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Sample records for rabies virus glycoprotein

  1. [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.

  2. 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

  3. 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.

  4. 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

  5. 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

  6. 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.

  7. 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

  8. 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

  9. 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

  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. 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.

  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

    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.

  14. 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

  15. 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

  16. 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.

  17. 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.

  18. 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.

  19. High level expression of surface glycoprotein of rabies virus in tobacco leaves and its immunoprotective activity in mice.

    PubMed

    Ashraf, Shadma; Singh, P K; Yadav, Dinesh K; Shahnawaz, Md; Mishra, Satish; Sawant, Samir V; Tuli, Rakesh

    2005-09-22

    A synthetic gene coding for the surface glycoprotein (G protein) of rabies virus was strategically designed to achieve high-level expression in transgenic plants. The native signal peptide was replaced by that of the pathogenesis related protein, PR-S of Nicotiana tabacum. An endoplasmic reticulum retention signal was included at C-terminus of the G protein. Tobacco plants were genetically engineered by nuclear transformation. Selected transgenic lines expressed the chimeric G protein at 0.38% of the total soluble leaf protein. Mice immunized intraperitoneally with the G protein purified from tobacco leaf microsomal fraction elicited high level of immune response as compared to the inactivated commercial viral vaccine. The plant-derived G protein induced complete protective immunity in mice against intracerebral lethal challenge with live rabies virus. The results establish that plants can provide a safe and effective production system for the expression of immunoprotective rabies virus surface protein. PMID:16038998

  20. 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

  1. Development and evaluation of a recombinant-glycoprotein-based latex agglutination test for rabies virus antibody assessment.

    PubMed

    Jemima, Ebenezer Angel; Manoharan, Seeralan; Kumanan, Kathaperumal

    2014-08-01

    The measurement of neutralizing antibodies induced by the glycoprotein of rabies virus is indispensable for assessing the level of neutralizing antibodies in animals or humans. A rapid fluorescent focus inhibition test (RFFIT) has been approved by WHO and is the most widely used method to measure the virus-neutralizing antibody content in serum, but a rapid test system would be of great value to screen large numbers of serum samples. To develop and evaluate a latex agglutination test (LAT) for measuring rabies virus antibodies, a recombinant glycoprotein was expressed in an insect cell system and purified, and the protein was coated onto latex beads at concentrations of 0.1, 0.25, 0.5, 0.75, and 1 mg/ml to find out the optimal concentration for coating latex beads. It was found that 0.5 mg/ml of recombinant protein was optimal for coating latex beads, and this concentration was used to sensitize the latex beads for screening of dog serum samples. Grading of LAT results was done with standard reference serum with known antibody titers. A total of 228 serum samples were tested, out of which 145 samples were positive by both RFFIT and LAT, and the specificity was found to be 100 %. In RFFIT, 151 samples were positive, the sensitivity was found to be 96.03 %, and the accuracy/concordance was found to be 97.39 %. A rapid field test-a latex agglutination test (LAT)-was developed and evaluated for rabies virus antibody assessment using recombinant glycoprotein of rabies virus expressed in an insect cell system.

  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.

  3. 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

  4. Phylogenetic analysis of Indian rabies virus isolates targeting the complete glycoprotein gene.

    PubMed

    Cherian, Susan; Singh, Rajendra; Singh, K P; Manjunatha Reddy, G B; Anjaneya; Ravi Kumar, G V P P S; Sumithra, T G; Singh, R P

    2015-12-01

    Rabies a fatal viral zoonosis is endemic in India. There is no report on phylogenetic study of Indian rabies virus isolates based on the complete G gene. In the present study, a total of 25 rabies positive brain samples collected during 2001-2014 from North India (UP, MP, Delhi, Rajasthan), South India (Kerala and Karnataka) and Gujarat states belonging to six different host species were subjected to G gene amplification by RT-PCR as three overlapping fragments of 881 bp, 991 bp and 618 bp. Phylogenetic analysis revealed that all Indian rabies virus isolates are genetically closely related with Arctic-like 1a lineage viruses. However, two distinct clusters were identified namely, India South and India North. All the Indian rabies isolates had 95.5-100% homology related to geography, but not to host species. Deduced amino acids on comparison revealed two amino acid changes, aa 356 in ECTO; N→K and aa 458; M→I, which were found to distinguish between the India South and India North isolates.

  5. Phylogenetic analysis of Indian rabies virus isolates targeting the complete glycoprotein gene.

    PubMed

    Cherian, Susan; Singh, Rajendra; Singh, K P; Manjunatha Reddy, G B; Anjaneya; Ravi Kumar, G V P P S; Sumithra, T G; Singh, R P

    2015-12-01

    Rabies a fatal viral zoonosis is endemic in India. There is no report on phylogenetic study of Indian rabies virus isolates based on the complete G gene. In the present study, a total of 25 rabies positive brain samples collected during 2001-2014 from North India (UP, MP, Delhi, Rajasthan), South India (Kerala and Karnataka) and Gujarat states belonging to six different host species were subjected to G gene amplification by RT-PCR as three overlapping fragments of 881 bp, 991 bp and 618 bp. Phylogenetic analysis revealed that all Indian rabies virus isolates are genetically closely related with Arctic-like 1a lineage viruses. However, two distinct clusters were identified namely, India South and India North. All the Indian rabies isolates had 95.5-100% homology related to geography, but not to host species. Deduced amino acids on comparison revealed two amino acid changes, aa 356 in ECTO; N→K and aa 458; M→I, which were found to distinguish between the India South and India North isolates. PMID:26427850

  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. The influence of the type of immunosorbent on rabies antibody EIA; advantages of purified glycoprotein over whole virus.

    PubMed

    Perrin, P; Versmisse, P; Delagneau, J F; Lucas, G; Rollin, P E; Sureau, P

    1986-04-01

    Two types of in vitro assay (enzyme immunoassay and sero-neutralization test) for the titration of rabies antibodies were used to assay sera from mice and humans immunized with cell culture vaccines or neural tissue vaccines. Enzyme immunoassays (EIA) were performed in plates sensitized with whole virus, purified glycoprotein or purified nucleocapsid. Neutralizing antibody titres were determined by the rapid fluorescent focus inhibition test (REFIT) and by an in vitro seroneutralization test including a rapid enzyme immunotitration of intracellular antigens (REITICA). The results obtained with sera of immunized mice and humans showed that (1) cell culture vaccines mainly induced the synthesis of antiglycoprotein neutralizing antibodies; and (2) neural tissue vaccines induced a high synthesis of antinucleocapsid non-neutralizing antibodies and a more or less important synthesis of antiglycoprotein antibodies depending on the origin of the tissue used for their preparation. Consequently, it was emphasized that when using EIA, the antibody titration must be run in glycoprotein-coated plates rather than in whole virus-coated plates to appreciate correctly the immunizing potency of a rabies vaccine, especially neural tissue vaccine.

  8. Rabies virus glycoprotein is an important determinant for the induction of innate immune responses and the pathogenic mechanisms.

    PubMed

    Zhang, Guoqing; Wang, Hualei; Mahmood, Fazal; Fu, Zhen F

    2013-03-23

    Our previous studies have suggested that street and fixed rabies viruses (RABVs) induce diseases in the mouse model via different mechanisms. In the present study, attempts were made to determine if it is the glycoprotein (G) that is responsible for the observed differences in the pathogenic mechanisms. To this end, an infectious clone from fixed virus B2c was established and used as a backbone for exchange of the G from street viruses. The rate of viral replication, expression of viral proteins, and the induction of innate immune responses were compared in cells or in mice infected with each of the viruses. Furthermore, the infiltration of inflammatory cells into the CNS and the enhancement of blood-brain barrier (BBB) permeability were also compared. It was found that fixed viruses induced stronger innate immune responses (expression of chemokines, infiltration of inflammatory cells, and enhancement of BBB permeability) than street RABV or recombinant viruses expressing the G from street RABVs. Fixed viruses induce disease via an immune-mediated pathogenic mechanism while street viruses or recombinant viruses expressing the G from street RABVs induce diseases via a mechanism other than immune-mediated pathogenesis. Therefore, RABV G is an important determinant for the induction of innate immune responses and consequently the pathogenic mechanisms.

  9. [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.

  10. [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

  11. Growth of recombinant Drosophila melanogaster Schneider 2 cells producing rabies virus glycoprotein in bioreactor employing serum-free medium

    PubMed Central

    Galesi, Adriana L. L.; Aguiar, Marcelo A.; Astray, Renato M.; Augusto, Elisabeth F. P.

    2008-01-01

    Drosophila melanogaster Schneider 2 (S2) cells have been increasingly used as a suitable expression system for the production of different recombinant proteins, and the employment of bioreactors for large-scale culture is an important tool for this purpose. In this work, Drosophila S2 cells producing the rabies virus glycoprotein RVGP were cultivated in bioreactor, employing a serum-free medium, aiming an improvement in cell growth and in glycoprotein production. To overcome cell growth limitation commonly observed in stirred flasks, different experiments in bioreactor were performed, in which some system modifications were carried out to attain the desired goal. The study showed that this cell line is considerably sensitive to hydrodynamic forces, and a high cell density (about 16.0 × 106 cells mL−1) was only obtained when Pluronic F68® percentage was increased to 0.6% (w/v). Despite ammonium concentration affected RVGP production, and also cell growth, an elevated amount of the target protein was obtained, attaining 563 ng 10−7 cells. PMID:19003175

  12. Structure-function relationships of curaremimetic neurotoxin loop 2 and of a structurally similar segment of rabies virus glycoprotein in their interaction with the nicotinic acetylcholine receptor

    SciTech Connect

    Lentz, T.L. )

    1991-11-12

    Peptides corresponding to portions of curaremimetic neurotoxin loop 2 and to a structurally similar segment of rabies virus glycoprotein were synthetically modified in order to gain information on structure-function relationships of neurotoxin loop 2 interactions with the acetylcholine receptor. Binding of synthetic peptides to the acetylcholine receptor of Torpedo electric organ membranes was assessed by measuring their ability to inhibit the binding of {sup 125}I-{alpha}-bungarotoxin to the receptor. The peptides showing the highest affinity for the receptor were a peptide corresponding to the sequence of loop 2 (residues 25-44) of Ophiophagus hannah (king cobra) toxin b and the structurally similar segment of CVS rabies virus glycoprotein. These affinities were comparable to those of d-tubocurarine and suberyldicholine. These results demonstrate the importance of loop 2 in the neurotoxin interaction with the receptor. N- and C-terminal deletions of the loop 2 peptides and substitution of residues invariant or highly conserved among neurotoxins were performed in order to determine the role of individual residues in binding. Residues 25-40 are the most crucial in the interaction with the acetylcholine receptor. Since this region of the glycoprotein contains residues corresponding to all of the functionally invariant neurotoxin residues, it may interact with the acetylcholine receptor through a mechanism similar to that of the neurotoxins.

  13. Molecular genetic characterization of rabies virus glycoprotein gene sequences from rabid dogs in Bangkok and neighboring provinces in Thailand, 2013-2014.

    PubMed

    Benjathummarak, Surachet; Fa-Ngoen, Chanon; Pipattanaboon, Chonlatip; Boonha, Khwanchit; Ramasoota, Pongrama; Pitaksajjakul, Pannamthip

    2016-05-01

    Because of its association with dogs, rabies virus (RABV) is still endemic in Thailand, where it is a serious public health problem. The genetic characterization of RABV in Thailand is limited. Therefore, in this study, we investigated the molecular epidemiology and genetic diversity of RABV in the endemic area. Viral RNA from 48 brain specimens from rabid dogs, collected in Bangkok and seven neighboring provinces in 2013-2014, was extracted and sequenced. The complete rabies glycoprotein (G) gene sequences (1575 nt) were aligned, and a phylogenetic analysis was performed using the maximum-likelihood method. All of the Thai rabies virus isolates belonged to lyssavirus genotype 1 and clustered in the same lineage as isolates from South East Asia (SEA) and China. The Thai rabies virus isolates formed two distinct clades, THA-1 and THA-2. Clade THA-1 was the predominant clade and could be divided into two subclades, THA-1A and THA-1B. Clade THA-2 was closely associated with human Thai isolates collected in a previous study. The overall mean rate of evolution based on the G gene was approximately 1.56 × 10(-4) substitutions/site/year. The genetic identities among the isolates from Thailand and other SEA countries were >88.4 % at the nucleotide sequence level and 95 % at the amino acid sequence level. The deduced amino acid sequences of the G proteins of the RABV isolates were compared. A single amino acid change (N194T) in subclade THA-1A distinguished the Thai RABV isolates from other RABV isolates. Our results suggest that these Thai dog RABV isolates share a common ancestor with the RABV isolates circulating in the endemic regions of SEA countries and China. Furthermore, there were strong genetic relationship to RABV from Cambodia, Vietnam and Laos. These data extend our understanding of the relatedness and genetic variation of RABV in Thailand. PMID:26887972

  14. Molecular genetic characterization of rabies virus glycoprotein gene sequences from rabid dogs in Bangkok and neighboring provinces in Thailand, 2013-2014.

    PubMed

    Benjathummarak, Surachet; Fa-Ngoen, Chanon; Pipattanaboon, Chonlatip; Boonha, Khwanchit; Ramasoota, Pongrama; Pitaksajjakul, Pannamthip

    2016-05-01

    Because of its association with dogs, rabies virus (RABV) is still endemic in Thailand, where it is a serious public health problem. The genetic characterization of RABV in Thailand is limited. Therefore, in this study, we investigated the molecular epidemiology and genetic diversity of RABV in the endemic area. Viral RNA from 48 brain specimens from rabid dogs, collected in Bangkok and seven neighboring provinces in 2013-2014, was extracted and sequenced. The complete rabies glycoprotein (G) gene sequences (1575 nt) were aligned, and a phylogenetic analysis was performed using the maximum-likelihood method. All of the Thai rabies virus isolates belonged to lyssavirus genotype 1 and clustered in the same lineage as isolates from South East Asia (SEA) and China. The Thai rabies virus isolates formed two distinct clades, THA-1 and THA-2. Clade THA-1 was the predominant clade and could be divided into two subclades, THA-1A and THA-1B. Clade THA-2 was closely associated with human Thai isolates collected in a previous study. The overall mean rate of evolution based on the G gene was approximately 1.56 × 10(-4) substitutions/site/year. The genetic identities among the isolates from Thailand and other SEA countries were >88.4 % at the nucleotide sequence level and 95 % at the amino acid sequence level. The deduced amino acid sequences of the G proteins of the RABV isolates were compared. A single amino acid change (N194T) in subclade THA-1A distinguished the Thai RABV isolates from other RABV isolates. Our results suggest that these Thai dog RABV isolates share a common ancestor with the RABV isolates circulating in the endemic regions of SEA countries and China. Furthermore, there were strong genetic relationship to RABV from Cambodia, Vietnam and Laos. These data extend our understanding of the relatedness and genetic variation of RABV in Thailand.

  15. 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

  16. 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

  17. 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.

  18. Purification and on-column refolding of a single-chain antibody fragment against rabies virus glycoprotein expressed in Escherichia coli.

    PubMed

    Xi, Hualong; Yuan, Ruosen; Chen, Xiaoxu; Gu, Tiejun; Cheng, Yue; Li, Zhuang; Jiang, Chunlai; Kong, Wei; Wu, Yongge

    2016-10-01

    An anti-rabies virus single-chain antibody fragment of an anti-glycoprotein with the VL-linker-VH orientation, designated scFv57RN, was successfully and conveniently prepared in this study. The scFv57RN protein was mainly expressed in inclusion bodies in Escherichia coli. After washing and purification, the inclusion bodies were finally obtained with an on-column refolding procedure. Further purification by gel exclusion chromatography was performed to remove inactive multimers. About 360 mg of final product was recovered from 1 L of bacterial culture. The final product showed a high neutralizing titer of 950 IU/mg to the CVS-11 strain as measured using the rapid fluorescent focus inhibition test. Our study demonstrated a highly efficient method to mass produce scFV57RN with activity from inclusion bodies, which may be applied in the purification of other insoluble proteins.

  19. Induction of a protective immune response to rabies virus in sheep after oral immunization with transgenic maize, expressing the rabies virus glycoprotein.

    PubMed

    Loza-Rubio, Elizabeth; Rojas-Anaya, Edith; López, Juan; Olivera-Flores, María Teresa; Gómez-Lim, Miguel; Tapia-Pérez, Graciela

    2012-08-10

    The introduction of exogenous genes into plants permits the development of a new generation of biological products, i.e., edible vaccines. Cereals, especially maize, have been the systems of choice for the expression of antigenic proteins because the proteins can be expressed at high levels in the kernel and stored for prolonged periods without excessive deterioration. The utilization of plant-derived antigens for oral delivery provides an alternative strategy for the control of pathogens in animals compared to the current vaccine administration methods, such as injection. However, there is some doubt about the efficacy of these types of vaccines in polygastric animals due to the features of their digestive system. Here, we report the efficacy of an edible vaccine against rabies evaluated in sheep. Kernels containing different doses of G protein (0.5, 1, 1.5 and 2mg) were given in a single dose by the oral route. Cumulative survival was better in groups that received 2mg of G protein and for the positive control (inactivated rabies vaccine); this observation was supported by the presence of neutralizing antibodies. Animals in the control group died after challenge. The degree of protection achieved for 2mg of G protein was comparable to that conferred by a commercial vaccine. In conclusion, this is the first study in which an orally administered edible vaccine showed efficacy in a polygastric model.

  20. Design and generation of recombinant rabies virus vectors.

    PubMed

    Osakada, Fumitaka; Callaway, Edward M

    2013-08-01

    Rabies viruses, negative-strand RNA viruses, infect neurons through axon terminals and spread trans-synaptically in a retrograde direction between neurons. Rabies viruses whose glycoprotein (G) gene is deleted from the genome cannot spread across synapses. Complementation of G in trans, however, enables trans-synaptic spreading of G-deleted rabies viruses to directly connected, presynaptic neurons. Recombinant rabies viruses can encode genes of interest for labeling cells, controlling gene expression and monitoring or manipulating neural activity. Cre-dependent or bridge protein-mediated transduction and single-cell electroporation via the EnvA-TVA or EnvB-TVB (envelope glycoprotein and its specific receptor for avian sarcoma leukosis virus subgroup A or B) system allow cell type-specific or single cell-specific targeting. These rabies virus-based approaches permit the linking of connectivity to cell morphology and circuit function for particular cell types or single cells. Here we describe methods for construction of rabies viral vectors, recovery of G-deleted rabies viruses from cDNA, amplification of the viruses, pseudotyping them with EnvA or EnvB and concentration and titration of the viruses. The entire protocol takes 6-8 weeks.

  1. Synthesis and characterization of rabies virus glycoprotein-tagged amphiphilic cyclodextrins for siRNA delivery in human glioblastoma cells: in vitro analysis.

    PubMed

    Gooding, Matt; Malhotra, Meenakshi; McCarthy, David J; Godinho, Bruno M D C; Cryan, John F; Darcy, Raphael; O'Driscoll, Caitriona M

    2015-04-25

    In man brain cancer is an aggressive, malignant form of tumour, it is highly infiltrative in nature, is associated with cellular heterogeneity and affects cerebral hemispheres of the brain. Current drug therapies are inadequate and an unmet clinical need exists to develop new improved therapeutics. The ability to silence genes associated with disease progression by using short interfering RNA (siRNA) presents the potential to develop safe and effective therapies. In this work, in order to protect the siRNA from degradation, promote cell specific uptake and enhance gene silencing efficiency, a PEGylated cyclodextrin (CD)-based nanoparticle, tagged with a CNS-targeting peptide derived from the rabies virus glycoprotein (RVG) was formulated and characterized. The modified cyclodextrin derivatives were synthesized and co-formulated to form nanoparticles containing siRNA which were analysed for size, surface charge, stability, cellular uptake and gene-knockdown in brain cancer cells. The results identified an optimised co-formulation prototype at a molar ratio of 1:1.5:0.5 (cationic cyclodextrin:PEGylated cyclodextrin:RVG-tagged PEGylated cyclodextrin) with a size of 281 ± 39.72 nm, a surface charge of 26.73 ± 3 mV, with efficient cellular uptake and a 27% gene-knockdown ability. This CD-based formulation represents a potential nanocomplex for systemic delivery of siRNA targeting brain cancer.

  2. 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.

  3. Synthesis and characterization of rabies virus glycoprotein-tagged amphiphilic cyclodextrins for siRNA delivery in human glioblastoma cells: in vitro analysis.

    PubMed

    Gooding, Matt; Malhotra, Meenakshi; McCarthy, David J; Godinho, Bruno M D C; Cryan, John F; Darcy, Raphael; O'Driscoll, Caitriona M

    2015-04-25

    In man brain cancer is an aggressive, malignant form of tumour, it is highly infiltrative in nature, is associated with cellular heterogeneity and affects cerebral hemispheres of the brain. Current drug therapies are inadequate and an unmet clinical need exists to develop new improved therapeutics. The ability to silence genes associated with disease progression by using short interfering RNA (siRNA) presents the potential to develop safe and effective therapies. In this work, in order to protect the siRNA from degradation, promote cell specific uptake and enhance gene silencing efficiency, a PEGylated cyclodextrin (CD)-based nanoparticle, tagged with a CNS-targeting peptide derived from the rabies virus glycoprotein (RVG) was formulated and characterized. The modified cyclodextrin derivatives were synthesized and co-formulated to form nanoparticles containing siRNA which were analysed for size, surface charge, stability, cellular uptake and gene-knockdown in brain cancer cells. The results identified an optimised co-formulation prototype at a molar ratio of 1:1.5:0.5 (cationic cyclodextrin:PEGylated cyclodextrin:RVG-tagged PEGylated cyclodextrin) with a size of 281 ± 39.72 nm, a surface charge of 26.73 ± 3 mV, with efficient cellular uptake and a 27% gene-knockdown ability. This CD-based formulation represents a potential nanocomplex for systemic delivery of siRNA targeting brain cancer. PMID:25703259

  4. Rabies

    MedlinePlus

    ... animals that can spread the rabies virus include: Foxes Skunks Very rarely, rabies has been transmitted without ... after being exposed to animals such as bats, foxes, and skunks. They may carry rabies. Call even ...

