the 5' side of the 3'long terminal repeat sequence. The open reading frame predicts the primary translation product has a total of 409 amino acids with a Mr of 44,752. Comparisons of the deduced amino acid sequence of gp55 with those of the envelope (env) gene products of murine leukemia viruses (MuLVs) revealed that gp55 is composed of three distinct regions. The amino-terminal 80% of the molecule has a high degree of sequence homology with the amino-terminal portion of the gp70 of the Moloney mink cell focus-forming virus (BALB/Mo-MCFV). This portion of the BALB/Mo-MCFV gp70 is known to be coded for by the acquired xenotropic env-like sequence. The sequence of the following 66 amino acids of gp55 is highly homologous to that of the middle portion of