  5. The Inability of Wild-Type Rabies Virus To Activate Dendritic Cells Is Dependent on the Glycoprotein and Correlates with Its Low Level of the De Novo-Synthesized Leader RNA

    PubMed Central

    Yang, Yang; Huang, Ying; Gnanadurai, Clement W.; Cao, Shengbo; Liu, Xueqin

    2014-01-01

    ABSTRACT Dendritic cells (DCs) are the most efficient antigen-presenting cells, playing a key role in the adaptive immune responses to viral infections. Our studies demonstrate that wild-type (wt) rabies virus (RABV) does not activate DCs. Adoptive transfer of DCs primed with wt RABV did not activate DCs, stimulate virus neutralizing antibodies (VNA), or protect recipients against challenge. However, adoptive transfer of DCs primed with laboratory-attenuated RABV resulted in DC activation, production of VNA, and protection against challenge. In vitro studies with recombinant RABV (laboratory-attenuated RABV expressing the glycoprotein or the phosphoprotein from wt RABV) demonstrate that DC activation is dependent on the glycoprotein and involves the IPS-1 pathway. Furthermore, binding to and entry into DCs by wt RABV is severely blocked, and the copy number of de novo-synthesized leader RNA was two logs lower in DCs infected with the wt than in DCs treated with laboratory-attenuated RABV. However, transient transfection of DCs with synthesized leader RNA from either wt or attenuated RABV is capable of activating DCs in a dose-dependent manner. Thus, the inability of wt RABV to activate DCs correlates with its low level of the de novo-synthesized leader RNA. IMPORTANCE Rabies remains a public health threat, with more than 55,000 fatalities each year around the world. Since DCs play a key role in the adaptive immune responses to viral infections, we investigated the ability of rabies virus (RABV) to activate DCs. It was found that the adoptive transfer of DCs primed with wt RABV did not activate DCs, stimulate VNA, or protect mice against lethal challenge. However, laboratory-attenuated RABV mediates the activation of DCs via the IPS-1 pathway and is glycoprotein dependent. We further show that wt RABV evades DC-mediated immune activation by inefficient binding/entry into DCs and as a result of a reduced level of de novo-synthesized leader RNA. These findings may

  6. Transplacental transmission of rabies virus from a naturally infected skunk.

    PubMed

    Howard, D R

    1981-04-01

    A female skunk (Mephitis mephitis) was submitted to the Veterinary Diagnostic Laboratory for routine rabies diagnosis. Rabies infection was confirmed by fluorescent rabies antibody examination on brain tissue. Additional tissues, including uterus, ovaries, and 6 embryos, were collected to study rabies pathogenesis. The fluorescent rabies antibody examination showed rabies virus antigen in 1 embryo, the uterus, and ovaries.

  7. Epidemic and maintenance of rabies in Chinese ferret badgers (Melogale moschata) indicated by epidemiology and the molecular signatures of rabies viruses.

    PubMed

    Zhang, Shoufeng; Liu, Ye; Hou, Yanli; Zhao, Jinghui; Zhang, Fei; Wang, Ying; Hu, Rongliang

    2013-06-01

    An epidemic of Chinese ferret badger-associated human rabies was investigated in Wuyuan county, Jiangxi province and rabies viruses isolates from ferret badgers in different districts in Jiangxi and Zhejiang provinces were sequenced with their nucleotides and amino acids and aligned for epidemiological analysis. The results showed that the human rabies in Wuyuan are only associated with ferret badger bites; the rabies virus can be isolated in a high percentage of ferret badgers in the epidemic areas in Jiangxi and Zhejiang provinces; the isolates share the same molecular features in nucleotides and have characteristic amino acid signatures, i.e., 2 sites in the nucleoprotein and 3 sites in the glycoprotein, that are distinct from virus isolates from dogs in the same region. We conclude that rabies in Chinese ferret badgers has formed an independent transmission cycle and ferret badgers may serve as another important rabies reservoir independent of dog rabies in China.

  8. Rabies virus neuritic paralysis: immunopathogenesis of nonfatal paralytic rabies.

    PubMed Central

    Weiland, F; Cox, J H; Meyer, S; Dahme, E; Reddehase, M J

    1992-01-01

    Two pathogenetically distinct disease manifestations are distinguished in a murine model of primary rabies virus infection with the Evelyn-Rokitnicky-Abelseth strain, rabies virus neuritic paralysis (RVNP) and fatal encephalopathogenic rabies. RVNP develops with high incidence in immunocompetent mice after intraplantar infection as a flaccid paralysis restricted to the infected limb. The histopathologic correlate of this monoplegia is a degeneration of the myelinated motor neurons of the peripheral nerve involved. While, in this model, fatal encephalopathogenic rabies develops only after depletion of the CD4 subset of T lymphocytes and without contribution of the CD8 subset, RVNP is identified as an immunopathological process in which both the CD4 and CD8 subsets of T lymphocytes are critically implicated. Images PMID:1629964

  9. A liquid-phase blocking ELISA for the detection of antibodies to rabies virus.

    PubMed

    Esterhuysen, J J; Prehaud, C; Thomson, G R

    1995-01-01

    A liquid-phase blocking ELISA was adapted to the detection and titration of antibodies to principally the nucleoprotein of rabies virus. Sera from animals that had either been vaccinated against rabies or inoculated with street rabies viruses, as well as sera from animals that had no recorded contact with rabies, were tested. These included sera from people, cattle, sheep, goats, dogs, laboratory mice, rabbits, yellow mongooses, wild dogs and lions. Where possible, the results were compared with those obtained with a commercial kit incorporating an indirect ELISA that measures antibody to the rabies glycoprotein. There was a high correlation (r = 0.79) between the two tests. The blocking ELISA provides a single test suitable for the rapid detection of antibodies against rabies virus in the sera of any animal species and for that reason is particularly apt for epidemiological investigations in regions where species diversity is important, as in southern Africa.

  10. 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.

  11. Rabies virus quasispecies: implications for pathogenesis.

    PubMed

    Morimoto, K; Hooper, D C; Carbaugh, H; Fu, Z F; Koprowski, H; Dietzschold, B

    1998-03-17

    Passage of the mouse-adapted rabies virus strain CVS-24 (where CVS is challenge virus standard) in BHK cells results in the rapid selection of a dominant variant designated CVS-B2c that differs genotypically and phenotypically from the dominant variant CVS-N2c present in mouse-brain- or neuroblastoma-cell-passaged CVS-24. The glycoprotein of CVS-B2c has 10 amino acid substitutions compared with that of CVS-N2c. Because CVS-B2c can be reproducibly selected in BHK cells, it is likely to be a conserved minor subpopulation of CVS-24. CVS-N2c is more neurotropic in vitro and in vivo than CVS-B2c, which replicates more readily in nonneuronal cells in vitro and in vivo. These characteristics appear to be relevant to the pathogenicity of the two variants. CVS-N2c is more pathogenic for adult mice than CVS-B2c. In contrast, CVS-B2c is more pathogenic for neonatal mice. These differences in pathogenicity are reflected in the selection pattern when mixtures of CVS-N2c and CVS-B2c were used to infect neonatal and adult mice. Although CVS-N2c was highly selected in adult mice, no selection for either variant was seen in neonates, suggesting that certain aspects of development, such as maturation of the nervous and immune systems, may contribute to the selection process. We speculate that the existence of different variants within a rabies virus strain may facilitate the virus in overcoming barriers to its spread, both within the host and between species.

  12. Molecular characterization of Korean rabies virus isolates

    PubMed Central

    Park, Young-Nam; Hong, Gyeong-Soo; Kang, Hee-Kyung; Oh, Yoon-I; Cho, Soo-Dong; Song, Jae-Young

    2011-01-01

    The nucleoprotein (N) and glycoprotein (G) of 11 Korean rabies virus (RABV) isolates collected from animals diagnosed with rabies between 2008 and 2009 were subjected to molecular and phylogenetic analyses. Six isolates originated from domestic animals (cattle and dogs) and five were obtained from wild free-ranging raccoon dogs. The similarities in the nucleotide sequences of the N gene among all Korean isolates ranged from 98.1 to 99.8%, while those of the G gene ranged from 97.9 to 99.3%. Based on the nucleotide analysis of the N and G genes, the Korean RABV isolates were confirmed as genotype I of Lyssavirus and classified into four distinct subgroups with high similarity. Phylogenetic analysis showed that the Korean isolates were most closely related to the non-Korean NeiMeng1025B and 857r strains, which were isolated from rabid raccoon dogs in Eastern China and Russia, respectively. These findings suggest that the Korean RABV isolates originated from a rabid raccoon dog in Northeastern Asia. Genetic analysis of the Korean RABV isolates revealed no substitutions at several antigenic sites, indicating that the isolates circulating in Korea may be pathogenic in several hosts. PMID:21368564

  13. Rabies.

    PubMed

    Burnett, Nark

    2013-01-01

    Rabies has been a scourge of mankind since antiquity. The name itself, ?rabies? is derived from the ancient Sanskrit rabhas meaning ?to do violence? and has been found described in medical writings several thousand years old. The rabies virus is an RNA virus of the family Rhabdoviridae (Greek for ?rod-shaped virus?), genus Lyssavirus (Lyssa being the Greek God of frenzy and rage). Rabies infections have a worldwide spread, with only a few, mostly island nations laying claim to being ?rabies free.? PMID:24049000

  14. Inactivation of rabies virus by hydrogen peroxide.

    PubMed

    Abd-Elghaffar, Asmaa A; Ali, Amal E; Boseila, Abeer A; Amin, Magdy A

    2016-02-01

    Development of safe and protective vaccines against infectious pathogens remains a challenge. Inactivation of rabies virus is a critical step in the production of vaccines and other research reagents. Beta-propiolactone (βPL); the currently used inactivating agent for rabies virus is expensive and proved to be carcinogenic in animals. This study aimed to investigate the ability of hydrogen peroxide (H2O2) to irreversibly inactivate rabies virus without affecting its antigenicity and immunogenicity in pursuit of finding safe, effective and inexpensive alternative inactivating agents. H2O2 3% rapidly inactivated a Vero cell adapted fixed rabies virus strain designated as FRV/K within 2h of exposure without affecting its antigenicity or immunogenicity. No residual infectious virus was detected and the H2O2-inactivated vaccine proved to be safe and effective when compared with the same virus harvest inactivated with the classical inactivating agent βPL. Mice immunized with H2O2-inactivated rabies virus produced sufficient level of antibodies and were protected when challenged with lethal CVS virus. These findings reinforce the idea that H2O2 can replace βPL as inactivating agent for rabies virus to reduce time and cost of inactivation process. PMID:26731189

  15. Inactivation of rabies virus by hydrogen peroxide.

    PubMed

    Abd-Elghaffar, Asmaa A; Ali, Amal E; Boseila, Abeer A; Amin, Magdy A

    2016-02-01

    Development of safe and protective vaccines against infectious pathogens remains a challenge. Inactivation of rabies virus is a critical step in the production of vaccines and other research reagents. Beta-propiolactone (βPL); the currently used inactivating agent for rabies virus is expensive and proved to be carcinogenic in animals. This study aimed to investigate the ability of hydrogen peroxide (H2O2) to irreversibly inactivate rabies virus without affecting its antigenicity and immunogenicity in pursuit of finding safe, effective and inexpensive alternative inactivating agents. H2O2 3% rapidly inactivated a Vero cell adapted fixed rabies virus strain designated as FRV/K within 2h of exposure without affecting its antigenicity or immunogenicity. No residual infectious virus was detected and the H2O2-inactivated vaccine proved to be safe and effective when compared with the same virus harvest inactivated with the classical inactivating agent βPL. Mice immunized with H2O2-inactivated rabies virus produced sufficient level of antibodies and were protected when challenged with lethal CVS virus. These findings reinforce the idea that H2O2 can replace βPL as inactivating agent for rabies virus to reduce time and cost of inactivation process.

  16. Two African viruses serologically and morphologically related to rabies virus.

    PubMed

    Shope, R E; Murphy, F A; Harrison, A K; Causey, O R; Kemp, G E; Simpson, D I; Moore, D L

    1970-11-01

    Lagos bat virus and an isolate from shrews (IbAn 27377), both from Nigeria, were found to be bullet-shaped and to mature intracytoplasmically in association with a distinct matrix. They were related to, but readily distinguishable from, rabies virus and each other by complement fixation and neutralization tests. The three viruses, including rabies, form a subgrouping within the rhabdoviruses. PMID:5530013

  17. Rabies virus isolates of India - simultaneous existence of two distinct evolutionary lineages.

    PubMed

    Reddy, R V Chandrasekhar; Mohana Subramanian, B; Surendra, K S N L; Babu, R P Aravindh; Rana, S K; Manjari, K Sunitha; Srinivasan, V A

    2014-10-01

    Rabies is a fatal viral disease of serious public health implication. The disease is enzootic in India. In the present study, thirty six rabies virus isolates were obtained from terrestrial mammals of India during 2002-2012. Ecto-domain coding region of the glycoprotein gene from all the isolates were sequenced and the phylogenetic analysis was performed in relation to the global rabies and rabies related virus isolates. The Indian isolates grouped into two distinctly separate lineages with majority of the Indian isolates in Arctic like 1 lineage and the remaining isolates in sub-continental lineage. Isolates of the two distinct lineages were identified simultaneously from the same geographical region. Time scaled phylogenetic tree indicated that the sub-continental lineage of the virus is one of the earliest clade of rabies virus that diverged from bat rabies virus. On the contrary, the Arctic-like 1 lineage of India appeared to be a more recent divergence event. The amino acid sequence comparison revealed that all the major antigenic sites were almost conserved among the Indian isolates whereas few amino acid variations could be identified around site IIa, minor site I and IV. The dN/dS study based on G ecto-domain is in support of the earlier reports of strong purifying selection. In conclusion, it is evident that the Indian rabies virus isolates are of two major distinct lineages with distant phylogenetic and evolutionary relationship. PMID:25077994

  18. Chronic rabies virus infection of cell cultures.

    PubMed

    Wiktor, T J; Clark, H F

    1972-12-01

    Exposure of both mammalian and reptilian cells in tissue culture to different strains of fixed rabies virus resulted in a carrier type of infection. No cytopathic effect was observed in either type of culture; infected cultures could be maintained by cell transfer for unlimited numbers of passages. A consistent pattern of cyclically rising and falling levels of viral infection was observed by fluorescent-antibody staining techniques and by titration of released infectious virus. Resistance to super-infection by vesicular stomatis virus and the production of an interferon-like substance by infected cells indicated that the maintenance of a carrier type of infection may be interferon-mediated. The degree of susceptibility of rabies-infected cells to immunolysis by antirabies antibody in the presence of complement was found to be correlated with the amount of virus maturation occurring by budding through the cell membrane and not with the presence of immunofluorescent antigen in the cytoplasm of infected cells.

  19. [Viruses and bats: rabies and Lyssavirus].

    PubMed

    Tordo, N; Marianneau, M Ph

    2009-01-01

    Recent emerging zoonoses (hemorrhagic fevers due to Ebola or Marburg virus, encephalitis due to Nipah virus, severe acute respiratory syndrome due to SRAS virus...) outline the potential of bats as vectors for transmission of infectious disease to humans. Such a potential is already known for rabies encephalitis since seven out of the eight genotypes of Lyssavirus are transmitted by bats. In addition, phylogenetic reconstructions indicate that Lyssavirus have evolved in chiropters before their emergence in carnivores. Nevertheless, carnivores remain the most critical vectors for public health, in particular dogs that are originating 55.000 rabies deaths per year, essentially in developing countries. Rabies control in carnivores by parenteral (dog) or oral (wild carnivores) vaccination is efficacious and campaigns start to be more widely applied. On the other hand, rabies control in bat still remains non realistic, particularly as the pathogenicity of bat Lyssavirus for bats is still under debate, suggesting that a "diplomatic relationship" between partners would have arisen from a long term cohabitation. While comparing the interactions that humans and bats establish with Lyssavirus, scientists try to understand the molecular basis ofpathogenicity in man, a indispensable prerequisite to identify antiviral targets in a perspective of therapy. PMID:19718950

  20. Emergence of a sylvatic enzootic formosan ferret badger-associated rabies in Taiwan and the geographical separation of two phylogenetic groups of rabies viruses.

    PubMed

    Tsai, K J; Hsu, W C; Chuang, W C; Chang, J C; Tu, Y C; Tsai, H J; Liu, H F; Wang, F I; Lee, S H

    2016-01-01

    Taiwan had been declared rabies-free in humans and domestic animals for five decades until July 2013, when surprisingly, three Formosan ferret badgers (FB) were diagnosed with rabies. Since then, a variety of wild carnivores and other wildlife species have been found dead, neurologically ill, or exhibiting aggressive behaviors around the island. To determine the affected animal species, geographic areas, and environments, animal bodies were examined for rabies by direct fluorescent antibody test (FAT). The viral genomes from the brains of selected rabid animals were sequenced for the phylogeny of rabies viruses (RABV). Out of a total of 1016 wild carnivores, 276/831 (33.2%) Formosan FBs were FAT positive, with occasional biting incidents in 1 dog and suspected spillover in 1 house shrew. All other animals tested, including dogs, cats, bats, mice, house shrews, and squirrels, were rabies-negative. The rabies was badger-associated and confined to nine counties/cities in sylvatic environments. Phylogeny of nucleoprotein and glycoprotein genes from 59 Formosan FB-associated RABV revealed them to be clustered in two distinct groups, TWI and TWII, consistent with the geographic segregation into western and eastern Taiwan provided by the Central Mountain Range and into northern rabies-free and central-southern rabies-affected regions by a river bisecting western Taiwan. The unique features of geographic and genetic segregation, sylvatic enzooticity, and FB-association of RABV suggest a logical strategy for the control of rabies in this nation.

  1. Emergence of a sylvatic enzootic formosan ferret badger-associated rabies in Taiwan and the geographical separation of two phylogenetic groups of rabies viruses.

    PubMed

    Tsai, K J; Hsu, W C; Chuang, W C; Chang, J C; Tu, Y C; Tsai, H J; Liu, H F; Wang, F I; Lee, S H

    2016-01-01

    Taiwan had been declared rabies-free in humans and domestic animals for five decades until July 2013, when surprisingly, three Formosan ferret badgers (FB) were diagnosed with rabies. Since then, a variety of wild carnivores and other wildlife species have been found dead, neurologically ill, or exhibiting aggressive behaviors around the island. To determine the affected animal species, geographic areas, and environments, animal bodies were examined for rabies by direct fluorescent antibody test (FAT). The viral genomes from the brains of selected rabid animals were sequenced for the phylogeny of rabies viruses (RABV). Out of a total of 1016 wild carnivores, 276/831 (33.2%) Formosan FBs were FAT positive, with occasional biting incidents in 1 dog and suspected spillover in 1 house shrew. All other animals tested, including dogs, cats, bats, mice, house shrews, and squirrels, were rabies-negative. The rabies was badger-associated and confined to nine counties/cities in sylvatic environments. Phylogeny of nucleoprotein and glycoprotein genes from 59 Formosan FB-associated RABV revealed them to be clustered in two distinct groups, TWI and TWII, consistent with the geographic segregation into western and eastern Taiwan provided by the Central Mountain Range and into northern rabies-free and central-southern rabies-affected regions by a river bisecting western Taiwan. The unique features of geographic and genetic segregation, sylvatic enzooticity, and FB-association of RABV suggest a logical strategy for the control of rabies in this nation. PMID:26711025

  2. Salivary excretion of rabies virus by healthy vampire bats.

    PubMed

    Aguilar-Setien, A; Loza-Rubio, E; Salas-Rojas, M; Brisseau, N; Cliquet, F; Pastoret, P P; Rojas-Dotor, S; Tesoro, E; Kretschmer, R

    2005-06-01

    Salivary excretion of rabies virus was evaluated in 14 adult vampire bats (Desmodus rotundus) intramuscularly injected with a large dose (10(6) MICLD50) of vampire rabies virus variant CASS88. Saliva samples were obtained from surviving bats every other day for 30 days, then weekly for 2 months, and finally 1 and 2 years later. Rabies virus was isolated in murine neuroblastoma cells and in randomly selected cases by PCR. Rabies virus was not detected in the saliva of any of the 11 animals that succumbed (somewhat early) to rabies challenge, nor in the control bats. In contrast, virus was detected early, and only once (days 6, 6 and 21) in each of the three animals that survived rabies challenge and remained healthy for at least 2 years after challenge. At that time even vigorous dexamethasone and cyclosporine administration failed to provoke further viral excretion.

  3. Salivary excretion of rabies virus by healthy vampire bats.

    PubMed Central

    Aguilar-Setien, A.; Loza-Rubio, E.; Salas-Rojas, M.; Brisseau, N.; Cliquet, F.; Pastoret, P. P.; Rojas-Dotor, S.; Tesoro, E.; Kretschmer, R.

    2005-01-01

    Salivary excretion of rabies virus was evaluated in 14 adult vampire bats (Desmodus rotundus) intramuscularly injected with a large dose (10(6) MICLD50) of vampire rabies virus variant CASS88. Saliva samples were obtained from surviving bats every other day for 30 days, then weekly for 2 months, and finally 1 and 2 years later. Rabies virus was isolated in murine neuroblastoma cells and in randomly selected cases by PCR. Rabies virus was not detected in the saliva of any of the 11 animals that succumbed (somewhat early) to rabies challenge, nor in the control bats. In contrast, virus was detected early, and only once (days 6, 6 and 21) in each of the three animals that survived rabies challenge and remained healthy for at least 2 years after challenge. At that time even vigorous dexamethasone and cyclosporine administration failed to provoke further viral excretion. PMID:15966107

  4. Salivary excretion of rabies virus by healthy vampire bats.

    PubMed

    Aguilar-Setien, A; Loza-Rubio, E; Salas-Rojas, M; Brisseau, N; Cliquet, F; Pastoret, P P; Rojas-Dotor, S; Tesoro, E; Kretschmer, R

    2005-06-01

    Salivary excretion of rabies virus was evaluated in 14 adult vampire bats (Desmodus rotundus) intramuscularly injected with a large dose (10(6) MICLD50) of vampire rabies virus variant CASS88. Saliva samples were obtained from surviving bats every other day for 30 days, then weekly for 2 months, and finally 1 and 2 years later. Rabies virus was isolated in murine neuroblastoma cells and in randomly selected cases by PCR. Rabies virus was not detected in the saliva of any of the 11 animals that succumbed (somewhat early) to rabies challenge, nor in the control bats. In contrast, virus was detected early, and only once (days 6, 6 and 21) in each of the three animals that survived rabies challenge and remained healthy for at least 2 years after challenge. At that time even vigorous dexamethasone and cyclosporine administration failed to provoke further viral excretion. PMID:15966107

  5. Neurotropism of rabies virus. An in vitro study.

    PubMed

    Tsiang, H; Koulakoff, A; Bizzini, B; Berwald-Netter, Y

    1983-07-01

    The relative susceptibility of neurons and glia, grown as monolayers in vitro, to rabies virus infection was explored. Established cell lines of neuronal or glial phenotype and primary cultures of cells derived from mouse dorsal root ganglia (DRC) or brain were used as homologues of the targets of rabies virus in the nervous system. Fixed rabies virus (CVS) strain was used in most experiments; other fixed rabies strains (PV, HEP, ERA) and a street rabies virus isolate were used in some. Virus-cell tropism was determined by immunofluorescence assay for rabies nucleocapsid antigen and cell permissivity was assessed by titration of virus yields. Neuronal cells always exhibited a much greater susceptibility to infection and a greater propensity to sustain viral growth. By immunofluorescence, 90-100% of neurons commonly had viral inclusion bodies, while doses of the virus three to four orders of magnitude higher still left greater than 99% of astrocytes, in brain cell cultures and 90 +/- 5% of the non-neuronal cells in DRG cultures without any obvious signs of rabies virus. Neuroblastoma cells (95 +/- 5% with viral antigens) produced viral yields about four orders of magnitude higher than glioma cells (10 +/- 5% with viral antigens). Though the overall infectivity of street virus was lower than that of fixed virus strains, a significantly higher viral tropism for neurons than for glia was maintained. Thus, primary neuronal cultures offer a means of exploring molecular events in rabies virus infection and their role in pathogenesis.

  6. Vaccination of vampire bats using recombinant vaccinia-rabies virus.

    PubMed

    Aguilar-Setién, Alvaro; Leon, Yolanda Campos; Tesoro, Emiliano Cruz; Kretschmer, Roberto; Brochier, Bernard; Pastoret, Paul-Pierre

    2002-07-01

    Adult vampire bats (Desmodus rotundus) were vaccinated by intramuscular, scarification, oral, or aerosol routes (n = 8 in each group) using a vaccinia-rabies glycoprotein recombinant virus. Sera were obtained before and 30 days after vaccination. All animals were then challenged intramuscularly with a lethal dose of rabies virus. Neutralizing antirabies antibodies were measured by rapid fluorescent focus inhibition test (RFFIT). Seroconversion was observed with each of the routes employed, but some aerosol and orally vaccinated animals failed to seroconvert. The highest antibody titers were observed in animals vaccinated by intramuscular and scarification routes. All animals vaccinated by intramuscular, scarification, and oral routes survived the viral challenge, but one of eight vampire bats receiving aerosol vaccination succumbed to the challenge. Of 31 surviving vaccinated and challenged animals, nine lacked detectable antirabies antibodies by RFFIT (five orally and four aerosol immunized animals). In contrast, nine of 10 non-vaccinated control bats succumbed to viral challenge. The surviving control bat had antiviral antibodies 90 days after viral challenge. These results suggest that the recombinant vaccine is an adequate and safe immunogen for bats by all routes tested.

  7. Vaccination of vampire bats using recombinant vaccinia-rabies virus.

    PubMed

    Aguilar-Setién, Alvaro; Leon, Yolanda Campos; Tesoro, Emiliano Cruz; Kretschmer, Roberto; Brochier, Bernard; Pastoret, Paul-Pierre

    2002-07-01

    Adult vampire bats (Desmodus rotundus) were vaccinated by intramuscular, scarification, oral, or aerosol routes (n = 8 in each group) using a vaccinia-rabies glycoprotein recombinant virus. Sera were obtained before and 30 days after vaccination. All animals were then challenged intramuscularly with a lethal dose of rabies virus. Neutralizing antirabies antibodies were measured by rapid fluorescent focus inhibition test (RFFIT). Seroconversion was observed with each of the routes employed, but some aerosol and orally vaccinated animals failed to seroconvert. The highest antibody titers were observed in animals vaccinated by intramuscular and scarification routes. All animals vaccinated by intramuscular, scarification, and oral routes survived the viral challenge, but one of eight vampire bats receiving aerosol vaccination succumbed to the challenge. Of 31 surviving vaccinated and challenged animals, nine lacked detectable antirabies antibodies by RFFIT (five orally and four aerosol immunized animals). In contrast, nine of 10 non-vaccinated control bats succumbed to viral challenge. The surviving control bat had antiviral antibodies 90 days after viral challenge. These results suggest that the recombinant vaccine is an adequate and safe immunogen for bats by all routes tested. PMID:12243138

  8. Preclinical Development of Inactivated Rabies Virus-Based Polyvalent Vaccine Against Rabies and Filoviruses.

    PubMed

    Willet, Mallory; Kurup, Drishya; Papaneri, Amy; Wirblich, Christoph; Hooper, Jay W; Kwilas, Steve A; Keshwara, Rohan; Hudacek, Andrew; Beilfuss, Stefanie; Rudolph, Grit; Pommerening, Elke; Vos, Adriaan; Neubert, Andreas; Jahrling, Peter; Blaney, Joseph E; Johnson, Reed F; Schnell, Matthias J

    2015-10-01

    We previously described the generation of a novel Ebola virus (EBOV) vaccine based on inactivated rabies virus (RABV) containing EBOV glycoprotein (GP) incorporated in the RABV virion. Our results demonstrated safety, immunogenicity, and protective efficacy in mice and nonhuman primates (NHPs). Protection against viral challenge depended largely on the quality of the humoral immune response against EBOV GP.Here we present the extension and improvement of this vaccine by increasing the amount of GP incorporation into virions via GP codon-optimization as well as the addition of Sudan virus (SUDV) and Marburg virus (MARV) GP containing virions. Immunogenicity studies in mice indicate similar immune responses for both SUDV GP and MARV GP compared to EBOV GP. Immunizing mice with multiple antigens resulted in immune responses similar to immunization with a single antigen. Moreover, immunization of NHP with the new inactivated RABV EBOV vaccine resulted in high titer neutralizing antibody levels and 100% protection against lethal EBOV challenge when applied with adjuvant.Our results indicate that an inactivated polyvalent vaccine against RABV filoviruses is achievable. Finally, the novel vaccines are produced on approved VERO cells and a clinical grade RABV/EBOV vaccine for human trials has been produced.

  9. Reverse genetics of rabies virus: new strategies to attenuate virus virulence for vaccine development.

    PubMed

    Zhu, Shimao; Li, Hui; Wang, Chunhua; Luo, Farui; Guo, Caiping

    2015-08-01

    Rabies is an ancient neurological disease that is almost invariably fatal once the clinical symptoms develop. Currently, prompt wound cleansing after exposing to a potentially rabid animal and vaccination using rabies vaccine combined with administration of rabies immune globulin are the only effective methods for post-exposure prophylaxis against rabies. Reverse genetic technique is a novel approach to investigate the function of a specific gene by analyzing the phenotypic effects through directly manipulating the gene sequences. It has revolutionized and provided a powerful tool to study the molecular biology of RNA viruses and has been widely used in rabies virus research. The attenuation of rabies virus virulence is the prerequisite for rabies vaccine development. Given the current challenge that sufficient and affordable high-quality vaccines are limited and lacking for global rabies prevention and control, highly cell-adapted, stable, and attenuated rabies viruses with broad cross-reactivity against different viral variants are ideal candidates for consideration to meet the need for human rabies control in the future. A number of approaches have been pursued to reduce the virulence of the virus and improve the safety of rabies vaccines. The application of reverse genetic technique has greatly advanced the engineering of rabies virus and paves the avenue for utilizing rabies virus for vaccine against rabies, viral vectors for exogenous antigen expression, and gene therapy in the future.

  10. Complete Genome Sequences of Six South African Rabies Viruses.

    PubMed

    Sabeta, Claude; Phahladira, Baby; Marston, Denise A; Wise, Emma L; Ellis, Richard J; Fooks, Anthony R

    2015-01-01

    South African rabies viruses (RABVs) from dogs and jackals (canid viruses) are highly related and most likely originated from a single progenitor. RABV is the cause of most global human rabies cases. The complete genome sequences of 3 RABVs from South Africa and Zimbabwe are reported here. PMID:26430028

  11. Complete Genome Sequences of Six South African Rabies Viruses

    PubMed Central

    Phahladira, Baby; Marston, Denise A.; Wise, Emma L.; Ellis, Richard J.; Fooks, Anthony R.

    2015-01-01

    South African rabies viruses (RABVs) from dogs and jackals (canid viruses) are highly related and most likely originated from a single progenitor. RABV is the cause of most global human rabies cases. The complete genome sequences of 3 RABVs from South Africa and Zimbabwe are reported here. PMID:26430028

  12. Complete Genome Sequences of Six South African Rabies Viruses.

    PubMed

    Sabeta, Claude; Phahladira, Baby; Marston, Denise A; Wise, Emma L; Ellis, Richard J; Fooks, Anthony R

    2015-10-01

    South African rabies viruses (RABVs) from dogs and jackals (canid viruses) are highly related and most likely originated from a single progenitor. RABV is the cause of most global human rabies cases. The complete genome sequences of 3 RABVs from South Africa and Zimbabwe are reported here.

  13. Recombinant canine distemper virus serves as bivalent live vaccine against rabies and canine distemper.

    PubMed

    Wang, Xijun; Feng, Na; Ge, Jinying; Shuai, Lei; Peng, Liyan; Gao, Yuwei; Yang, Songtao; Xia, Xianzhu; Bu, Zhigao

    2012-07-20

    Effective, safe, and affordable rabies vaccines are still being sought. Attenuated live vaccine has been widely used to protect carnivores from canine distemper. In this study, we generated a recombinant canine distemper virus (CDV) vaccine strain, rCDV-RVG, expressing the rabies virus glycoprotein (RVG) by using reverse genetics. The recombinant virus rCDV-RVG retained growth properties similar to those of vector CDV in Vero cell culture. Animal studies demonstrated that rCDV-RVG was safe in mice and dogs. Mice inoculated intracerebrally or intramuscularly with rCDV-RVG showed no apparent signs of disease and developed a strong rabies virus (RABV) neutralizing antibody response, which completely protected mice from challenge with a lethal dose of street virus. Canine studies showed that vaccination with rCDV-RVG induced strong and long-lasting virus neutralizing antibody responses to RABV and CDV. This is the first study demonstrating that recombinant CDV has the potential to serve as bivalent live vaccine against rabies and canine distemper in animals. PMID:22698451

  14. Phylogeographic analysis of rabies viruses in the Philippines.

    PubMed

    Tohma, Kentaro; Saito, Mariko; Kamigaki, Taro; Tuason, Laarni T; Demetria, Catalino S; Orbina, Jun Ryan C; Manalo, Daria L; Miranda, Mary E; Noguchi, Akira; Inoue, Satoshi; Suzuki, Akira; Quiambao, Beatriz P; Oshitani, Hitoshi

    2014-04-01

    Rabies still remains a public health threat in the Philippines. A significant number of human rabies cases, about 200-300 cases annually, have been reported, and the country needs an effective strategy for rabies control. To develop an effective control strategy, it is important to understand the transmission patterns of the rabies viruses. We conducted phylogenetic analyses by considering the temporal and spatial evolution of rabies viruses to reveal the transmission dynamics in the Philippines. After evaluating the molecular clock and phylogeographic analysis, we estimated that the Philippine strains were introduced from China around the beginning of 20th century. Upon this introduction, the rabies viruses evolved within the Philippines to form three major clades, and there was no indication of introduction of other rabies viruses from any other country. However, within the Philippines, island-to-island migrations were observed. Since then, the rabies viruses have diffused and only evolved within each island group. The evolutionary pattern of these viruses was strongly shaped by geographical boundaries. The association index statistics demonstrated a strong spatial structure within the island group, indicating that the seas were a significant geographical barrier for viral dispersal. Strong spatial structure was also observed even at a regional level, and most of the viral migrations (79.7% of the total median number) in Luzon were observed between neighboring regions. Rabies viruses were genetically clustered at a regional level, and this strong spatial structure suggests a geographical clustering of transmission chains and the potential effectiveness of rabies control that targets geographical clustering. Dog vaccination campaigns have been conducted independently by local governments in the Philippines, but it could be more effective to implement a coordinated vaccination campaign among neighboring areas to eliminate geographically-clustered rabies

  15. A single immunization with recombinant rabies virus (ERAG3G) confers complete protection against rabies in mice

    PubMed Central

    2014-01-01

    Purpose New alternative bait rabies vaccines applicable to pet dogs and wild animals are needed to eradicate rabies in Korea. In this study, recombinant rabies virus, ERAG3G strain was constructed using reverse genetic system and the safety, efficacy and immunogenicity of the ERAG3G strain was evaluated in mice and dogs. Materials and Methods Using the full-length genome mutated amino acid at position 333 of glycoprotein of rabies virus (RABV) and helper plasmids, the ERAG3G strain was rescued in BHK/T7-9 cells successfully. Mice were inoculated with the ERAG3G strain for safety and efficacy. Safety and immunogenicity of the dog inoculated with the ERAG3G strain (1 mL, 108.0 FAID50/mL) via intramuscular route was evaluated for 28 days after inoculation. Results The ERAG3G strain rescued by reverse genetic system was propagated well in the mouse neuroblastoma cells revealing titer of 108.5 FAID50/mL and was not pathogenic to 4- or 6-week-old mice that received by intramuscular or intracranical route. Immunization with the ERAG3G strain conferred complete protection from lethal RABV in mice. Dogs inoculated with the vaccine candidate via intramuscular route showed high neutralizing antibody titer ranging from 2.62 to 23.9 IU/mL at 28 days postinoculation. Conclusion Our findings suggest that the ERAG3G strain plays an important role in inducing protective efficacy in mice and causes to arise anti-rabies neutralizing antibody in dogs. PMID:25003091

  16. The Phylogeography and Spatiotemporal Spread of South-Central Skunk Rabies Virus

    PubMed Central

    Kuzmina, Natalia A.; Lemey, Philippe; Kuzmin, Ivan V.; Mayes, Bonny C.; Ellison, James A.; Orciari, Lillian A.; Hightower, Dillon; Taylor, Steven T.; Rupprecht, Charles E.

    2013-01-01

    The south-central skunk rabies virus (SCSK) is the most broadly distributed terrestrial viral lineage in North America. Skunk rabies has not been efficiently targeted by oral vaccination campaigns and represents a natural system of pathogen invasion, yielding insights to rabies emergence. In the present study we reconstructed spatiotemporal spread of SCSK in the whole territory of its circulation using a combination of Bayesian methods. The analysis based on 241 glycoprotein gene sequences demonstrated that SCSK is much more divergent phylogenetically than was appreciated previously. According to our analyses the SCSK originated in the territory of Texas ~170 years ago, and spread geographically during the following decades. The wavefront velocity in the northward direction was significantly greater than in the eastward and westward directions. Rivers (except the Mississippi River and Rio Grande River) did not constitute significant barriers for epizootic spread, in contrast to deserts and mountains. The mean dispersal rate of skunk rabies was lower than that of the raccoon and fox rabies. Viral lineages circulate in their areas with limited evidence of geographic spread during decades. However, spatiotemporal reconstruction shows that after a long period of stability the dispersal rate and wavefront velocity of SCSK are increasing. Our results indicate that there is a need to develop control measures for SCSK, and suggest how such measure can be implemented most efficiently. Our approach can be extrapolated to other rabies reservoirs and used as a tool for investigation of epizootic patterns and planning interventions towards disease elimination. PMID:24312657

  17. The phylogeography and spatiotemporal spread of south-central skunk rabies virus.

    PubMed

    Kuzmina, Natalia A; Lemey, Philippe; Kuzmin, Ivan V; Mayes, Bonny C; Ellison, James A; Orciari, Lillian A; Hightower, Dillon; Taylor, Steven T; Rupprecht, Charles E

    2013-01-01

    The south-central skunk rabies virus (SCSK) is the most broadly distributed terrestrial viral lineage in North America. Skunk rabies has not been efficiently targeted by oral vaccination campaigns and represents a natural system of pathogen invasion, yielding insights to rabies emergence. In the present study we reconstructed spatiotemporal spread of SCSK in the whole territory of its circulation using a combination of Bayesian methods. The analysis based on 241 glycoprotein gene sequences demonstrated that SCSK is much more divergent phylogenetically than was appreciated previously. According to our analyses the SCSK originated in the territory of Texas ~170 years ago, and spread geographically during the following decades. The wavefront velocity in the northward direction was significantly greater than in the eastward and westward directions. Rivers (except the Mississippi River and Rio Grande River) did not constitute significant barriers for epizootic spread, in contrast to deserts and mountains. The mean dispersal rate of skunk rabies was lower than that of the raccoon and fox rabies. Viral lineages circulate in their areas with limited evidence of geographic spread during decades. However, spatiotemporal reconstruction shows that after a long period of stability the dispersal rate and wavefront velocity of SCSK are increasing. Our results indicate that there is a need to develop control measures for SCSK, and suggest how such measure can be implemented most efficiently. Our approach can be extrapolated to other rabies reservoirs and used as a tool for investigation of epizootic patterns and planning interventions towards disease elimination. PMID:24312657

  18. The phylogeography and spatiotemporal spread of south-central skunk rabies virus.

    PubMed

    Kuzmina, Natalia A; Lemey, Philippe; Kuzmin, Ivan V; Mayes, Bonny C; Ellison, James A; Orciari, Lillian A; Hightower, Dillon; Taylor, Steven T; Rupprecht, Charles E

    2013-01-01

    The south-central skunk rabies virus (SCSK) is the most broadly distributed terrestrial viral lineage in North America. Skunk rabies has not been efficiently targeted by oral vaccination campaigns and represents a natural system of pathogen invasion, yielding insights to rabies emergence. In the present study we reconstructed spatiotemporal spread of SCSK in the whole territory of its circulation using a combination of Bayesian methods. The analysis based on 241 glycoprotein gene sequences demonstrated that SCSK is much more divergent phylogenetically than was appreciated previously. According to our analyses the SCSK originated in the territory of Texas ~170 years ago, and spread geographically during the following decades. The wavefront velocity in the northward direction was significantly greater than in the eastward and westward directions. Rivers (except the Mississippi River and Rio Grande River) did not constitute significant barriers for epizootic spread, in contrast to deserts and mountains. The mean dispersal rate of skunk rabies was lower than that of the raccoon and fox rabies. Viral lineages circulate in their areas with limited evidence of geographic spread during decades. However, spatiotemporal reconstruction shows that after a long period of stability the dispersal rate and wavefront velocity of SCSK are increasing. Our results indicate that there is a need to develop control measures for SCSK, and suggest how such measure can be implemented most efficiently. Our approach can be extrapolated to other rabies reservoirs and used as a tool for investigation of epizootic patterns and planning interventions towards disease elimination.

  19. A single immunization with a recombinant canine adenovirus expressing the rabies virus G protein confers protective immunity against rabies in mice

    SciTech Connect

    Li Jianwei; Faber, Milosz; Papaneri, Amy; Faber, Marie-Luise; McGettigan, James P.; Schnell, Matthias J.; Dietzschold, Bernhard . E-mail: bernhard.dietzschold@jefferson.edu

    2006-12-20

    Rabies vaccines based on live attenuated rabies viruses or recombinant pox viruses expressing the rabies virus (RV) glycoprotein (G) hold the greatest promise of safety and efficacy, particularly for oral immunization of wildlife. However, while these vaccines induce protective immunity in foxes, they are less effective in other animals, and safety concerns have been raised for some of these vaccines. Because canine adenovirus 2 (CAV2) is licensed for use as a live vaccine for dogs and has an excellent efficacy and safety record, we used this virus as an expression vector for the RVG. The recombinant CAV2-RV G produces virus titers similar to those produced by wild-type CAV2, indicating that the RVG gene does not affect virus replication. Comparison of RVG expressed by CAV2-RV G with that of vaccinia-RV G recombinant virus (V-RG) revealed similar amounts of RV G on the cell surface. A single intramuscular or intranasal immunization of mice with CAV2-RVG induced protective immunity in a dose-dependent manner, with no clinical signs or discomfort from the virus infection regardless of the route of administration or the amount of virus.

  20. Efficacy and bait acceptance of vaccinia vectored rabies glycoprotein vaccine in captive foxes (Vulpes vulpes), raccoon dogs (Nyctereutes procyonoides) and dogs (Canis familiaris).

    PubMed

    Cliquet, F; Barrat, J; Guiot, A L; Caël, N; Boutrand, S; Maki, J; Schumacher, C L

    2008-08-26

    The red fox, dog, and raccoon dog are known to play a major role in the global epidemiology of rabies. These three canid species were used to compare the appetency and efficacy of two commercial bait formats, each containing a single dose of vaccinia-rabies glycoprotein (V-RG) vaccine. Square and rectangular RABORAL V-RG baits were fed to individual caged animal, and results were evaluated using three parameters: bait consumption, induction of rabies virus neutralizing antibodies and protection after a virulent rabies challenge. The rectangular and square RABORAL V-RG baits were found to deliver the oral rabies vaccine in a similar manner to all three species resulting in acceptable seroconversion and effective protection levels after the rabies challenge. Appetency of each bait type was measured by bait consumption and found to be similar for both RABORAL V-RG bait formats in the fox and dog. The square RABORAL V-RG bait, however, was consumed more effectively than the rectangular RABORAL V-RG bait by the raccoon dog. PMID:18620017

  1. Infection of Bergmann glia in the cerebellum of a skunk experimentally infected with street rabies virus.

    PubMed

    Jackson, A C; Phelan, C C; Rossiter, J P

    2000-10-01

    Rabies virus is a highly neuronotropic virus and glial cell infection is not prominent in the central nervous system (CNS). Paraffin-embedded tissues from the cerebella of skunks experimentally infected with either a skunk salivary gland isolate of street rabies virus or the challenge virus standard (CVS) strain of fixed rabies virus were examined with immunoperoxidase staining for rabies virus antigen by using an anti-rabies virus nucleocapsid protein monoclonal antibody. A skunk infected with street rabies virus showed prominent infection of Bergmann glia. Although infected Purkinje cells were observed, they usually demonstrated a relatively small amount of antigen in their perikarya. A CVS-infected skunk showed many intensely labeled Purkinje cells and a relatively small number of infected Bergmann glia. These findings indicate that although rabies virus is a highly neuronotropic virus, street rabies virus strains do not always demonstrate strict neuronotropism in the central nervous system.

  2. λ-Carrageenan P32 Is a Potent Inhibitor of Rabies Virus Infection

    PubMed Central

    Luo, Zhaochen; Tian, Dayong; Zhou, Ming; Xiao, Wenjie; Zhang, Yachun; Li, Mingming; Sui, Baokun; Wang, Wei; Guan, Huashi; Chen, Huanchun; Fu, Zhen F.; Zhao, Ling

    2015-01-01

    Rabies, caused by rabies virus (RABV), is an acute, fatal encephalitic disease that affects many warm-blooded mammals. Currently, post-exposure prophylaxis regimens are effective for most rabies cases, but once the clinical signs of the disease appear, current treatment options become ineffective. Carrageenan has been reported as a potent inhibitor of many viruses. In this study, the λ-carrageenan (λ-CG) P32 was investigated for its potential role in inhibiting RABV infection. Our results show that P32 specifically inhibits the replication of several RABV strains but not vesicular stomatitis virus in multiple cell lines and shows low cytotoxicity. P32 mainly abrogated viral replication during the early stage of the post-adsorption period. Further studies demonstrated that P32 could affect not only viral internalization but also viral uncoating by blocking cell fusion mediated by RABV glycoprotein. Moreover, P32 can fully inhibit RABV infection in vitro during the post-adsorption period, whereas heparin and heparan sulfate, which possess similar structures to P32, showed significant but not complete inhibition of RABV infectivity. Collectively, our results indicate that λ-CG P32 is a promising agent that can inhibit RABV infection mainly by inhibiting viral internalization and glycoprotein-mediated cell fusion and can be used for the development of novel anti-RABV drugs. PMID:26465753

  3. Multicenter comparative study of a new ELISA, PLATELIA RABIES II, for the detection and titration of anti-rabies glycoprotein antibodies and comparison with the rapid fluorescent focus inhibition test (RFFIT) on human samples from vaccinated and non-vaccinated people.

    PubMed

    Feyssaguet, M; Dacheux, L; Audry, L; Compoint, A; Morize, J L; Blanchard, I; Bourhy, H

    2007-03-01

    The envelope glycoprotein G of rabies virus induces the production of neutralising antibodies, which are important in protection against rabies. Therefore, titration of anti-envelope glycoprotein antibodies is a good indicator of the degree of immunity in people during anti-rabies treatment or after vaccination. According to the World Health Organization (WHO) guidelines, a booster vaccine dose should be given if the rabies antibody titre falls below 0.5 IU/ml. Titration of anti-rabies antibodies is also useful for plasma centers in the preparation and standardization of human anti-rabies gamma-globulins for therapeutic use and to a lesser extent for the diagnosis of rabies in human sera and cerebrospinal fluid (CSF). This paper presents a new enzyme-linked immunosorbent assay (ELISA), PLATELIA RABIES II, developed for rabies envelope glycoprotein antibody detection or titration and its comparison to the current reference method (RFFIT). The data collected during validation of the test in a multicenter study are analysed to give a sound overall knowledge of the capabilities of the PLATELIA RABIES II, for instance specificity, linearity, accuracy, precision, detection limit and quantitation limit. To this aim, human serum samples from a total of 1348 vaccinated or non-vaccinated people were tested in parallel using the new ELISA and the RFFIT for the presence of anti-rabies antibodies. Data generated indicate a linear relationship across the range of titration between the two methods. The sensitivity reaches 98.6% and the specificity 99.4%. This study indicates that this new ELISA test is as sensitive and specific as the current standardized reference method. The method is simple, safe, rapid and can be considered as a useful alternative to the neutralisation test.

  4. Two potential recombinant rabies vaccines expressing canine parvovirus virion protein 2 induce immunogenicity to canine parvovirus and rabies virus.

    PubMed

    Luo, Jun; Shi, Hehe; Tan, Yeping; Niu, Xuefeng; Long, Teng; Zhao, Jing; Tian, Qin; Wang, Yifei; Chen, Hao; Guo, Xiaofeng

    2016-08-17

    Both rabies virus (RABV) and canine parvovirus (CPV) cause lethal diseases in dogs. In this study, both high egg passage Flury (HEP-Flury) strains of RABV and recombinant RABV carrying double RABV glycoprotein (G) gene were used to express the CPV virion protein 2 (VP2) gene, and were designated rHEP-VP2 and, rHEP-dG-VP2 respectively. The two recombinant RABVs maintained optimal virus titration according to their viral growth kinetics assay compared with the parental strain HEP-Flury. Western blotting indicated that G protein and VP2 were expressed in vitro. The expression of VP2 in Crandell feline kidney cells post-infection by rHEP-VP2 and rHEP-dG-VP2 was confirmed by indirect immunofluorescence assay with antibody against VP2. Immunogenicity of recombinant rabies viruses was tested in Kunming mice. Both rHEP-VP2 and rHEP-dG-VP2 induced high levels of rabies antibody compared with HEP-Flury. Mice immunized with rHEP-VP2 and rHEP-dG-VP2 both had a high level of antibodies against VP2, which can protect against CPV infection. A challenge experiment indicated that more than 80% mice immunized with recombinant RABVs survived after infection of challenge virus standard 24 (CVS-24). Together, this study showed that recombinant RABVs expressing VP2 induced protective immune responses to RABV and CPV. Therefore, rHEP-VP2 and rHEP-dG-VP2 might be potential combined vaccines for RABV and CPV.

  5. Two potential recombinant rabies vaccines expressing canine parvovirus virion protein 2 induce immunogenicity to canine parvovirus and rabies virus.

    PubMed

    Luo, Jun; Shi, Hehe; Tan, Yeping; Niu, Xuefeng; Long, Teng; Zhao, Jing; Tian, Qin; Wang, Yifei; Chen, Hao; Guo, Xiaofeng

    2016-08-17

    Both rabies virus (RABV) and canine parvovirus (CPV) cause lethal diseases in dogs. In this study, both high egg passage Flury (HEP-Flury) strains of RABV and recombinant RABV carrying double RABV glycoprotein (G) gene were used to express the CPV virion protein 2 (VP2) gene, and were designated rHEP-VP2 and, rHEP-dG-VP2 respectively. The two recombinant RABVs maintained optimal virus titration according to their viral growth kinetics assay compared with the parental strain HEP-Flury. Western blotting indicated that G protein and VP2 were expressed in vitro. The expression of VP2 in Crandell feline kidney cells post-infection by rHEP-VP2 and rHEP-dG-VP2 was confirmed by indirect immunofluorescence assay with antibody against VP2. Immunogenicity of recombinant rabies viruses was tested in Kunming mice. Both rHEP-VP2 and rHEP-dG-VP2 induced high levels of rabies antibody compared with HEP-Flury. Mice immunized with rHEP-VP2 and rHEP-dG-VP2 both had a high level of antibodies against VP2, which can protect against CPV infection. A challenge experiment indicated that more than 80% mice immunized with recombinant RABVs survived after infection of challenge virus standard 24 (CVS-24). Together, this study showed that recombinant RABVs expressing VP2 induced protective immune responses to RABV and CPV. Therefore, rHEP-VP2 and rHEP-dG-VP2 might be potential combined vaccines for RABV and CPV. PMID:27449079

  6. Presence of neutralizing antibodies to rabies virus in striped skunks from areas free of skunk rabies in Alberta.

    PubMed

    Rosatte, R C; Gunson, J R

    1984-07-01

    Nine percent of 198 serum samples from striped skunks, Mephitis mephitis (Schreber) from five areas of Alberta were positive for rabies neutralizing antibody. Positive samples were minimal (2%) from specimens sampled in an area enzootic for rabies and occurred at greater rates in areas negative for skunk rabies. Transmission of rabies virus to skunks may have been from a source other than skunks in those areas, most probably from bats.

  7. Antigenic and molecular characterization of rabies virus in Argentina.

    PubMed

    Cisterna, Daniel; Bonaventura, Romina; Caillou, Susana; Pozo, Oscar; Andreau, Maria Lidia; Fontana, Liliana Dalla; Echegoyen, Cristina; de Mattos, Carlos; de Mattos, Cecilia; Russo, Susana; Novaro, Laura; Elberger, Diana; Freire, María Cecilia

    2005-05-01

    The nucleoprotein genes of 54 human, domestic and wild animals rabies isolates obtained in Argentina between 1995 and 2002 were characterized using monoclonal antibodies and partial gene sequence analysis. The antigenic and genetic diversities of rabies virus in samples from bat and bat-related cases were studied, leading to the identification of five distinct genetic variants. Rabies viruses isolated from vampire bat related cases were very similar to each other, showing 98.9% overall similarity. Specific antigenic variants (AgV) were detected associated with different insectivorous bats species, in samples from Tadarida brasiliensis and Eumops patagonicus bats. In contrast, isolates from Myotis sp. and Histiotus sp. bats could not be matched to any antigenic type. Additionally, bat rabies cases were also detected in southern provinces previously considered rabies-free. Finally, two independent antigenic and genetic variants co-circulating in northern Argentina were found in isolates obtained from dogs and dog-related cases, suggesting two independent cycles of virus transmission. This is the first national coordinated study of antigenic as well as molecular epidemiology of rabies in Argentina. The information presented here will improve our knowledge about rabies epidemiology and therefore, will assist preventing fatal human cases. PMID:15763144

  8. Further studies on the replication of rabies and rabies-like viruses in organized cultures of mammalian neural tissues.

    PubMed

    Matsumoto, S; Schneider, L G; Kawai, A; Yonezawa, T

    1974-10-01

    Organized cultures of mammalian spinal and dorsal root ganglions were used for a comparative study of the neurocytopathology caused by rabies and so-called rabies-like viruses. Electron microscopy and titration of infectivity revised earlier data in which no difference could be demonstrated between street and fixed-virus infection. In the present study, fixed virus produced inclusion bodies without apparent virus assembly. Sequential electron microscopy revealed that the main sites of virus assembly were the membranes of the Golgi complex. In contrast, rabies-like viruses freshly isolated from wild rodents produced inclusion bodies all of which were associated with virus replication. Electron microscopic evidence has led us to classify these strains as street virus. Nonneural cell elements from cultivated ganglions were susceptible to fixed virus and the cultures yielded higher titers of infectivity as compared to those of rabies-like viruses. Virus budding was shown to occur at the cell surface as well as at intracytoplasmic membranes.

  9. Rabies Virus Maintained by Dogs in Humans and Terrestrial Wildlife, Ceará State, Brazil

    PubMed Central

    de Mattos, Cecília C.; de Morais, Nélio B.; Carrieri, Maria Luíza; Rolim, Benedito N.; Silva, Lucia M.; Rupprecht, Charles E.; Durigon, Edison L.; de Mattos, Carlos A.

    2006-01-01

    Rabies viruses circulating in Ceará, Brazil, were identified by molecular analysis to be related to variants maintained by dogs, bats, and other wildlife. Most of these viruses are associated with human rabies cases. We document the emergence of a rabies virus variant responsible for an independent epidemic cycle in the crab-eating fox (Cerdocyon thous). PMID:17326958

  10. First Case of Human Rabies in Chile Caused by an Insectivorous Bat Virus Variant

    PubMed Central

    Favi, Myriam; Yung, Verónica; Chala, Evelyn; López, Luis R.

    2002-01-01

    The first human rabies case in Chile since 1972 occurred in March 1996 in a patient without history of known exposure. Antigenic and genetic characterization of the rabies isolate indicated that its reservoir was the insectivorous bat Tadarida brasiliensis. This is the first human rabies case caused by an insectivorous bat rabies virus variant reported in Latin America. PMID:11749754

  11. Identification of New Rabies Virus Variant in Mexican Immigrant

    PubMed Central

    Messenger, Sharon L.; Orciari, Lillian A.; Niezgoda, Michael; Blanton, Jesse D.; Fukagawa, Chris; Rupprecht, Charles E.

    2008-01-01

    A novel rabies virus was identified after death in a man who had immigrated from Oaxaca, Mexico, to California, USA. Despite the patient’s history of exposure to domestic and wild carnivores, molecular and phylogenetic characterizations suggested that the virus originated from insectivorous bats. Enhanced surveillance is needed to elucidate likely reservoirs. PMID:19046517

  12. Rabies virus matrix protein interplay with eIF3, new insights into rabies virus pathogenesis.

    PubMed

    Komarova, Anastassia V; Real, Eléonore; Borman, Andrew M; Brocard, Michèle; England, Patrick; Tordo, Noël; Hershey, John W B; Kean, Katherine M; Jacob, Yves

    2007-01-01

    Viral proteins are frequently multifunctional to accommodate the high density of information encoded in viral genomes. Matrix (M) protein of negative-stranded RNA viruses such as Rhabdoviridae is one such example. Its primary function is virus assembly/budding but it is also involved in the switch from viral transcription to replication and the concomitant down regulation of host gene expression. In this study we undertook a search for potential rabies virus (RV) M protein's cellular partners. In a yeast two-hybrid screen the eIF3h subunit was identified as an M-interacting cellular factor, and the interaction was validated by co-immunoprecipitation and surface plasmon resonance assays. Upon expression in mammalian cell cultures, RV M protein was localized in early small ribosomal subunit fractions. Further, M protein added in trans inhibited in vitro translation on mRNA encompassing classical (Kozak-like) 5'-UTRs. Interestingly, translation of hepatitis C virus IRES-containing mRNA, which recruits eIF3 via a different noncanonical mechanism, was unaffected. Together, the data suggest that, as a complement to its functions in virus assembly/budding and regulation of viral transcription, RV M protein plays a role in inhibiting translation in virus-infected cells through a protein-protein interaction with the cellular translation machinery.

  13. Recombinant rabies virus expressing dog GM-CSF is an efficacious oral rabies vaccine for dogs.

    PubMed

    Zhou, Ming; Wang, Lei; Zhou, Songqin; Wang, Zhao; Ruan, Juncheng; Tang, Lijun; Jia, Ziming; Cui, Min; Zhao, Ling; Fu, Zhen F

    2015-11-17

    Developing efficacious oral rabies vaccines is an important step to increase immunization coverage for stray dogs, which are not accessible for parenteral vaccination. Our previous studies have demonstrated that recombinant rabies virus (RABV) expressing cytokines/chemokines induces robust protective immune responses after oral immunization in mice by recruiting and activating dendritic cells (DCs) and B cells. To develop an effective oral rabies vaccine for dogs, a recombinant attenuated RABV expressing dog GM-CSF, designated as LBNSE-dGM-CSF was constructed and used for oral vaccination in a dog model. Significantly more DCs or B cells were activated in the peripheral blood of dogs vaccinated orally with LBNSE-dGM-CSF than those vaccinated with the parent virus LBNSE, particularly at 3 days post immunization (dpi). As a result, significantly higher levels of virus neutralizing antibodies (VNAs) were detected in dogs immunized with LBNSE-dGM-CSF than with the parent virus. All the immunized dogs were protected against a lethal challenge with 4500 MICLD50 of wild-type RABV SXTYD01. LBNSE-dGM-CSF was found to replicate mainly in the tonsils after oral vaccination as detected by nested RT-PCR and immunohistochemistry. Taken together, our results indicate that LBNSE-dGM-CSF could be a promising oral rabies vaccine candidate for dogs.

  14. Recombinant rabies virus expressing dog GM-CSF is an efficacious oral rabies vaccine for dogs.

    PubMed

    Zhou, Ming; Wang, Lei; Zhou, Songqin; Wang, Zhao; Ruan, Juncheng; Tang, Lijun; Jia, Ziming; Cui, Min; Zhao, Ling; Fu, Zhen F

    2015-11-17

    Developing efficacious oral rabies vaccines is an important step to increase immunization coverage for stray dogs, which are not accessible for parenteral vaccination. Our previous studies have demonstrated that recombinant rabies virus (RABV) expressing cytokines/chemokines induces robust protective immune responses after oral immunization in mice by recruiting and activating dendritic cells (DCs) and B cells. To develop an effective oral rabies vaccine for dogs, a recombinant attenuated RABV expressing dog GM-CSF, designated as LBNSE-dGM-CSF was constructed and used for oral vaccination in a dog model. Significantly more DCs or B cells were activated in the peripheral blood of dogs vaccinated orally with LBNSE-dGM-CSF than those vaccinated with the parent virus LBNSE, particularly at 3 days post immunization (dpi). As a result, significantly higher levels of virus neutralizing antibodies (VNAs) were detected in dogs immunized with LBNSE-dGM-CSF than with the parent virus. All the immunized dogs were protected against a lethal challenge with 4500 MICLD50 of wild-type RABV SXTYD01. LBNSE-dGM-CSF was found to replicate mainly in the tonsils after oral vaccination as detected by nested RT-PCR and immunohistochemistry. Taken together, our results indicate that LBNSE-dGM-CSF could be a promising oral rabies vaccine candidate for dogs. PMID:26436700

  15. Safety and immunogenicity of recombinant rabies virus (ERAGS) in mice and raccoon dogs

    PubMed Central

    2016-01-01

    Purpose The development of a genetically modified live rabies vaccine applicable to wild raccoon dogs is necessary for the eradication of rabies in Korea. Thus, we constructed a recombinant rabies virus (RABV) called the ERAGS strain, using a reverse genetic system and evaluated its safety and efficacy in mice and its safety and immunogenicity in raccoon dogs. Materials and Methods ERAGS, which has Asn194Ser and Arg333Glu substitutions in the glycoprotein, was constructed using site-directed mutagenesis. Mice were inoculated with the ERAGS strain (either 105.0 or 107.0 FAID50/mL) via intramuscular (IM) or intracranial injections and then challenged with a virulent RABV. Raccoon dogs were administered the ERAGS strain (108.0 FAID50/mL) either orally or via the IM route and the immunogenicity of the strain was evaluated using fluorescent antibody virus neutralization tests. Results The ERAGS strain inoculated into murine neuroblastoma cells reached 107.8 FAID50/mL at 96-hour post-inoculation. The virus was not pathogenic and induced complete protection from virulent RABV in immunized 4- and 6-week-old mice. Korean raccoon dogs immunized with the ERAGS strain via IM or oral route were also safe from the virus and developed high titer levels (26.4-32.8 IU/mL) of virus-neutralizing antibody (VNA) at 4 weeks post-inoculation. Conclusion The ERAGS RABV strain was effectively protective against rabies in mice and produced a high VNA titer in raccoon dogs. PMID:27489806

  16. Typing of the rabies virus in Chile, 2002-2008.

    PubMed

    Yung, V; Favi, M; Fernandez, J

    2012-12-01

    In Chile, dog rabies has been controlled and insectivorous bats have been identified as the main rabies reservoir. This study aimed to determine the rabies virus (RABV) variants circulating in the country between 2002 and 2008. A total of 612 RABV isolates were tested using a panel with eight monoclonal antibodies against the viral nucleoprotein (N-mAbs) for antigenic typing, and a product of 320-bp of the nucleoprotein gene was sequenced from 99 isolates. Typing of the isolates revealed six different antigenic variants but phylogenetic analysis identified four clusters associated with four different bat species. Tadarida brasiliensis bats were confirmed as the main reservoir. This methodology identified several independent rabies enzootics maintained by different species of insectivorous bats in Chile. PMID:22458941

  17. The distribution of Challenge virus standard rabies virus versus skunk street rabies virus in the brains of experimentally infected rabid skunks.

    PubMed

    Smart, N L; Charlton, K M

    1992-01-01

    The proposal that the bizarre behavioral changes which occur during rabies infection are due to selective infection of limbic system neurons was further studied in skunks (a species important in naturally occurring disease). A detailed immunohistochemical study of brains of skunks experimentally infected with either Challenge virus standard (CVS) or street rabies virus revealed only trace amounts of viral antigen in many limbic system neurons and marked differences in viral distribution between street and CVS virus. These data were collected during early stage rabies when behavioral changes occur. Areas which contained heavy accumulations of street rabies virus but low amounts of CVS rabies virus were the neuronal perikarya and processes of the dorsal motor nucleus of the vagus, midbrain raphe, hypoglossal and red nuclei. In contrast, large accumulations of CVS virus were found in the Purkinje cells of the cerebellum, the habenular nuclei and in pyramidal cells throughout the cerebral cortex, while corresponding areas in all street virus-infected skunks contained minimal antigen. These findings were very consistent for animals of the same experimental group and between skunks inoculated both intramuscularly and intranasally with skunk street virus. Skunks inoculated intramuscularly with CVS rabies virus failed to develop rabies. Since, in this model, street virus infection generally produces furious rabies and CVS infection results in dumb rabies, we speculate that the behavioral changes which occur in these two different clinical syndromes are due to the heavy and specific accumulation of virus in different regions of the CNS. These results show that regions other than those of the limbic system may also be involved in the pathogenesis of behavior changes in rabid animals.

  18. The Activity of Rabies Vaccines against Genetic Clusters of Rabies Virus Circulating at the Territory of Ukraine

    PubMed Central

    Ivanov, Mykola; Polupan, Ivan; Deryabin, Oleg

    2013-01-01

    Objective To identify the presence of genetic clusters of rabies virus at the territory of Ukraine and to determine the degree of activity of rabies vaccines against these genetic clusters. Introduction To develop and implement an effective program of rabies eradication in Ukraine in 2008 was founded the unique collection of samples of pathological materials confirmed as positive in rabies at the regional veterinary laboratories of Ukraine. The collection is constantly updated and to present moment it includes 1389 samples from all regions of Ukraine, selected from 17 animal species and humans. Methods Identification of the rabies virus in samples of pathological material for their further selection was carried out using the test developed by us which based on RT-PCR with primers complementary to the conservative fragments of the 5’-end of nucleoprotein gene of rabies virus. For the study of the street rabies virus isolates from the collection we use RT-PCR with the primers pair (509, 304) flanking the variable 3’-end part of nucleoprotein gene of the reference strain of rabies virus CVS (fragment in 377 bp). Studies of rabies vaccines activity were carried out with modified method of U.S. National Institutes of Health using rabies virus street isolates of both genetic clusters instead of the Challenge Virus Standard (CVS). All isolates of street rabies virus were inoculated in a dose of 5–50 LD50. The criteria for evaluation of protective activity of rabies vaccine was effective dose (− lg ED50). Results In molecular genetic studies with variant-specific primers we established the presence in Ukraine of two clusters of rabies virus. Clusters I circulates on the right bank of the Dnipro river (the largest water barrier that divides the country into eastern and western side), and cluster II – on the left bank of the Dnieper. The relationship of these variants with the epizootic situation was researched. For this purpose epizootological zoning of Ukraine

  19. Overwintering of Rabies Virus in Silver Haired Bats (Lasionycteris noctivagans)

    PubMed Central

    Davis, April D.; Morgan, Shannon M. D.; Dupuis, Michelle; Poulliott, Craig E.; Jarvis, Jodie A.; Franchini, Rhianna; Clobridge, Anne; Rudd, Robert J.

    2016-01-01

    Silver-haired bats, (Lasionycteris noctivagans) are semi-colonial, migratory tree bats that have infrequent contact with humans. Despite the species rarity, the L. noctivagans rabies variant is the most commonly reported rabies virus variant (RABV) in domestically acquired human rabies cases in the US. Unlike big brown bats (Eptesicus fuscus) and little brown bats (Myotis lucifugus), L. noctivagans are not considered true hibernators. It is unknown if RABV can overwinter in hibernating L. noctivagans or is only maintained in members of this taxa that migrate to warmer climates. To better understand RABV overwintering in this species, L. noctivagans were inoculated intramuscularly with either a homologous RABV (L. noctivagans Virus 1) or one of two heterologous RABV (Eptesicus fuscus Virus 2 and Myotis lucifugus Virus 1). Five days following inoculation, L. noctivagans were placed in a hibernation chamber for 6 weeks. Our results demonstrate that rabies virus can overwinter in L. noctivagans yet the incubation period was extended 6 weeks when compared to bats maintained at ambient temperatures. Additionally, we found that the longer the incubation period, the greater the viral dissemination to the salivary glands. Similar to our previous studies, L. noctivagans were most susceptible to a homologous variant. In summary, we found that RABV incubation is extended following a subcutaneous exposure or maintenance in hibernation and longer incubation times increase dissemination and potential for transmission. PMID:27195489

  20. Overwintering of Rabies Virus in Silver Haired Bats (Lasionycteris noctivagans).

    PubMed

    Davis, April D; Morgan, Shannon M D; Dupuis, Michelle; Poulliott, Craig E; Jarvis, Jodie A; Franchini, Rhianna; Clobridge, Anne; Rudd, Robert J

    2016-01-01

    Silver-haired bats, (Lasionycteris noctivagans) are semi-colonial, migratory tree bats that have infrequent contact with humans. Despite the species rarity, the L. noctivagans rabies variant is the most commonly reported rabies virus variant (RABV) in domestically acquired human rabies cases in the US. Unlike big brown bats (Eptesicus fuscus) and little brown bats (Myotis lucifugus), L. noctivagans are not considered true hibernators. It is unknown if RABV can overwinter in hibernating L. noctivagans or is only maintained in members of this taxa that migrate to warmer climates. To better understand RABV overwintering in this species, L. noctivagans were inoculated intramuscularly with either a homologous RABV (L. noctivagans Virus 1) or one of two heterologous RABV (Eptesicus fuscus Virus 2 and Myotis lucifugus Virus 1). Five days following inoculation, L. noctivagans were placed in a hibernation chamber for 6 weeks. Our results demonstrate that rabies virus can overwinter in L. noctivagans yet the incubation period was extended 6 weeks when compared to bats maintained at ambient temperatures. Additionally, we found that the longer the incubation period, the greater the viral dissemination to the salivary glands. Similar to our previous studies, L. noctivagans were most susceptible to a homologous variant. In summary, we found that RABV incubation is extended following a subcutaneous exposure or maintenance in hibernation and longer incubation times increase dissemination and potential for transmission. PMID:27195489

  1. Pre- and post-exposure safety and efficacy of attenuated rabies virus vaccines are enhanced by their expression of IFNγ

    SciTech Connect

    Barkhouse, Darryll A.; Faber, Milosz; Hooper, D. Craig

    2015-01-01

    Consistent with evidence of a strong correlation between interferon gamma (IFNγ) production and rabies virus (RABV) clearance from the CNS, we recently demonstrated that engineering a pathogenic RABV to express IFNγ highly attenuates the virus. Reasoning that IFNγ expression by RABV vaccines would enhance their safety and efficacy, we reverse-engineered two proven vaccine vectors, GAS and GASGAS, to express murine IFNγ. Mortality and morbidity were monitored during suckling mice infection, immunize/challenge experiments and mixed intracranial infections. We demonstrate that GASγ and GASγGAS are significantly attenuated in suckling mice compared to the GASGAS vaccine. GASγ better protects mice from lethal DRV4 RABV infection in both pre- and post-exposure experiments compared to GASGAS. Finally, GASγGAS reduces post-infection neurological sequelae, compared to control, during mixed intracranial infection with DRV4. These data show IFNγ expression by a vaccine vector can enhance its safety while increasing its efficacy as pre- and post-exposure treatment. - Highlights: • IFNγ expression improves attenuated rabies virus safety and immunogenicity. • IFNγ expression is safer and more immunogenic than doubling glycoprotein expression. • Co-infection with IFNγ-expressing RABV prevents wild-type rabies virus lethality. • Vaccine safety and efficacy is additive for IFNγ and double glycoprotein expression.

  2. Rabies

    MedlinePlus

    ... has rabies, quick treatment can prevent the illness. Animal Bites Rabies is very serious and can make ... important for someone who's been bitten by an animal to see a doctor. This is especially important ...

  3. Innate immune responses in raccoons after raccoon rabies virus infection.

    PubMed

    Srithayakumar, Vythegi; Sribalachandran, Hariharan; Rosatte, Rick; Nadin-Davis, Susan A; Kyle, Christopher J

    2014-01-01

    Zoonotic wildlife diseases pose significant health risks not only to their primary vectors but also to humans and domestic animals. Rabies is a lethal encephalitis caused by rabies virus (RV). This RNA virus can infect a range of terrestrial mammals but each viral variant persists in a particular reservoir host. Active management of these host vectors is needed to minimize the negative impacts of this disease, and an understanding of the immune response to RV infection aids strategies for host vaccination. Current knowledge of immune responses to RV infection comes primarily from rodent models in which an innate immune response triggers activation of several genes and signalling pathways. It is unclear, however, how well rodent models represent the immune response of natural hosts. This study investigates the innate immune response of a primary host, the raccoon, to a peripheral challenge using the raccoon rabies virus (RRV). The extent and temporal course of this response during RRV infection was analysed using genes predicted to be upregulated during infection (IFNs; IFN regulatory factors; IL-6; Toll like receptor-3; TNF receptor). We found that RRV activated components of the innate immune system, with changes in levels of transcripts correlated with presence of viral RNA. Our results suggest that natural reservoirs of rabies may not mimic the immune response triggered in rodent models, highlighting the need for further studies of infection in primary hosts.

  4. Epidemiology and molecular virus characterization of reemerging rabies, South Africa.

    PubMed

    Cohen, Cheryl; Sartorius, Benn; Sabeta, Claude; Zulu, Gugulethu; Paweska, Janusz; Mogoswane, Mamokete; Sutton, Chris; Nel, Louis H; Swanepoel, Robert; Leman, Patricia A; Grobbelaar, Antoinette A; Dyason, Edwin; Blumberg, Lucille

    2007-12-01

    The incidence of dog rabies in Limpopo Province, South Africa, increased from 5 cases in 2004 to 100 in 2006. Human rabies had last been confirmed in 1981, but investigations instituted after an index case was recognized in February 2006 identified 21 confirmed, 4 probable, and 5 possible human cases between August 5, 2005, and December 31, 2006. Twelve of these case-patients were identified retrospectively because the diagnosis of rabies was not considered: 6 of these patients consulted a traditional healer, 6 had atypical manifestations with prominent abdominal symptoms, and 6 of 7 patients tested had elevated liver enzyme activity. Molecular genetic analysis indicated that outbreak virus strains were most closely related to recent canine strains from southern Zimbabwe. Delayed recognition of the human cases may have resulted from decreased clinical suspicion after many years of effective control of the disease and the occurrence of atypical clinical presentations. PMID:18258039

  5. Epidemiology and molecular virus characterization of reemerging rabies, South Africa.

    PubMed

    Cohen, Cheryl; Sartorius, Benn; Sabeta, Claude; Zulu, Gugulethu; Paweska, Janusz; Mogoswane, Mamokete; Sutton, Chris; Nel, Louis H; Swanepoel, Robert; Leman, Patricia A; Grobbelaar, Antoinette A; Dyason, Edwin; Blumberg, Lucille

    2007-12-01

    The incidence of dog rabies in Limpopo Province, South Africa, increased from 5 cases in 2004 to 100 in 2006. Human rabies had last been confirmed in 1981, but investigations instituted after an index case was recognized in February 2006 identified 21 confirmed, 4 probable, and 5 possible human cases between August 5, 2005, and December 31, 2006. Twelve of these case-patients were identified retrospectively because the diagnosis of rabies was not considered: 6 of these patients consulted a traditional healer, 6 had atypical manifestations with prominent abdominal symptoms, and 6 of 7 patients tested had elevated liver enzyme activity. Molecular genetic analysis indicated that outbreak virus strains were most closely related to recent canine strains from southern Zimbabwe. Delayed recognition of the human cases may have resulted from decreased clinical suspicion after many years of effective control of the disease and the occurrence of atypical clinical presentations.

  6. Population structure of two rabies hosts relative to the known distribution of rabies virus variants in Alaska.

    PubMed

    Goldsmith, Elizabeth W; Renshaw, Benjamin; Clement, Christopher J; Himschoot, Elizabeth A; Hundertmark, Kris J; Hueffer, Karsten

    2016-02-01

    For pathogens that infect multiple species, the distinction between reservoir hosts and spillover hosts is often difficult. In Alaska, three variants of the arctic rabies virus exist with distinct spatial distributions. We tested the hypothesis that rabies virus variant distribution corresponds to the population structure of the primary rabies hosts in Alaska, arctic foxes (Vulpes lagopus) and red foxes (Vulpes vulpes) to possibly distinguish reservoir and spillover hosts. We used mitochondrial DNA (mtDNA) sequence and nine microsatellites to assess population structure in those two species. mtDNA structure did not correspond to rabies virus variant structure in either species. Microsatellite analyses gave varying results. Bayesian clustering found two groups of arctic foxes in the coastal tundra region, but for red foxes it identified tundra and boreal types. Spatial Bayesian clustering and spatial principal components analysis identified 3 and 4 groups of arctic foxes, respectively, closely matching the distribution of rabies virus variants in the state. Red foxes, conversely, showed eight clusters comprising two regions (boreal and tundra) with much admixture. These results run contrary to previous beliefs that arctic fox show no fine-scale spatial population structure. While we cannot rule out that the red fox is part of the maintenance host community for rabies in Alaska, the distribution of virus variants appears to be driven primarily by the arctic fox. Therefore, we show that host population genetics can be utilized to distinguish between maintenance and spillover hosts when used in conjunction with other approaches.

  7. Rabies

    MedlinePlus

    ... your pet. Rabies vaccines are available for dogs, cats and farm animals Don't let pets roam Don't approach stray animals. Animals with rabies might be aggressive and vicious, or tired and weak Centers for Disease Control and Prevention

  8. Diversity of currently circulating rabies virus strains in Croatia.

    PubMed

    Lojkić, Ivana; Cac, Zeljko; Bedeković, Tomislav; Lemo, Nina; Brstilo, Mate; Müller, Thomas; Freuling, Conrad M

    2012-01-01

    Sylvatic rabies has been present in Croatia for more than three decades, with the red fox (Vulpes vulpes) as the main reservoir. The present epidemic of sylvatic rabies in Croatia started already in 1977 and in the past ten years the disease has become enzootic in the entire country and thus represents a considerable veterinary and public health threat. A genetic characterization and phylogenetic analysis of rabies virus isolates (RABV) from Croatia was performed using panel of 32 selected rabies-positive brain samples from domestic and wild animals collected between 2008 and 2010. Based on the comparison of 367-nucleotide sequences of a conserved region of the nucleoprotein (N) gene (nucleotides 75-441), the phylogenetic analysis revealed a low genetic diversity of currently circulating RABV strains in Croatia. 18 RABV isolates mainly originating from Eastern Croatia clustered with the formerly established Eastern European (EE) lineage, and the rest (14) were identical with the West European (WE) group. Both phylogenetic groups seem to coincide in central regions on both sides along the Save River. A high sequence identity in the N gene of the RABV isolates from neighbouring countries was found.

  9. Molecular diversity of rabies viruses associated with bats in Mexico and other countries of the Americas.

    PubMed

    Velasco-Villa, Andrés; Orciari, Lillian A; Juárez-Islas, Víctor; Gómez-Sierra, Mauricio; Padilla-Medina, Irma; Flisser, Ana; Souza, Valeria; Castillo, Amanda; Franka, Richard; Escalante-Mañe, Maribel; Sauri-González, Isaias; Rupprecht, Charles E

    2006-05-01

    Bat rabies and its transmission to humans and other species in Mexico were investigated. Eighty-nine samples obtained from rabid livestock, cats, dogs, and humans in Mexico were studied by antigenic typing and partial sequence analysis. Samples were further compared with enzootic rabies associated with different species of bats in the Americas. Patterns of nucleotide variation allowed the definition of at least 20 monophyletic clusters associated with 9 or more different bat species. Several lineages associated with distinctive antigenic patterns were found in rabies viruses related to rabies in vampire bats in Mexico. Vampire bat rabies virus lineages associated with antigenic variant 3 are widely spread from Mexico to South America, suggesting these lineages as the most likely ancestors of vampire bat rabies and the ones that have been moved by vampire bat populations throughout the Americas. Rabies viruses related to Lasiurus cinereus, Histiotus montanus, and some other not yet identified species of the genus Lasiurus were found circulating in Mexico. Long-range dissemination patterns of rabies are not necessarily associated with migratory bat species, as in the case of rabies in Desmodus rotundus and Histiotus montanus. Human rabies was associated with vampire bat transmission in most cases, and in one case, rabies transmission from free-tailed bats was inferred. The occurrence of rabies spillover from bats to domestic animals was also demonstrated. Genetic typing of rabies viruses allowed us to distinguish trends of disease dissemination and to address, in a preliminary fashion, aspects of the complex evolution of rabies viruses in different host-reservoir species.

  10. Molecular Diversity of Rabies Viruses Associated with Bats in Mexico and Other Countries of the Americas

    PubMed Central

    Velasco-Villa, Andrés; Orciari, Lillian A.; Juárez-Islas, Víctor; Gómez-Sierra, Mauricio; Padilla-Medina, Irma; Flisser, Ana; Souza, Valeria; Castillo, Amanda; Franka, Richard; Escalante-Mañe, Maribel; Sauri-González, Isaias; Rupprecht, Charles E.

    2006-01-01

    Bat rabies and its transmission to humans and other species in Mexico were investigated. Eighty-nine samples obtained from rabid livestock, cats, dogs, and humans in Mexico were studied by antigenic typing and partial sequence analysis. Samples were further compared with enzootic rabies associated with different species of bats in the Americas. Patterns of nucleotide variation allowed the definition of at least 20 monophyletic clusters associated with 9 or more different bat species. Several lineages associated with distinctive antigenic patterns were found in rabies viruses related to rabies in vampire bats in Mexico. Vampire bat rabies virus lineages associated with antigenic variant 3 are widely spread from Mexico to South America, suggesting these lineages as the most likely ancestors of vampire bat rabies and the ones that have been moved by vampire bat populations throughout the Americas. Rabies viruses related to Lasiurus cinereus, Histiotus montanus, and some other not yet identified species of the genus Lasiurus were found circulating in Mexico. Long-range dissemination patterns of rabies are not necessarily associated with migratory bat species, as in the case of rabies in Desmodus rotundus and Histiotus montanus. Human rabies was associated with vampire bat transmission in most cases, and in one case, rabies transmission from free-tailed bats was inferred. The occurrence of rabies spillover from bats to domestic animals was also demonstrated. Genetic typing of rabies viruses allowed us to distinguish trends of disease dissemination and to address, in a preliminary fashion, aspects of the complex evolution of rabies viruses in different host-reservoir species. PMID:16672396

  11. Molecular diversity of rabies viruses associated with bats in Mexico and other countries of the Americas.

    PubMed

    Velasco-Villa, Andrés; Orciari, Lillian A; Juárez-Islas, Víctor; Gómez-Sierra, Mauricio; Padilla-Medina, Irma; Flisser, Ana; Souza, Valeria; Castillo, Amanda; Franka, Richard; Escalante-Mañe, Maribel; Sauri-González, Isaias; Rupprecht, Charles E

    2006-05-01

    Bat rabies and its transmission to humans and other species in Mexico were investigated. Eighty-nine samples obtained from rabid livestock, cats, dogs, and humans in Mexico were studied by antigenic typing and partial sequence analysis. Samples were further compared with enzootic rabies associated with different species of bats in the Americas. Patterns of nucleotide variation allowed the definition of at least 20 monophyletic clusters associated with 9 or more different bat species. Several lineages associated with distinctive antigenic patterns were found in rabies viruses related to rabies in vampire bats in Mexico. Vampire bat rabies virus lineages associated with antigenic variant 3 are widely spread from Mexico to South America, suggesting these lineages as the most likely ancestors of vampire bat rabies and the ones that have been moved by vampire bat populations throughout the Americas. Rabies viruses related to Lasiurus cinereus, Histiotus montanus, and some other not yet identified species of the genus Lasiurus were found circulating in Mexico. Long-range dissemination patterns of rabies are not necessarily associated with migratory bat species, as in the case of rabies in Desmodus rotundus and Histiotus montanus. Human rabies was associated with vampire bat transmission in most cases, and in one case, rabies transmission from free-tailed bats was inferred. The occurrence of rabies spillover from bats to domestic animals was also demonstrated. Genetic typing of rabies viruses allowed us to distinguish trends of disease dissemination and to address, in a preliminary fashion, aspects of the complex evolution of rabies viruses in different host-reservoir species. PMID:16672396

  12. Spatial Temporal Dynamics and Molecular Evolution of Re-Emerging Rabies Virus in Taiwan.

    PubMed

    Lin, Yung-Cheng; Chu, Pei-Yu; Chang, Mei-Yin; Hsiao, Kuang-Liang; Lin, Jih-Hui; Liu, Hsin-Fu

    2016-03-17

    Taiwan has been recognized by the World Organization for Animal Health as rabies-free since 1961. Surprisingly, rabies virus (RABV) was identified in a dead Formosan ferret badger in July 2013. Later, more infected ferret badgers were reported from different geographic regions of Taiwan. In order to know its evolutionary history and spatial temporal dynamics of this virus, phylogeny was reconstructed by maximum likelihood and Bayesian methods based on the full-length of glycoprotein (G), matrix protein (M), and nucleoprotein (N) genes. The evolutionary rates and phylogeographic were determined using Beast and SPREAD software. Phylogenetic trees showed a monophyletic group containing all of RABV isolates from Taiwan and it further separated into three sub-groups. The estimated nucleotide substitution rates of G, M, and N genes were between 2.49 × 10(-4)-4.75 × 10(-4) substitutions/site/year, and the mean ratio of dN/dS was significantly low. The time of the most recent common ancestor was estimated around 75, 89, and 170 years, respectively. Phylogeographic analysis suggested the origin of the epidemic could be in Eastern Taiwan, then the Formosan ferret badger moved across the Central Range of Taiwan to western regions and separated into two branches. In this study, we illustrated the evolution history and phylogeographic of RABV in Formosan ferret badgers.

  13. Spatial Temporal Dynamics and Molecular Evolution of Re-Emerging Rabies Virus in Taiwan.

    PubMed

    Lin, Yung-Cheng; Chu, Pei-Yu; Chang, Mei-Yin; Hsiao, Kuang-Liang; Lin, Jih-Hui; Liu, Hsin-Fu

    2016-01-01

    Taiwan has been recognized by the World Organization for Animal Health as rabies-free since 1961. Surprisingly, rabies virus (RABV) was identified in a dead Formosan ferret badger in July 2013. Later, more infected ferret badgers were reported from different geographic regions of Taiwan. In order to know its evolutionary history and spatial temporal dynamics of this virus, phylogeny was reconstructed by maximum likelihood and Bayesian methods based on the full-length of glycoprotein (G), matrix protein (M), and nucleoprotein (N) genes. The evolutionary rates and phylogeographic were determined using Beast and SPREAD software. Phylogenetic trees showed a monophyletic group containing all of RABV isolates from Taiwan and it further separated into three sub-groups. The estimated nucleotide substitution rates of G, M, and N genes were between 2.49 × 10(-4)-4.75 × 10(-4) substitutions/site/year, and the mean ratio of dN/dS was significantly low. The time of the most recent common ancestor was estimated around 75, 89, and 170 years, respectively. Phylogeographic analysis suggested the origin of the epidemic could be in Eastern Taiwan, then the Formosan ferret badger moved across the Central Range of Taiwan to western regions and separated into two branches. In this study, we illustrated the evolution history and phylogeographic of RABV in Formosan ferret badgers. PMID:26999115

  14. Spatial Temporal Dynamics and Molecular Evolution of Re-Emerging Rabies Virus in Taiwan

    PubMed Central

    Lin, Yung-Cheng; Chu, Pei-Yu; Chang, Mei-Yin; Hsiao, Kuang-Liang; Lin, Jih-Hui; Liu, Hsin-Fu

    2016-01-01

    Taiwan has been recognized by the World Organization for Animal Health as rabies-free since 1961. Surprisingly, rabies virus (RABV) was identified in a dead Formosan ferret badger in July 2013. Later, more infected ferret badgers were reported from different geographic regions of Taiwan. In order to know its evolutionary history and spatial temporal dynamics of this virus, phylogeny was reconstructed by maximum likelihood and Bayesian methods based on the full-length of glycoprotein (G), matrix protein (M), and nucleoprotein (N) genes. The evolutionary rates and phylogeographic were determined using Beast and SPREAD software. Phylogenetic trees showed a monophyletic group containing all of RABV isolates from Taiwan and it further separated into three sub-groups. The estimated nucleotide substitution rates of G, M, and N genes were between 2.49 × 10−4–4.75 × 10−4 substitutions/site/year, and the mean ratio of dN/dS was significantly low. The time of the most recent common ancestor was estimated around 75, 89, and 170 years, respectively. Phylogeographic analysis suggested the origin of the epidemic could be in Eastern Taiwan, then the Formosan ferret badger moved across the Central Range of Taiwan to western regions and separated into two branches. In this study, we illustrated the evolution history and phylogeographic of RABV in Formosan ferret badgers. PMID:26999115

  15. Report of isolations of unusual lyssaviruses (rabies and Mokola virus) identified retrospectively from Zimbabwe.

    PubMed

    Bingham, J; Javangwe, S; Sabeta, C T; Wandeler, A I; Nel, L H

    2001-06-01

    Rabies isolates that had been stored between 1983 and 1997 were examined with a panel of anti-lyssavirus nucleocapsid monoclonal antibodies. Out of 56 isolates from cats and various wild carnivore species, 1 isolate of Mokola virus and 5 other non-typical rabies viruses were identified. The Mokola virus isolate was diagnosed as rabies in 1993 from a cat. Genetic analysis of this isolate suggests that it falls in a distinct subgroup of the Mokola virus genotype. The 5 non-typical rabies viruses were isolated from honey badgers (Mellivora capensis), African civets (Civettictis civetta) and an unidentified mongoose (Herpestidae). These isolates are representatives of rarely-reported wildlife-associated strains of rabies, probably maintained by the slender mongoose (Galerella sanguinea). These findings indicate that both Mokola virus and the mongoose-associated variant may be more common in Zimbabwe than is apparent from routine surveillance. PMID:11513267

  16. iTRAQ protein profile analysis of neuroblastoma (NA) cells infected with the rabies viruses rHep-Flury and Hep-dG

    PubMed Central

    Yang, Youtian; Liu, Wenjun; Yan, Guangrong; Luo, Yongwen; Zhao, Jing; Yang, Xianfeng; Mei, Mingzhu; Wu, Xiaowei; Guo, Xiaofeng

    2015-01-01

    The rabies virus (RABV) glycoprotein (G) is the principal contributor to the pathogenicity and protective immunity of RABV. In a previous work, we reported that recombinant rabies virus Hep-dG, which was generated by reverse genetics to carry two copies of the G-gene, showed lower virulence than the parental virus rHep-Flury in suckling mice with a better immune protection effect. To better understand the mechanisms underlying rabies virus attenuation and the role of glycoprotein G, isobaric tags for relative and absolute quantitation (iTRAQ) was performed to identify and quantify distinct proteins. 10 and 111 differentially expressed proteins were obtained in rHep-Flury and Hep-dG infection groups, respectively. Selected data were validated by western blot and qRT-PCR. Bioinformatics analysis of the distinct protein suggested that glycoprotein over-expression in the attenuated RABV strain can induce activation of the interferon signaling. Furthermore, it may promote the antiviral response, MHC-I mediated antigen-specific T cell immune response, apoptosis and autophagy in an IFN-dependent manner. These findings might not only improve the understanding of the dynamics of RABV and host interaction, but also help understand the mechanisms underlying innate and adaptive immunity during RABV infection. PMID:26217322

  17. 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.

  18. [Study on the B cell linear epitopes of rabies virus CVS-11 nucleoprotein].

    PubMed

    Lv, Xin-Jun; Shen, Xin-Xin; Yu, Peng-Cheng; Li, Hao; Wang, Li-Hua; Tang, Qing; Liang, Guo-Dong

    2014-05-01

    To study the B cell linear epitopes of rabies virus CVS-11 nucleoprotein, peptides were synthesized according to the amino acid sequences of B cell linear epitopes. Linear epitopes predicted by bioinformatics analysis were evaluated with immunological techniques. Indirect enzyme-linked immunosorbent assay showed that titers of antibodies to peptides (355-369 and 385-400 residues of rabies virus CVS-11 nucleoprotein) were above 1:12 800 in mouse sera. The antibodies recognized denatured rabies virus CVS-11 nucleoprotein in Western blot analysis. Purified anti-peptide antibodies recognized natural rabies virus CVS-11 nucleoprotein in BHK-21 cells in indirect fluorescent antibody test. The 355-369 and 385-400 residues of rabies virus CVS-11 nucleoprotein were validated as B cell linear epitopes.

  19. [Analysis of full-length gene sequence of rabies vaccine virus aG strain].

    PubMed

    Li, Jia; Cao, Shou-Chun; Shi, Lei-Tai; Wu, Xiao-Hong; Liu, Jing-Hua; Wang, Yun-Peng; Tang, Jian-Rong; Yu, Yong-Xin; Dong, Guan-Mu

    2013-06-01

    To sequence and analyze the full-length gene sequence of rabies vaccine virus aG strain. The full-length gene sequence of aG strain was amplified by RT-PCR by 8 fragments,each PCR product was cloned into vector pGEM-T respectively, sequenced and assemblied; The 5' leader sequence was sequenced with method of 5' RACE. The homology between aG and other rabies vaccine virus was analyzed by using DNAstar and Mega4. 0 software. aG strain was 11 925nt(GenBank accession number: JN234411) in length and belonged to the genotype I . The Bioinformatics revealed that the homology showed disparation form different rabies vaccine virus. the full-length gene sequence of rabies vaccine virus aG strain provided a support for perfecting the standard for quality control of virus strains for production of rabies vaccine for human use in China.

  20. Hsp70 protein positively regulates rabies virus infection.

    PubMed

    Lahaye, Xavier; Vidy, Aurore; Fouquet, Baptiste; Blondel, Danielle

    2012-05-01

    The Hsp70 chaperone plays a central role in multiple processes within cells, including protein translation, folding, intracellular trafficking, and degradation. This protein is implicated in the replication of numerous viruses. We have shown that rabies virus infection induced the cellular expression of Hsp70, which accumulated in Negri body-like structures, where viral transcription and replication take place. In addition, Hsp70 is present in both nucleocapsids purified from infected cells and in purified virions. Hsp70 has been shown to interact with the nucleoprotein N. The downregulation of Hsp70, using specific chaperone inhibitors, such as quercetin or RNA interference, resulted in a significant decrease of the amount of viral mRNAs, viral proteins, and virus particles. These results indicate that Hsp70 has a proviral function during rabies virus infection and suggest that Hsp70 is involved in at least one stage(s) of the viral life cycle, such as viral transcription, translation, and/or production. The mechanism by which Hsp70 controls viral infection will be discussed.

  1. Antigenic and genetic characterization of rabies virus isolates from Uruguay.

    PubMed

    Guarino, Helena; Castilho, Juliana Galera; Souto, Juanita; Oliveira, Rafael de Novaes; Carrieri, Maria Luiza; Kotait, Ivanete

    2013-05-01

    After 25 years without any reported cases of rabies in Uruguay, the northern region of the country experienced an epizootic of bovine paralytic rabies in October 2007. The outbreak affected bovines and equines, and the main source of infection was the bat Desmodus rotundus, the only hematophagous species in the country. From October 2007 to July 2008, 42 bovine, 3 equine and 120 chiropteran samples were submitted to the National Veterinary Diagnostic Laboratory for rabies testing. A total of 12 samples (7 bovine, 2 equine and 3 from D. rotundus) were positive by the fluorescent antibody test, and viruses were isolated by the mouse inoculation test. The objective of this study was to compare the antigenic and genetic characteristics of these isolates and three isolates from insectivorous bats from other regions. Antigenic typing using a panel of eight monoclonal antibodies identified all 12 viruses as variant 3 (AgV3), a variant associated with D. rotundus. Two isolates from insectivorous bats (Tadarida brasiliensis and Molossus sp.) were characterized as antigenic variant 4 (AgV4) while the third, from Myotis sp., could not be characterized using this panel as its reactivity pattern did not match that of any of the known antigenic variants. Partial N-gene sequences (nt 149-1420) of these isolates were aligned with homologous sequences derived from GenBank by the CLUSTAL/W method and used to build a neighbor-joining distance tree with the Kimura 2-parameter model. All 12 isolates were genetically grouped into the D. rotundus cluster as they shared 100% identity. In the phylogenetic analysis, the three isolates from insectivorous bats segregated into three clusters: one related to T. brasiliensis, one to Myotis sp. and the other to Lasiurus sp., although the isolate associated with the latter came from a Molossus sp. specimen. These results indicate that AgV3 was associated with the outbreak of bovine paralytic rabies in Uruguay. This is the first report of rabies

  2. Genetic characterization and phylogenetic analysis of skunk-associated rabies viruses in North America with special emphasis on the central plains.

    PubMed

    Davis, Rolan; Nadin-Davis, Susan A; Moore, Michael; Hanlon, Cathleen

    2013-06-01

    Across North America the skunk acts as a reservoir for several rabies virus variants. Some of these variants are geographically restricted in range as is the case for the California skunk variant and two distinct variants present in Mexico. In contrast the North Central and South Central skunk rabies viruses are dispersed in overlapping ranges over large areas of the Midwestern region of the United States with the former extending into southern parts of the Canadian prairies. Despite this extensive range, there has been only very limited molecular characterization of these two viral variants. This study has examined the genetic diversity of the rabies viruses associated with North American skunks, with particular emphasis on the South Central skunk variant which was found to comprise three distinct geographically restricted groups of viruses that could in some cases be further sub-divided. The phylogenetic relationships of these groups and sub-groups allowed us to infer the likely direction of spread of these variants in some instances. Patterns of amino acid replacement of North American skunk-associated rabies viruses for both the nucleoprotein and glycoprotein products are also examined. These patterns reflect the virus phylogeny but no amino acid residues associated specifically with the skunk host were identified. PMID:23524137

  3. Genetic characterization and phylogenetic analysis of skunk-associated rabies viruses in North America with special emphasis on the central plains.

    PubMed

    Davis, Rolan; Nadin-Davis, Susan A; Moore, Michael; Hanlon, Cathleen

    2013-06-01

    Across North America the skunk acts as a reservoir for several rabies virus variants. Some of these variants are geographically restricted in range as is the case for the California skunk variant and two distinct variants present in Mexico. In contrast the North Central and South Central skunk rabies viruses are dispersed in overlapping ranges over large areas of the Midwestern region of the United States with the former extending into southern parts of the Canadian prairies. Despite this extensive range, there has been only very limited molecular characterization of these two viral variants. This study has examined the genetic diversity of the rabies viruses associated with North American skunks, with particular emphasis on the South Central skunk variant which was found to comprise three distinct geographically restricted groups of viruses that could in some cases be further sub-divided. The phylogenetic relationships of these groups and sub-groups allowed us to infer the likely direction of spread of these variants in some instances. Patterns of amino acid replacement of North American skunk-associated rabies viruses for both the nucleoprotein and glycoprotein products are also examined. These patterns reflect the virus phylogeny but no amino acid residues associated specifically with the skunk host were identified.

  4. 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.

  5. Subversion of the Immune Response by Rabies Virus

    PubMed Central

    Scott, Terence P.; Nel, Louis H.

    2016-01-01

    Rabies has affected mankind for several centuries and is one of the oldest known zoonoses. It is peculiar how little is known regarding the means by which rabies virus (RABV) evades the immune response and kills its host. This review investigates the complex interplay between RABV and the immune system, including the various means by which RABV evades, or advantageously utilizes, the host immune response in order to ensure successful replication and spread to another host. Different factors that influence immune responses—including age, sex, cerebral lateralization and temperature—are discussed, with specific reference to RABV and the effects on host morbidity and mortality. We also investigate the role of apoptosis and discuss whether it is a detrimental or beneficial mechanism of the host’s response to infection. The various RABV proteins and their roles in immune evasion are examined in depth with reference to important domains and the downstream effects of these interactions. Lastly, an overview of the means by which RABV evades important immune responses is provided. The research discussed in this review will be important in determining the roles of the immune response during RABV infections as well as to highlight important therapeutic target regions and potential strategies for rabies treatment. PMID:27548204

  6. Subversion of the Immune Response by Rabies Virus.

    PubMed

    Scott, Terence P; Nel, Louis H

    2016-01-01

    Rabies has affected mankind for several centuries and is one of the oldest known zoonoses. It is peculiar how little is known regarding the means by which rabies virus (RABV) evades the immune response and kills its host. This review investigates the complex interplay between RABV and the immune system, including the various means by which RABV evades, or advantageously utilizes, the host immune response in order to ensure successful replication and spread to another host. Different factors that influence immune responses-including age, sex, cerebral lateralization and temperature-are discussed, with specific reference to RABV and the effects on host morbidity and mortality. We also investigate the role of apoptosis and discuss whether it is a detrimental or beneficial mechanism of the host's response to infection. The various RABV proteins and their roles in immune evasion are examined in depth with reference to important domains and the downstream effects of these interactions. Lastly, an overview of the means by which RABV evades important immune responses is provided. The research discussed in this review will be important in determining the roles of the immune response during RABV infections as well as to highlight important therapeutic target regions and potential strategies for rabies treatment. PMID:27548204

  7. Subversion of the Immune Response by Rabies Virus.

    PubMed

    Scott, Terence P; Nel, Louis H

    2016-08-19

    Rabies has affected mankind for several centuries and is one of the oldest known zoonoses. It is peculiar how little is known regarding the means by which rabies virus (RABV) evades the immune response and kills its host. This review investigates the complex interplay between RABV and the immune system, including the various means by which RABV evades, or advantageously utilizes, the host immune response in order to ensure successful replication and spread to another host. Different factors that influence immune responses-including age, sex, cerebral lateralization and temperature-are discussed, with specific reference to RABV and the effects on host morbidity and mortality. We also investigate the role of apoptosis and discuss whether it is a detrimental or beneficial mechanism of the host's response to infection. The various RABV proteins and their roles in immune evasion are examined in depth with reference to important domains and the downstream effects of these interactions. Lastly, an overview of the means by which RABV evades important immune responses is provided. The research discussed in this review will be important in determining the roles of the immune response during RABV infections as well as to highlight important therapeutic target regions and potential strategies for rabies treatment.

  8. Evaluation of cytokines concentration and percentage of survival of rabies virus-infected mice submitted to anti-rabies Vero-cell propagated vaccine and P. acnes.

    PubMed

    Megid, J; Appolinario, C M; Mazzini, A M; Almeida, M F

    2006-11-15

    Previously, survival of rabies infection was shown to correlate with low IL-6 serum concentration in mice subjected to post-exposure treatment with the Fuenzalida Palacios rabies vaccine in conjunction with the immunomodulator Propionibacterium acnes, previously Corynebacterium parvum. Considering the substitution of the Fuenzalida Palacios rabies vaccine by the Vero cell raised anti-rabies vaccine in almost all countries, the objective of this work was to evaluate the survival and cytokine serum concentration of rabies virus-infected mice treated with P. acnes in conjunction with or the anti-rabies-VERO vaccine. For this, Swiss mice were experimentally infected with street rabies virus and subjected to vaccine and/or P. acnes following infection. Animals were killed at different times and serum was collected to evaluate cytokines. The greatest survival was observed in animals given one or two does of P. acnes in the absence of vaccination. Animals given anti-rabies VERO vaccine alone or with three doses of P. acnes had the second highest survival rate. The group that had the highest percentage of mortality also had the highest IL-6 concentration on the 10th day, a time correlating with clinical symptoms of the animals. The results reinforce the inefficacy of anti-rabies vaccine in only one dose as a post-exposure treatment irrespective of the type of vaccine used, the immunomodulation activity of P. acnes in rabies post-exposure treatment and suggest a role for IL-6 in rabies virus pathogenesis. PMID:16930720

  9. Intracellular Spread of Rabies Virus Is Reduced in the Paralytic Form of Canine Rabies Compared to the Furious Form

    PubMed Central

    Shuangshoti, Shanop; Thorner, Paul Scott; Teerapakpinyo, Chinachote; Thepa, Nisachol; Phukpattaranont, Pornchai; Intarut, Nirun; Lumlertdacha, Boonlert; Tepsumethanon, Veera; Hemachudha, Thiravat

    2016-01-01

    Studies of the furious and paralytic forms of canine rabies at the early stage of disease have shown a more rapid viral colonization of the cerebral hemispheres in the furious form, as measured by viral antigen within neuronal cell bodies and viral RNA levels. Measurement of cellular processes separate from neuronal cell body provides a visual record of the spread of rabies virus which occurs across synapses. In this study, the amount of rabies viral antigen within cell processes was quantitatively assessed by image analysis in a cohort of naturally rabies infected non-vaccinated dogs (5 furious and 5 paralytic) that were sacrificed shortly after developing illness. Measurements were taken at different levels of the spinal cord, brain stem, and cerebrum. Results were compared to the amount of rabies viral antigen in neuronal cell bodies. Generally, the amount of rabies viral antigen in cell processes decreased in a rostral direction, following the pattern for the amount of rabies viral antigen in neuronal cell bodies and the percentage of involved cell bodies. However, there was a delay in cell process involvement following cell body involvement, consistent with replication occurring in the cell body region and subsequent transport out to cell processes. Greater amounts of antigen were seen in cell processes in dogs with the furious compared to paralytic form, at all anatomic levels examined. This difference was even evident when comparing (1) neurons with similar amounts of antigen, (2) similar percentages of involved neurons, and (3) anatomic levels that showed 100% positive neurons. These findings suggest that intracellular transport of the virus may be slower in the paralytic form, resulting in slower viral propagation. Possible mechanisms might involve host-specific differences in intracellular virus transport. The latter could be cytokine-mediated, since previous studies have documented greater inflammation in the paralytic form. PMID:27253394

  10. Infection of cultured rat myotubes and neurons from the spinal cord by rabies virus.

    PubMed

    Tsiang, H; de la Porte, S; Ambroise, D J; Derer, M; Koenig, J

    1986-01-01

    Rabies virus multiplication was investigated in cultured primary rat myotubes and neurons. The susceptibility of these two cell types to fixed rabies challenge virus strain (CVS) was monitored by fluorescence and virus titration. Differentiated rat myotubes were susceptible to rabies virus infection, and showed an increasing accumulation of viral material from day one to day four. However, these cells did not release infective viral particles, nor did they accumulate infectious virions in the cytoplasm. In contrast, infected neurons released large amounts of infectious particles. Electron microscopy observation of infected myotubes showed minor alterations and the presence of typical viral inclusions in the cytoplasm without mature virions assembling viral membranes. Competition binding experiments show that alpha-bungarotoxin inhibits rabies virus infection from 10(-5) to 10(-7) M, whereas lower toxin concentrations failed to have any effect. These data do not confirm the hypothesis of a fixed rabies virus amplification step at the site of the viral entry. On the other hand, the high susceptibility of peripheral neurons to rabies virus infection is an argument for the direct uptake of virions by these cells. The restrictive viral multiplication in the myotubes is an alternative explanation for the local persistence of rabies virus at the site of inoculation.

  11. Uptake of Rabies Virus into Epithelial Cells by Clathrin-Mediated Endocytosis Depends upon Actin

    PubMed Central

    Piccinotti, Silvia

    2013-01-01

    Rabies virus (RABV) causes a fatal zoonotic encephalitis. Disease symptoms require replication and spread of the virus within neuronal cells; however, in infected animals as well as in cell culture the virus replicates in a broad range of cell types. Here we use a single-cycle RABV and a recombinant vesicular stomatitis virus (rVSV) in which the glycoprotein (G) was replaced with that of RABV (rVSV RABV G) to examine RABV uptake into the African green monkey kidney cell line BS-C-1. Combining biochemical studies and real-time spinning-disk confocal fluorescence microscopy, we show that the predominant entry pathway of RABV particles into BS-C-1 cells is clathrin dependent. Viral particles enter cells in pits with elongated structures and incomplete clathrin coats which depend upon actin to complete the internalization process. By measuring the time of internalization and the abundance of the clathrin adaptor protein AP2, we further show that the pits that internalize RABV particles are similar to those that internalize VSV particles. Pharmacological perturbations of dynamin or of actin polymerization inhibit productive infection, linking our observations on particle uptake with viral infectivity. This work extends to RABV particles the finding that clathrin-mediated endocytosis of rhabdoviruses proceeds through incompletely coated pits which depend upon actin. PMID:23966407

  12. Rabies Epidemiology and Control in Ecuador

    PubMed Central

    Ortiz-Prado, Esteban; Ponce-Zea, Jorge; Ramirez, Dario; Stewart-Ibarra, Anna M.; Armijos, Luciana; Yockteng, Jaime; Cárdenas, Washington B.

    2016-01-01

    Objective: Describe the epidemiology and the control effort for rabies in Ecuador. Methods: This observational study included data from the Ecuadorian National Institute of Census and Statistics (INEC), and mortality and morbidity data reported by the Ministry of Public Health and the National Institute for Social Security. We conducted a phylogeny analyses to compare the N gene from the Challenge Virus Standard (CVS) vaccine strain used in Ecuador with published Cosmopolitan, Asian and Sylvatic strains. Descriptive and inferential statistics were used to determine the significance of the data. Results: In 1996 Ecuador suffered the highest rate of rabies per capita in the Americas, with an incidence rate of 0.56 cases per 100 000 people per year. Human and canine rabies showed a sharp decline until 2012. Between 1994 and 2014, we found a correlation of 0.925 (p<0.01) between annual cases of dog and human rabies. In 2011, there was an epidemic of sylvatic rabies transmitted to people by vampire bats (Desmodus rotundus) in the Amazon region, specifically in Morona Santiago, leading to 11 fatalities. Phylogenetic analyses of the CVS vaccine N gene showed an association with urban canine rabies strains (the Cosmopolitan lineage and Asian strains), whereas sylvatic rabies, like those reported in the Amazon region, were found to be grouped in a different clade represented mainly by bat-derived strains. Conclusions: This study presents the first compilation of epidemiological data on rabies in Ecuador. The incidence of human and canine rabies, also known as urban rabies, has clearly decreased due to massive canine vaccination campaigns. Phylogenetic analysis of the prevailing vaccine used in the country showed a clear separation from bat-derived rabies, the source of recent rabies outbreaks. Efforts are ongoing to develop rabies vaccines that are highly specific to the rabies virus genotype circulating in the region, including sylvatic rabies. These efforts include the

  13. Experimental inoculation of raccoons (Procyon lotor) with rabies virus of skunk origin.

    PubMed

    Hill, R E; Beran, G W

    1992-01-01

    To determine raccoon (Procyon lotor) susceptibility and serum neutralizing antibody response to a skunk salivary gland rabies virus, raccoons were inoculated with a rabies virus isolated from a naturally-infected striped skunk (Mephitis mephitis). Raccoons were divided into four groups of three animals each. A dilution of the rabies virus suspension, 10(2.4), 10(3.4), or 10(4.8), mouse intracerebral lethal dose50 (MICLD50), was administered into the masseter muscles of each animal. Three negative control animals received only diluent. Saliva and sera were collected on post-inoculation days 35, 63 and 92 for virus isolation and determination of serum neutralizing antibody titer. All animals survived the 92 day observation period and none exhibited the behavioral changes classically associated with clinical rabies virus infections. Rabies virus was not detected in the saliva of any raccoon and two of the three animals receiving the highest inoculum developed serum neutralizing antibodies (SNA). On day 92, a challenge suspension of New York City/Georgia (NYC/GA) strain rabies virus in fox salivary glands (10(3.2) MICLD50) was administered to all 12 raccoons. All animals succumbed to rabies virus except the two animals that had earlier developed SNA. The results of this study provided evidence about the susceptibility of raccoons to a skunk rabies virus and demonstrated that exposed raccoons could survive for at least 92 days following exposure. Furthermore, animals developing SNA under such circumstances were capable of withstanding challenge with rabies virus that was fatal for seronegative raccoons.

  14. Genetic characterization of rabies viruses isolated from frugivorous bat (Artibeus spp.) in Brazil.

    PubMed

    Shoji, Youko; Kobayashi, Yuki; Sato, Go; Itou, Takuya; Miura, Yasuo; Mikami, Takeshi; Cunha, Elenice M S; Samara, Samir I; Carvalho, Adlorata A B; Nocitti, Darci P; Ito, Fumio H; Kurane, Ichiro; Sakai, Takeo

    2004-10-01

    In Latin America, rabies cases related to frugivorous bats have been reported since 1930's. Recently, two viruses isolated from Artibeus lituratus were proved to be vampire bat variants by monoclonal antibodies panels [2], but their genetic information is not well known. In this report, four rabies viruses were isolated from frugivorous bats (Artibeus spp.) in Brazil and their nucleoprotein gene sequences were determined. These isolates were found to be genotype 1 of lyssavirus and showed the maximum nucleotide sequence homology of 97.6-99.4% with vampire bat-related viruses in Brazil [6]. These results indicate that the Brazilian frugivorous bat rabies viruses in this study are closely related to vampire bat-related viruses that play a main role in rabies virus transmission to livestock in Brazil. PMID:15528863

  15. Attenuated rabies virus activates, while pathogenic rabies virus evades, the host innate immune responses in the central nervous system.

    PubMed

    Wang, Zhi W; Sarmento, Luciana; Wang, Yuhuan; Li, Xia-qing; Dhingra, Vikas; Tseggai, Tesfai; Jiang, Baoming; Fu, Zhen F

    2005-10-01

    Rabies virus (RV) induces encephalomyelitis in humans and animals. However, the pathogenic mechanism of rabies is not fully understood. To investigate the host responses to RV infection, we examined and compared the pathology, particularly the inflammatory responses, and the gene expression profiles in the brains of mice infected with wild-type (wt) virus silver-haired bat RV (SHBRV) or laboratory-adapted virus B2C, using a mouse genomic array (Affymetrix). Extensive inflammatory responses were observed in animals infected with the attenuated RV, but little or no inflammatory responses were found in mice infected with wt RV. Furthermore, attenuated RV induced the expression of the genes involved in the innate immune and antiviral responses, especially those related to the alpha/beta interferon (IFN-alpha/beta) signaling pathways and inflammatory chemokines. For the IFN-alpha/beta signaling pathways, many of the interferon regulatory genes, such as the signal transduction activation transducers and interferon regulatory factors, as well as the effector genes, for example, 2'-5'-oligoadenylate synthetase and myxovirus proteins, are highly induced in mice infected with attenuated RV. However, many of these genes were not up-regulated in mice infected with wt SHBRV. The data obtained by microarray analysis were confirmed by real-time PCR. Together, these data suggest that attenuated RV activates, while pathogenic RV evades, the host innate immune and antiviral responses.

  16. Resistance of mice vaccinated with rabies virus internal structural proteins to lethal infection.

    PubMed

    Takita-Sonoda, Y; Fujii, H; Mifune, K; Ito, Y; Hiraga, M; Nishizono, A; Mannen, K; Minamoto, N

    1993-01-01

    Mice were vaccinated with recombinant vaccinia virus (rVac) expressing the glycoprotein (G), nucleoprotein (N), phosphoprotein (NS) or matrix protein (M) of rabies virus and their resistance to peripheral lethal infection with street rabies virus was examined. Mice vaccinated with rVac-G or rVac-N developed strong antibody responses to the corresponding proteins and essentially all mice survived challenge infection. Mice vaccinated with rVac-NS or rVac-M developed only a slight antibody response, however, a significant protection (59%) was observed in the rVac-NS-vaccinated mice, whereas rVac-M-vaccinated mice were not protected. No anti-G antibodies were detected in the sera of mice which has been vaccinated with rVac-N or rVac-NS and survived challenge infection. Passive transfer of anti-N monoclonal antibodies (MAbs) recognizing an epitope located on amino acids 1-224 of the protein prior to challenge resulted in significant protection, although the protection was not complete even with a high amount of antibodies. In contrast, none of the mice given MAbs recognizing an epitope of amino acids 247-415 or F(ab')2 fragments from a protective MAb IgG were protected. Administration of anti-CD 8 MAb to rVac-N-vaccinated mice showed no significant effect on protection. Our observations suggest that a considerable part of the protection achieved by the vaccination with rVac-N can be ascribed to the intact anti-N antibodies recognizing an epitope located on amino acids 1-224 of the protein.

  17. Rabies (image)

    MedlinePlus

    ... messages between the brain and the body. The rabies virus spreads through the nerves, first causing flu- ... to hallucinations, delirium, and insomnia. If left untreated, rabies is nearly always fatal.

  18. Expression of rabies glycoprotein and ricin toxin B chain (RGP-RTB) fusion protein in tomato hairy roots: a step towards oral vaccination for rabies.

    PubMed

    Singh, Ankit; Srivastava, Subhi; Chouksey, Ankita; Panwar, Bhupendra Singh; Verma, Praveen C; Roy, Sribash; Singh, Pradhyumna K; Saxena, Gauri; Tuli, Rakesh

    2015-04-01

    Transgenic hairy roots of Solanum lycopersicum were engineered to express a recombinant protein containing a fusion of rabies glycoprotein and ricin toxin B chain (rgp-rtxB) antigen under the control of constitutive CaMV35S promoter. Asialofetuin-mediated direct ELISA of transgenic hairy root extracts was performed using polyclonal anti-rabies antibodies (Ab1) and epitope-specific peptidal anti-RGP (Ab2) antibodies which confirmed the expression of functionally viable RGP-RTB fusion protein. Direct ELISA based on asialofetuin-binding activity was used to screen crude protein extracts from five transgenic hairy root lines. Expressions of RGP-RTB fusion protein in different tomato hairy root lines varied between 1.4 and 8 µg in per gram of tissue. Immunoblotting assay of RGP-RTB fusion protein from these lines showed a protein band on monomeric size of ~84 kDa after denaturation. Tomato hairy root line H03 showed highest level of RGP-RTB protein expression (1.14 %) and was used further in bench-top bioreactor for the optimization of scale-up process to produce large quantity of recombinant protein. Partially purified RGP-RTB fusion protein was able to induce the immune response in BALB/c mice after intra-mucosal immunization. In the present investigation, we have not only successfully scaled up the hairy root culture but also established the utility of this system to produce vaccine antigen which subsequently will reduce the total production cost for implementing rabies vaccination programs in developing nations. This study in a way aims to provide consolidated base for low-cost preparation of improved oral vaccine against rabies. PMID:25519901

  19. PATHOLOGY AND MOLECULAR DETECTION OF RABIES VIRUS IN FERRET BADGERS ASSOCIATED WITH A RABIES OUTBREAK IN TAIWAN.

    PubMed

    Chiou, Hue-Ying; Jeng, Chian-Ren; Wang, Hurng-Yi; Inoue, Satoshi; Chan, Fang-Tse; Liao, Jiunn-Wang; Chiou, Ming-Tang; Pang, Victor Fei

    2016-01-01

    Until Rabies virus (RABV) infection in Taiwan ferret badgers (TWFB; Melogale moschata subaurantiaca) was diagnosed in mid-June 2013, Taiwan had been considered rabies free for >50 yr. Although rabies has also been reported in ferret badgers in China, the pathologic changes and distribution of viral antigens of ferret badger-associated rabies have not been described. We performed a comprehensive pathologic study and molecular detection of rabies virus in three necropsied rabid TWFBs and evaluated archival paraffin-embedded tissue blocks of six other TWFBs necropsied during 2004 and 2012. As in other RABV-infected species, the characteristic pathologic changes in TWFBs were nonsuppurative meningoencephalomyelitis, ganglionitis, and the formation of typical intracytoplasmic Negri bodies, with the brain stem most affected. There was also variable spongiform degeneration, primarily in the perikaryon of neurons and neuropil, in the cerebral cortex, thalamus, and brain stem. In nonnervous system tissues, representative lesions included adrenal necrosis and lymphocytic interstitial sialadenitis. Immunohistochemical staining and fluorescent antibody test demonstrated viral antigens in the perikaryon of the neurons and axonal or dendritic processes throughout the nervous tissue and in the macrophages in various tissues. Similar to raccoons (Procyon lotor) and skunks (Mephitidae), the nervous tissue of rabid TWFBs displayed widely dispersed lesions, RABV antigens, and large numbers of Negri bodies. We traced the earliest rabid TWFB case back to 2004. PMID:26560756

  20. PATHOLOGY AND MOLECULAR DETECTION OF RABIES VIRUS IN FERRET BADGERS ASSOCIATED WITH A RABIES OUTBREAK IN TAIWAN.

    PubMed

    Chiou, Hue-Ying; Jeng, Chian-Ren; Wang, Hurng-Yi; Inoue, Satoshi; Chan, Fang-Tse; Liao, Jiunn-Wang; Chiou, Ming-Tang; Pang, Victor Fei

    2016-01-01

    Until Rabies virus (RABV) infection in Taiwan ferret badgers (TWFB; Melogale moschata subaurantiaca) was diagnosed in mid-June 2013, Taiwan had been considered rabies free for >50 yr. Although rabies has also been reported in ferret badgers in China, the pathologic changes and distribution of viral antigens of ferret badger-associated rabies have not been described. We performed a comprehensive pathologic study and molecular detection of rabies virus in three necropsied rabid TWFBs and evaluated archival paraffin-embedded tissue blocks of six other TWFBs necropsied during 2004 and 2012. As in other RABV-infected species, the characteristic pathologic changes in TWFBs were nonsuppurative meningoencephalomyelitis, ganglionitis, and the formation of typical intracytoplasmic Negri bodies, with the brain stem most affected. There was also variable spongiform degeneration, primarily in the perikaryon of neurons and neuropil, in the cerebral cortex, thalamus, and brain stem. In nonnervous system tissues, representative lesions included adrenal necrosis and lymphocytic interstitial sialadenitis. Immunohistochemical staining and fluorescent antibody test demonstrated viral antigens in the perikaryon of the neurons and axonal or dendritic processes throughout the nervous tissue and in the macrophages in various tissues. Similar to raccoons (Procyon lotor) and skunks (Mephitidae), the nervous tissue of rabid TWFBs displayed widely dispersed lesions, RABV antigens, and large numbers of Negri bodies. We traced the earliest rabid TWFB case back to 2004.

  1. Disease outbreaks caused by steppe-type rabies viruses in China.

    PubMed

    Feng, Y; Wang, W; Guo, J; Alatengheli; Li, Y; Yang, G; Su, N; Zhang, L; Xu, W; Sheng, Z; Ma, L; Gui, J; Dejide; Lin, H; Tu, C

    2015-04-01

    While rabies is a significant public health concern in China, the epidemiology of animal rabies in the north and northwest border provinces remains unknown. From February 2013 to March 2014, seven outbreaks of domestic animal rabies caused by wild carnivores in Xinjiang (XJ) and Inner Mongolia (IM) Autonomous Regions, China were reported and diagnosed in brain samples of infected animals by the fluorescent antibody test (FAT) and RT-PCR. Ten field rabies viruses were obtained. Sequence comparison and phylogenetic analysis based on the complete N gene (1353 bp) amplified directly from the original brain tissues showed that these ten strains were steppe-type viruses, closely related to strains reported in Russia and Mongolia. None had been identified previously in China. The viruses from XJ and IM clustered separately into two lineages showing their different geographical distribution. This study emphasizes the importance of wildlife surveillance and of cross-departmental cooperation in the control of transboundary rabies transmission. PMID:25078967

  2. Detection of rabies virus antibodies in Brazilian free-ranging wild carnivores.

    PubMed

    Jorge, Rodrigo Silva Pinto; Pereira, Monicque Silva; Morato, Ronaldo Gonçalves; Scheffer, Karin C; Carnieli, Pedro; Ferreira, Fernando; Furtado, Mariana Malzoni; Kashivakura, Cyntia Kayo; Silveira, Leandro; Jacomo, Anah T A; Lima, Edson Souza; de Paula, Rogério Cunha; May-Junior, Joares Adenílson

    2010-10-01

    Rabies virus is a pathogen of major concern in free-ranging wild carnivores in several regions of the world, but little is known about its circulation in Brazilian wild carnivores. Sera from 211 free-ranging wild carnivores, captured from 2000 to 2006 in four locations of two Brazilian biomes (Pantanal and Cerrado), were tested for rabies antibodies. Twenty-six individuals (12.3%) had neutralizing antibody titers ≥0.10 IU/ml. The four sampled locations had antibody-positive animals, suggesting that Rabies virus circulates in all of these regions. Results underscore the risk posed by rabies for conservation of Brazilian carnivores and the possibility of the animals acting as reservoirs for the Rabies virus.

  3. Phylogeography of the current rabies viruses in Indonesia.

    PubMed

    Dibia, I Nyoman; Sumiarto, Bambang; Susetya, Heru; Putra, Anak Agung Gde; Scott-Orr, Helen; Mahardika, Gusti Ngurah

    2015-01-01

    Rabies is a major fatal zoonotic disease in Indonesia. This study was conducted to determine the recent dynamics of rabies virus (RABV) in various areas and animal species throughout Indonesia. A total of 27 brain samples collected from rabid animals of various species in Bali, Sumatra, Kalimantan, Sulawesi, Java, and Flores in 2008 to 2010 were investigated. The cDNA of the nucleoprotein gene from each sample was generated and amplified by one-step reverse transcription-PCR, after which the products were sequenced and analyzed. The symmetric substitution model of a Bayesian stochastic search variable selection extension of the discrete phylogeographic model of the social network was applied in BEAST ver. 1.7.5 software. The spatial dispersal was visualized in Cartographica using Spatial Phylogenetic Reconstruction of Evolutionary Dynamics. We demonstrated inter-island introduction and reintroduction, and dog was found to be the only source of infection of other animals. Ancestors of Indonesian RABVs originated in Java and its descendants were transmitted to Kalimantan, then further to Sumatra, Flores, and Bali. The Flores descendent was subsequently transmitted to Sulawesi and back to Kalimantan. The viruses found in various animal species were transmitted by the dog.

  4. Phylogeography of the current rabies viruses in Indonesia

    PubMed Central

    Dibia, I Nyoman; Sumiarto, Bambang; Susetya, Heru; Putra, Anak Agung Gde; Scott-Orr, Helen

    2015-01-01

    Rabies is a major fatal zoonotic disease in Indonesia. This study was conducted to determine the recent dynamics of rabies virus (RABV) in various areas and animal species throughout Indonesia. A total of 27 brain samples collected from rabid animals of various species in Bali, Sumatra, Kalimantan, Sulawesi, Java, and Flores in 2008 to 2010 were investigated. The cDNA of the nucleoprotein gene from each sample was generated and amplified by one-step reverse transcription-PCR, after which the products were sequenced and analyzed. The symmetric substitution model of a Bayesian stochastic search variable selection extension of the discrete phylogeographic model of the social network was applied in BEAST ver. 1.7.5 software. The spatial dispersal was visualized in Cartographica using Spatial Phylogenetic Reconstruction of Evolutionary Dynamics. We demonstrated inter-island introduction and reintroduction, and dog was found to be the only source of infection of other animals. Ancestors of Indonesian RABVs originated in Java and its descendants were transmitted to Kalimantan, then further to Sumatra, Flores, and Bali. The Flores descendent was subsequently transmitted to Sulawesi and back to Kalimantan. The viruses found in various animal species were transmitted by the dog. PMID:25643792

  5. Cellular Chaperonin CCTγ Contributes to Rabies Virus Replication during Infection

    PubMed Central

    Zhang, Jinyang; Wu, Xiaopeng; Zan, Jie; Wu, Yongping; Ye, Chengjin; Ruan, Xizhen

    2013-01-01

    Rabies, as the oldest known infectious disease, remains a serious threat to public health worldwide. The eukaryotic cytosolic chaperonin TRiC/CCT complex facilitates the folding of proteins through ATP hydrolysis. Here, we investigated the expression, cellular localization, and function of neuronal CCTγ during neurotropic rabies virus (RABV) infection using mouse N2a cells as a model. Following RABV infection, 24 altered proteins were identified by using two-dimensional electrophoresis and mass spectrometry, including 20 upregulated proteins and 4 downregulated proteins. In mouse N2a cells infected with RABV or cotransfected with RABV genes encoding nucleoprotein (N) and phosphoprotein (P), confocal microscopy demonstrated that upregulated cellular CCTγ was colocalized with viral proteins N and P, which formed a hollow cricoid inclusion within the region around the nucleus. These inclusions, which correspond to Negri bodies (NBs), did not form in mouse N2a cells only expressing the viral protein N or P. Knockdown of CCTγ by lentivirus-mediated RNA interference led to significant inhibition of RABV replication. These results demonstrate that the complex consisting of viral proteins N and P recruits CCTγ to NBs and identify the chaperonin CCTγ as a host factor that facilitates intracellular RABV replication. This work illustrates how viruses can utilize cellular chaperonins and compartmentalization for their own benefit. PMID:23637400

  6. Rabies virus-induced apoptosis involves caspase-dependent and caspase-independent pathways.

    PubMed

    Sarmento, Luciana; Tseggai, Tesfai; Dhingra, Vikas; Fu, Zhen F

    2006-11-01

    Previously, it has been shown that the laboratory attenuated rabies virus CVS-B2C, but not the wild-type virus SHBRV, induces apoptosis in mice and the induction of apoptosis is mediated by viral glycoprotein. Induction of apoptosis by CVS-B2C limits the spread of the virus in the CNS. In the present study, we characterized the pathways by which CVS-B2C induces apoptosis. BSR cells were infected with CVS-B2C or SHBRV and harvested at different time points for detection of apoptosis by immunofluorescence and flow cytometry. Apoptosis was detected only in cells infected with CVS-B2C, but not SHBRV. Caspase activity and expression of several apoptotic proteins were analyzed by fluorometric assay and Western blotting. Activation of caspase-8 and -3, but not of caspase-9, was observed in CVS-B2C-infected cells. In addition, the level of expression of Apaf-1 did not change. Furthermore, PARP was cleaved confirming activation of downstream caspases. All these data suggest that CVS-B2C infection activates the extrinsic, but not the intrinsic, apoptotic pathway. In addition, AIF, a caspase-independent apoptotic protein was up-regulated and translocated from the cytoplasm to the nucleus post-infection, suggesting that apoptosis induced by CVS-B2C also involves the activation of a caspase-independent pathway.

  7. Unusual molecular architecture of the machupo virus attachment glycoprotein.

    PubMed

    Bowden, Thomas A; Crispin, Max; Graham, Stephen C; Harvey, David J; Grimes, Jonathan M; Jones, E Yvonne; Stuart, David I

    2009-08-01

    New World arenaviruses, which cause severe hemorrhagic fever, rely upon their envelope glycoproteins for attachment and fusion into their host cell. Here we present the crystal structure of the Machupo virus GP1 attachment glycoprotein, which is responsible for high-affinity binding at the cell surface to the transferrin receptor. This first structure of an arenavirus glycoprotein shows that GP1 consists of a novel alpha/beta fold. This provides a blueprint of the New World arenavirus attachment glycoproteins and reveals a new architecture of viral attachment, using a protein fold of unknown origins.

  8. [Proteomic Analyses of Purified Particles of the Rabies Virus].

    PubMed

    Tu, Zhongzhong; Gong, Wenjie; Zhang, Yan; Feng, Ye; Li, Nan; Tu, Changchun

    2015-05-01

    The rabies virus (RABV) is an enveloped RNA virus. It mainly damages the central nervous system and causes anencephaly in mammals and humans. There is now compelling evidence that enveloped virions released from infected cells can carry many host proteins, some of which may play an important part in viral replication. Several host proteins have been reported to be incorporated into RABV particles. However, a systematic study to reveal the proteomics of RABV particles has not been conducted. In the present study, after virus culture and purification by sucrose density gradient ultracentrifugation, a proteomics approach was used to analyze the protein composition of purified RABV particles to understand the molecular mechanisms of virus-cell interactions. Fifty host proteins, along with five virus-encoded structural proteins, were identified in purified RABV particles. These proteins could be classified into ten categories according to function: intracellular trafficking (14%), molecular chaperone (12%), cytoskeletal (24%), signal transduction (8%), transcription regulation (12%), calcium ion-binding (6%), enzyme binding (6%), metabolic process (2%), ubiquitin (2%) and other (14%). Of these, four proteins (beta-actin, p-tubulin, Cofilin, Hsc70) were validated by western blotting to be present in purified RABV particles. This novel study of the composition of host proteins in RABV particles may aid investigation of the mechanism of RABV replication. PMID:26470524

  9. [Proteomic Analyses of Purified Particles of the Rabies Virus].

    PubMed

    Tu, Zhongzhong; Gong, Wenjie; Zhang, Yan; Feng, Ye; Li, Nan; Tu, Changchun

    2015-05-01

    The rabies virus (RABV) is an enveloped RNA virus. It mainly damages the central nervous system and causes anencephaly in mammals and humans. There is now compelling evidence that enveloped virions released from infected cells can carry many host proteins, some of which may play an important part in viral replication. Several host proteins have been reported to be incorporated into RABV particles. However, a systematic study to reveal the proteomics of RABV particles has not been conducted. In the present study, after virus culture and purification by sucrose density gradient ultracentrifugation, a proteomics approach was used to analyze the protein composition of purified RABV particles to understand the molecular mechanisms of virus-cell interactions. Fifty host proteins, along with five virus-encoded structural proteins, were identified in purified RABV particles. These proteins could be classified into ten categories according to function: intracellular trafficking (14%), molecular chaperone (12%), cytoskeletal (24%), signal transduction (8%), transcription regulation (12%), calcium ion-binding (6%), enzyme binding (6%), metabolic process (2%), ubiquitin (2%) and other (14%). Of these, four proteins (beta-actin, p-tubulin, Cofilin, Hsc70) were validated by western blotting to be present in purified RABV particles. This novel study of the composition of host proteins in RABV particles may aid investigation of the mechanism of RABV replication.

  10. Regular exposure to rabies virus and lack of symptomatic disease in Serengeti spotted hyenas.

    PubMed

    East, M L; Hofer, H; Cox, J H; Wulle, U; Wiik, H; Pitra, C

    2001-12-18

    We report a previously unrecognized complexity to the ecology of rabies in wildlife. Rabies-specific virus-neutralizing antibodies in spotted hyenas, the most numerous large carnivore in the Serengeti ecosystem (Tanzania, East Africa), revealed a high frequency of exposure of 37.0% to rabies virus, and reverse transcriptase (RT) PCR demonstrated rabies RNA in 13.0% of hyenas. Despite this high frequency, exposure neither caused symptomatic rabies nor decreased survival among members of hyena social groups monitored for 9 to 13 years. Repeated, intermittent presence of virus in saliva of 45.5% of seropositive hyenas indicated a "carrier" state. Rabies isolates from Serengeti hyenas differed significantly (8.5% sequence divergence) from those isolated from other Serengeti carnivores, suggesting that at least two separate strains circulate within the Serengeti carnivore community. This finding is consistent with the fact that exposure in hyenas increased with age and social status, following a pattern predicted by intraspecific age and social-status-dependent oral and bite contact rates. High seroprevalence of rabies, low basic reproductive rate of the virus (R(0)) of 1.9, a carrier state, and the absence of symptomatic rabies in a carnivore in an ecosystem with multihost and multistrain maintenance has not been previously demonstrated for rabies. Because of the substantial differences between the hyena viral isolates and those from canids and viverrids in the Serengeti, it is unlikely that spotted hyenas were the source of rabies virus that killed several African wild dog packs in the Serengeti ecosystem in the 1990s.

  11. Expression of rabies virus G protein in carrots (Daucus carota).

    PubMed

    Rojas-Anaya, Edith; Loza-Rubio, Elizabeth; Olivera-Flores, Maria Teresa; Gomez-Lim, Miguel

    2009-12-01

    Antigens derived from various pathogens can readily be synthesized at high levels in plants in their authentic forms. Such antigens administered orally can induce an immune response and, in some cases, result in protection against a subsequent challenge. We here report the expression of rabies virus G protein into carrots. The G gene was subcloned into the pUCpSSrabG vector and then used to transform carrot embryogenic cells by particle bombardment. The carrot cells were selected in liquid medium, a method previously unreported. The presence of the transgene was verified by PCR, and by RT-PCR. By western blot, G protein transgene was identified in 93.3% of adult carrot roots. The G protein was quantified by densitometric analysis (range 0.4-1.2%). The expressed protein was antigenic in mice. This confirms that the carrot is an adequate system for antigen expression.

  12. Epidemiology and molecular diversity of rabies viruses in Bulgaria.

    PubMed

    Robardet, E; Ilieva, D; Iliev, E; Gagnev, E; Picard-Meyer, E; Cliquet, F

    2014-04-01

    A health emergency situation occurred in Bulgaria in 2007 when positive rabies cases were notified in Sofia district in the central-western part of the country, suggesting a southward spread of the disease for the first time in the last 10 years. Phylogenetic analysis on 49 isolates sampled between 2009 and 2011 showed, for the first time, evidence of the existence of NEE and D clustered lineages in Bulgaria. Their geographical distribution clearly reveals the permeability of natural barriers, as already suggested by the disease spread that occurred across the Balkan mountain range in 2007. The monitoring and passive surveillance programmes conducted since the first 2009 oral vaccination campaign, the spatio-temporal evolution of the disease in the country since 2007, and the need for further investigation of the role of jackals in virus dispersion are discussed. PMID:23830231

  13. Epidemiology and molecular diversity of rabies viruses in Bulgaria.

    PubMed

    Robardet, E; Ilieva, D; Iliev, E; Gagnev, E; Picard-Meyer, E; Cliquet, F

    2014-04-01

    A health emergency situation occurred in Bulgaria in 2007 when positive rabies cases were notified in Sofia district in the central-western part of the country, suggesting a southward spread of the disease for the first time in the last 10 years. Phylogenetic analysis on 49 isolates sampled between 2009 and 2011 showed, for the first time, evidence of the existence of NEE and D clustered lineages in Bulgaria. Their geographical distribution clearly reveals the permeability of natural barriers, as already suggested by the disease spread that occurred across the Balkan mountain range in 2007. The monitoring and passive surveillance programmes conducted since the first 2009 oral vaccination campaign, the spatio-temporal evolution of the disease in the country since 2007, and the need for further investigation of the role of jackals in virus dispersion are discussed.

  14. Rabies virus matrix protein induces apoptosis by targeting mitochondria.

    PubMed

    Zan, Jie; Liu, Juan; Zhou, Jian-Wei; Wang, Hai-Long; Mo, Kai-Kun; Yan, Yan; Xu, Yun-Bin; Liao, Min; Su, Shuo; Hu, Rong-Liang; Zhou, Ji-Yong

    2016-09-10

    Apoptosis, as an innate antiviral defense, not only functions to limit viral replication by eliminating infected cells, but also contribute to viral dissemination, particularly at the late stages of infection. A highly neurotropic CVS strain of rabies virus induces apoptosis both in vitro and in vivo. However, the detailed mechanism of CVS-mediated neuronal apoptosis is not entirely clear. Here, we show that CVS induces apoptosis through mitochondrial pathway by dissipating mitochondrial membrane potential, release of cytochrome c and AIF. CVS blocks Bax activation at the early stages of infection; while M protein partially targets mitochondria and induces mitochondrial apoptosis at the late stages of infection. The α-helix structure spanning 67-79 amino acids of M protein is essential for mitochondrial targeting and induction of apoptosis. These results suggest that CVS functions on mitochondria to regulate apoptosis at different stages of infection, so as to for viral replication and dissemination. PMID:27426727

  15. Persistence of Rabies Virus-Neutralizing Antibodies after Vaccination of Rural Population following Vampire Bat Rabies Outbreak in Brazil

    PubMed Central

    Medeiros, Rita; Jusot, Viviane; Houillon, Guy; Rasuli, Anvar; Martorelli, Luzia; Kataoka, Ana Paula; Mechlia, Mohamed Ben; Le Guern, Anne-Sophie; Rodrigues, Liliam; Assef, Rhomero; Maestri, Alvino; Bosch-Castells, Valérie; Tordo, Noël

    2016-01-01

    Background Animal control measures in Latin America have decreased the incidence of urban human rabies transmitted by dogs and cats; currently most cases of human rabies are transmitted by bats. In 2004–2005, rabies outbreaks in populations living in rural Brazil prompted widespread vaccination of exposed and at-risk populations. More than 3,500 inhabitants of Augusto Correa (Pará State) received either post-exposure (PEP) or pre-exposure (PrEP) prophylaxis. This study evaluated the persistence of rabies virus-neutralizing antibodies (RVNA) annually for 4 years post-vaccination. The aim was to evaluate the impact of rabies PrEP and PEP in a population at risk living in a rural setting to help improve management of vampire bat exposure and provide additional data on the need for booster vaccination against rabies. Methodology/Principal Findings This prospective study was conducted in 2007 through 2009 in a population previously vaccinated in 2005; study participants were followed-up annually. An RVNA titer >0.5 International Units (IU)/mL was chosen as the threshold of seroconversion. Participants with titers ≤0.5 IU/mL or Equivalent Units (EU)/mL at enrollment or at subsequent annual visits received booster doses of purified Vero cell rabies vaccine (PVRV). Adherence of the participants from this Amazonian community to the study protocol was excellent, with 428 of the 509 (84%) who attended the first interview in 2007 returning for the final visit in 2009. The long-term RVNA persistence was good, with 85–88.0% of the non-boosted participants evaluated at each yearly follow-up visit remaining seroconverted. Similar RVNA persistence profiles were observed in participants originally given PEP or PrEP in 2005, and the GMT of the study population remained >1 IU/mL 4 years after vaccination. At the end of the study, 51 subjects (11.9% of the interviewed population) had received at least one dose of booster since their vaccination in 2005. Conclusions

  16. 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.

  17. 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...

  18. Rabies serogroup viruses in neuroblastoma cells: propagation, "autointerference," and apparently random back-mutation of attenuated viruses to the virulent state.

    PubMed

    Clark, H F

    1980-03-01

    Each of several strains of fixed rabies virus was found to replicate to high titers in C1300 mouse neuroblastoma (clone NA) cells, without adaptation. Rabies serogroup Lagos bat, Mokola, and Duvenhage viruses also replicated efficiently in NA cells. Kotonkan and Obodhiang viruses replicated efficiently after adaptation, to titers not previously obtained in vitro. Infection in NA cells was frequently more cytopathic than in BHK-21 cells, allowing titration of Kotonkan and Obodhiang viruses by plaque assay. Duvenhage virus caused syncytium formation. Serial propagation of rabies viruses at a high multiplicity of infection in NA cells led to a rapid decline in virus yields; similar "autointerference" has not previously been demonstrated with rabies virus in other cell systems. Rabies virus infection in NA cells exhibited extreme sensitivity to interference by experimentally added defective interfering virions. Although several strains of attenuated rabies virus consistently reverted rapidly to virulence after propagation in NA cells, other strains of attenuated rabies and rabies serogroup viruses acquired increased virulence at a more gradual rate or not at all, suggesting that diverse characters may control virulence. When attenuated Flury HEP rabies virus was serially propagated at a low multiplicity of infection in either NA cells or suckling mouse brain, virulence appeared at a very variable rate, indicating that these systems may selectively enhance replication of randomly occurring virulent virus mutants.

  19. Complete Genome Sequence of a Vampire Bat Rabies Virus from French Guiana.

    PubMed

    Lavergne, Anne; Darcissac, Edith; Bourhy, Hervé; Tirera, Sourakhata; de Thoisy, Benoît; Lacoste, Vincent

    2016-04-07

    A rabies virus was detected in a common vampire bat (Desmodus rotundus) in French Guiana. Its genomic sequence was obtained and found to be closely related to other hematophagous bat-related viruses that widely circulate in the northern Amazon region. This virus is named AT6.

  20. Complete Genome Sequence of a Vampire Bat Rabies Virus from French Guiana

    PubMed Central

    Lavergne, Anne; Darcissac, Edith; Bourhy, Hervé; Tirera, Sourakhata; de Thoisy, Benoît

    2016-01-01

    A rabies virus was detected in a common vampire bat (Desmodus rotundus) in French Guiana. Its genomic sequence was obtained and found to be closely related to other hematophagous bat-related viruses that widely circulate in the northern Amazon region. This virus is named AT6. PMID:27056216

  1. Development of monoclonal antibodies suitable for rabies virus antibody and antigen detection.

    PubMed

    Chander, Vishal; Singh, R P; Verma, P C

    2012-12-01

    The control of an infectious viral disease as rabies is made easier by rapid and accurate diagnosis. Successful rabies prophylaxis is dependent upon the active immunization with vaccine along with passive administration of rabies virus neutralizing antibodies which together clear the virus before widespread infection of central nervous system occurs. The present study aimed at the development of monoclonal antibodies (MAbs) suitable for rabies virus antibody and antigen detection. For the production of rabies specific MAbs, immunization of Swiss albino mice with a commercially available vaccine was done and Polyethylene glycol mediated fusion of spleenocytes with myeloma cells was performed. The positive clones were selected on the basis of distinct reactivity by cell Enzyme linked immunosorbent assay and fluorescence in Indirect Fluorescent antibody test. The positive clones obtained were subjected to single cell cloning by limiting dilution method. The reactive clones were further titrated and employed for virus titration and virus neutralization. The neutralizing activity was evaluated using Fluorescence Activated Cell Sorter technique. Three MAb clones showed a distinct percent inhibition in the presence of positive serum. One of the MAb clone No. 5C3 was relatively more specific in detecting rabies antibodies and also found suitable for competitive ELISA to assess the antibody level in vaccinated subjects.

  2. Distribution of rabies virus in infected mice, vaccinated and submitted to P. acnes as immunomodulator.

    PubMed

    Megid, J; Cremonini, D N; Leomil, H

    2002-07-01

    The lethality and distribution of rabies virus were evaluated in swiss mice experimentally infected with street rabies virus, vaccinated and submitted to immunomodulation by P .acnes (formerly Corynebacterium parvum). The animals were sacrificed at different times,when the different tissues were collected and submitted to fluorescent antibody test (FAT) and mouse inoculation test (MIT). The group submitted to vaccination and P. acnes treatment presented a percentage of survival superior to that observed in infected mice only treated with P. acnes. Control infected animals had the lowest survival rates. The distribution of rabies virus in spleen of infected mice, vaccinated and submitted to P. acnes was superior to that verified in infected mice not treated with P.acnes. The increased survival correlated with the distribution of rabies virus in lymphoid tissues, could be interpreted as the consequence of P. acnes activity on macrophages. The results suggest the role of macrophages against rabies virus infection in mice and the importance of vaccination in the post expositive treatment of rabies. PMID:12135238

  3. Antibody Quality and Protection from Lethal Ebola Virus Challenge in Nonhuman Primates Immunized with Rabies Virus Based Bivalent Vaccine

    PubMed Central

    Papaneri, Amy B.; Wirblich, Christoph; Feldmann, Friederike; Holbrook, Michael; Jahrling, Peter; Feldmann, Heinz; Schnell, Matthias J.

    2013-01-01

    We have previously described the generation of a novel Ebola virus (EBOV) vaccine platform based on (a) replication-competent rabies virus (RABV), (b) replication-deficient RABV, or (c) chemically inactivated RABV expressing EBOV glycoprotein (GP). Mouse studies demonstrated safety, immunogenicity, and protective efficacy of these live or inactivated RABV/EBOV vaccines. Here, we evaluated these vaccines in nonhuman primates. Our results indicate that all three vaccines do induce potent immune responses against both RABV and EBOV, while the protection of immunized animals against EBOV was largely dependent on the quality of humoral immune response against EBOV GP. We also determined if the induced antibodies against EBOV GP differ in their target, affinity, or the isotype. Our results show that IgG1-biased humoral responses as well as high levels of GP-specific antibodies were beneficial for the control of EBOV infection after immunization. These results further support the concept that a successful EBOV vaccine needs to induce strong antibodies against EBOV. We also showed that a dual vaccine against RABV and filoviruses is achievable; therefore addressing concerns for the marketability of this urgently needed vaccine. PMID:23737747

  4. Bat rabies in Guatemala.

    PubMed

    Ellison, James A; Gilbert, Amy T; Recuenco, Sergio; Moran, David; Alvarez, Danilo A; Kuzmina, Natalia; Garcia, Daniel L; Peruski, Leonard F; Mendonça, Mary T; Lindblade, Kim A; Rupprecht, Charles E

    2014-01-01

    Rabies in bats is considered enzootic throughout the New World, but few comparative data are available for most countries in the region. As part of a larger pathogen detection program, enhanced bat rabies surveillance was conducted in Guatemala, between 2009 and 2011. A total of 672 bats of 31 species were sampled and tested for rabies. The prevalence of rabies virus (RABV) detection among all collected bats was low (0.3%). Viral antigens were detected and infectious virus was isolated from the brains of two common vampire bats (Desmodus rotundus). RABV was also isolated from oral swabs, lungs and kidneys of both bats, whereas viral RNA was detected in all of the tissues examined by hemi-nested RT-PCR except for the liver of one bat. Sequencing of the nucleoprotein gene showed that both viruses were 100% identical, whereas sequencing of the glycoprotein gene revealed one non-synonymous substitution (302T,S). The two vampire bat RABV isolates in this study were phylogenetically related to viruses associated with vampire bats in the eastern states of Mexico and El Salvador. Additionally, 7% of sera collected from 398 bats demonstrated RABV neutralizing antibody. The proportion of seropositive bats varied significantly across trophic guilds, suggestive of complex intraspecific compartmentalization of RABV perpetuation.

  5. Bat rabies in Guatemala.

    PubMed

    Ellison, James A; Gilbert, Amy T; Recuenco, Sergio; Moran, David; Alvarez, Danilo A; Kuzmina, Natalia; Garcia, Daniel L; Peruski, Leonard F; Mendonça, Mary T; Lindblade, Kim A; Rupprecht, Charles E

    2014-01-01

    Rabies in bats is considered enzootic throughout the New World, but few comparative data are available for most countries in the region. As part of a larger pathogen detection program, enhanced bat rabies surveillance was conducted in Guatemala, between 2009 and 2011. A total of 672 bats of 31 species were sampled and tested for rabies. The prevalence of rabies virus (RABV) detection among all collected bats was low (0.3%). Viral antigens were detected and infectious virus was isolated from the brains of two common vampire bats (Desmodus rotundus). RABV was also isolated from oral swabs, lungs and kidneys of both bats, whereas viral RNA was detected in all of the tissues examined by hemi-nested RT-PCR except for the liver of one bat. Sequencing of the nucleoprotein gene showed that both viruses were 100% identical, whereas sequencing of the glycoprotein gene revealed one non-synonymous substitution (302T,S). The two vampire bat RABV isolates in this study were phylogenetically related to viruses associated with vampire bats in the eastern states of Mexico and El Salvador. Additionally, 7% of sera collected from 398 bats demonstrated RABV neutralizing antibody. The proportion of seropositive bats varied significantly across trophic guilds, suggestive of complex intraspecific compartmentalization of RABV perpetuation. PMID:25080103

  6. Bat Rabies in Guatemala

    PubMed Central

    Ellison, James A.; Gilbert, Amy T.; Recuenco, Sergio; Moran, David; Alvarez, Danilo A.; Kuzmina, Natalia; Garcia, Daniel L.; Peruski, Leonard F.; Mendonça, Mary T.; Lindblade, Kim A.; Rupprecht, Charles E.

    2014-01-01

    Rabies in bats is considered enzootic throughout the New World, but few comparative data are available for most countries in the region. As part of a larger pathogen detection program, enhanced bat rabies surveillance was conducted in Guatemala, between 2009 and 2011. A total of 672 bats of 31 species were sampled and tested for rabies. The prevalence of rabies virus (RABV) detection among all collected bats was low (0.3%). Viral antigens were detected and infectious virus was isolated from the brains of two common vampire bats (Desmodus rotundus). RABV was also isolated from oral swabs, lungs and kidneys of both bats, whereas viral RNA was detected in all of the tissues examined by hemi-nested RT-PCR except for the liver of one bat. Sequencing of the nucleoprotein gene showed that both viruses were 100% identical, whereas sequencing of the glycoprotein gene revealed one non-synonymous substitution (302T,S). The two vampire bat RABV isolates in this study were phylogenetically related to viruses associated with vampire bats in the eastern states of Mexico and El Salvador. Additionally, 7% of sera collected from 398 bats demonstrated RABV neutralizing antibody. The proportion of seropositive bats varied significantly across trophic guilds, suggestive of complex intraspecific compartmentalization of RABV perpetuation. PMID:25080103

  7. Signs Observed Among Animal Species Infected with Raccoon Rabies Variant Virus, Massachusetts, USA, 1992–2010

    PubMed Central

    Wang, Xingtai; Werner, Barbara G.; Smole, Sandra; Pani, Vasil; Han, Linda L.

    2011-01-01

    Simple Summary We analyzed signs occurring among domestic and wild terrestrial animal species with raccoon rabies variant virus in Massachusetts, 1992–2010. While aggression is a useful predictor of rabies among wild animals, combinations of other signs such as ataxia, disorientation, and salivation are useful predictors of rabies among domestic animals. Abstract We analyzed signs occurring among domestic and wild terrestrial animal species infected with raccoon rabies variant virus (RRV) in Massachusetts, 1992–2010. The clinical sign of aggression was significantly associated with rabid stray cats (odds ratio, OR = 2.3) and RRV affected major wild terrestrial animal species individually, which included raccoons (OR = 2.8), skunks (OR = 8.0), gray foxes (OR = 21.3), red foxes (OR = 10.4), woodchucks (OR = 4.7) and coyotes (OR = 27.6). While aggression is a useful predictor of rabies among wild animals, combinations of other signs such as ataxia, disorientation, and salivation are useful predictors of rabies among domestic animals. Pets reported with multiple clinical signs had significantly higher rabies positive testing result than those reported with single clinical sign (p < 0.001). The result suggested the importance of avoiding aggressive terrestrial wild animals and giving additional attention to pets with multiple clinical signs. PMID:26486623

  8. A novel rabies virus lipopeptide provides a better protection by improving the magnitude of DCs activation and T cell responses.

    PubMed

    Liu, Rui; Wang, Jingbo; Yang, Yan; Khan, Inamullah; Dong, Yue; Zhu, Naishuo

    2016-08-01

    Besides rabies virus neutralizing antibody, non-neutralizing antibody to internal vital proteins, interferon, and possibly cell-mediated immunity also have a critical role in preventing the infection by rabies virus (RV). We identified novel CTL and Th epitopes which could induce lymphocyte proliferation and IFN-γ, IL-4 production, and designed linear and branched lipopeptides with these selected CTL and Th epitopes. Compared to linear construct, branched lipopeptides, especially Lipo C, stimulate stronger phenotypic and functional maturation of DCs, as well as more efficient CD8(+) T cell responses, evaluating by using FACS, G333-341 tetramer staining and specific CTL assay. Lipo C could also assist rabies vaccine to induce an instant rabies virus neutralizing antibody production, and better protection against rabies virus challenge at early stage. These data reveal that Lipo C could be a promising component for developing novel rabies vaccines. PMID:27182006

  9. Protection of non-human primates against rabies with an adenovirus recombinant vaccine

    SciTech Connect

    Xiang, Z.Q.; Greenberg, L.; Ertl, H.C.; Rupprecht, C.E.

    2014-02-15

    Rabies remains a major neglected global zoonosis. New vaccine strategies are needed for human rabies prophylaxis. A single intramuscular immunization with a moderate dose of an experimental chimpanzee adenovirus (Ad) vector serotype SAd-V24, also termed AdC68, expressing the rabies virus glycoprotein, resulted in sustained titers of rabies virus neutralizing antibodies and protection against a lethal rabies virus challenge infection in a non-human primate model. Taken together, these data demonstrate the safety, immunogenicity, and efficacy of the recombinant Ad-rabies vector for further consideration in human clinical trials. - Highlights: • Pre-exposure vaccination with vaccine based on a chimpanzee derived adenovirus protects against rabies. • Protection is sustained. • Protection is achieved with single low-dose of vaccine given intramuscularly. • Protection is not affected by pre-existing antibodies to common human serotypes of adenovirus.

  10. Analysis of Subcellular Prefoldin 1 Redistribution During Rabies Virus Infection

    PubMed Central

    Zhang, Jinyang; Han, Qinqin; Song, Yuzhu; Chen, Qiang; Xia, Xueshan

    2015-01-01

    Background: Rabies virus (RABV) is one of the old deadly zoonotic viruses. It attacks the central nervous system and causes acute encephalitis in humans and animals. Host factors are known to be essential for virus infection and replication in cells. The identification of the key host factors required for RABV infection may provide important information on RABV replication and may provide new potential targets for RABV drug discovery. Objectives: This study aimed to investigate the change in the subcellular distribution and expression of the host protein Prefoldin subunit 1 (PFDN1) in RABV-infected cells and the viral expression of plasmids in the transfected cells. Materials and Methods: Mouse Neuro-2a (N2a) cells were infected by RABV or transfected with the plasmids of the nucleoprotein (N) and/or phosphoprotein (P) gene of RABV. The subcellular distribution of PFDN1 was analyzed by confocal microscopy, and the transcription levels of PFDN1 in the N and/or P gene of the RABV-transfected or RABV-infected N2a cells were assessed via real-time quantitative polymerase chain reaction. Results: Confocal microscopy showed that PFDN1 was colocalized with the N protein of RABV in the infected N2a cells and was mainly recruited to the characteristic Negri-Body-Like (NBL) structures in the cytoplasm, as well as the cotransfection of the N and P genes of RABV. The transcription of PFDN1 in the RABV-infected N2a cells was upregulated, whereas the transfection of the N and/or P genes did not result in the upregulation of PFDN1. Conclusions: The results of this work demonstrated that the subcellular distribution of PFDN1 was altered in the RABV-infected N2a cells and colocalized with the N protein of RABV in the NBL structures. PMID:26421138

  11. In vitro rabies vaccine potency appraisal by ELISA: advantages of the immunocapture method with a neutralizing anti-glycoprotein monoclonal antibody.

    PubMed

    Perrin, P; Morgeaux, S; Sureau, P

    1990-10-01

    The replacement of the in vivo potency test (NIH test) for rabies vaccine evaluation by in vitro methods is at present discussed in many reports and also by WHO expert working groups. For this purpose, in vitro glycoprotein titration has been proposed. Among the different glycoprotein assays, we have studied two ELISA methods (immunocapture and direct plate coating with the antigen to be tested) using neutralizing mono- and polyclonal antibodies. In our view, the immunocapture method based on the use of a neutralizing monoclonal anti-glycoprotein antibody seems to be a convenient tool for the determination of the in vitro potency of rabies vaccine and of the products corresponding to the different steps of their production process.

  12. Everything You Always Wanted to Know About Rabies Virus (But Were Afraid to Ask).

    PubMed

    Davis, Benjamin M; Rall, Glenn F; Schnell, Matthias J

    2015-11-01

    The cultural impact of rabies, the fatal neurological disease caused by infection with rabies virus, registers throughout recorded history. Although rabies has been the subject of large-scale public health interventions, chiefly through vaccination efforts, the disease continues to take the lives of about 40,000-70,000 people per year, roughly 40% of whom are children. Most of these deaths occur in resource-poor countries, where lack of infrastructure prevents timely reporting and postexposure prophylaxis and the ubiquity of domestic and wild animal hosts makes eradication unlikely. Moreover, although the disease is rarer than other human infections such as influenza, the prognosis following a bite from a rabid animal is poor: There is currently no effective treatment that will save the life of a symptomatic rabies patient. This review focuses on the major unanswered research questions related to rabies virus pathogenesis, especially those connecting the disease progression of rabies with the complex dysfunction caused by the virus in infected cells. The recent applications of cutting-edge research strategies to this question are described in detail. PMID:26958924

  13. Immunogenic virus-like particles continuously expressed in mammalian cells as a veterinary rabies vaccine candidate.

    PubMed

    Fontana, Diego; Kratje, Ricardo; Etcheverrigaray, Marina; Prieto, Claudio

    2015-08-20

    Rabies is one of the most lethal infectious diseases in the world, with a mortality approaching 100%. There are between 60,000 and 70,000 reported annual deaths, but this is probably an underestimation. Despite the fact that there are vaccines available for rabies, there is a real need of developing more efficacious and cheaper vaccines. This is particularly true for veterinary vaccines because dogs are still the main vector for rabies transmission to human beings. In a previous work, we described the development and characterization of rabies virus-like particles (RV-VLPs) expressed in HEK293 cells. We showed that RV-VLPs are able to induce a specific antibodies response. In this work, we show that VLPs are able to protect mice against virus challenge. Furthermore, we developed a VLPs expressing HEK-293 clone (sP2E5) that grows in serum free medium (SFM) reaching high cell densities. sP2E5 was cultured in perfusion mode in a 5 L bioreactor for 20 days, and the RV-VLPs produced were capable of triggering a protective immune response without the need of concentration or adjuvant addition. Further, these VLPs are able to induce the production of rabies virus neutralizing antibodies. These results demonstrate that RV-VLPs are a promising rabies vaccine candidate.

  14. Molecular characterization of rabies virus isolates from Mexico: implications for transmission dynamics and human risk.

    PubMed

    De Mattos, C C; De Mattos, C A; Loza-Rubio, E; Aguilar-Setién, A; Orciari, L A; Smith, J S

    1999-10-01

    Twenty-eight samples from humans and domestic and wild animals collected in Mexico between 1990 and 1995 were characterized by using anti-nucleoprotein monoclonal antibodies and limited sequence analysis of the nucleoprotein gene. The variants of rabies viruses identified in these samples were compared with other isolates from Mexico and the rest of the Americas to establish epidemiologic links between cases and outbreaks and to increase the understanding of rabies epidemiology in the Western Hemisphere. Antigenic and genetic diversity was found in all samples from dogs and dog-related cases, suggesting a long-term endemic situation with multiple, independent cycles of virus transmission. Two isolates from bobcats were antigenically and genetically homologous to the rabies variant circulating in the Arizona gray fox population, indicating a wider distribution of this variant than previously reported. Rabies isolates from skunks were unrelated to any variant analyzed in this study and represent a previously unrecognized cycle of rabies transmission in skunks in Baja California Sur. Two antigenic and genetic variants co-circulating in southern and eastern Mexico were found in viruses obtained from cases epidemiologically related to vampire bats. These results serve as a baseline for the better understanding of the molecular epidemiology of rabies in Mexico. PMID:10548293

  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. Enzyme immunoassay for rabies antibody in hybridoma culture fluids and its application to differentiation of street and laboratory strains of rabies virus.

    PubMed Central

    Smith, J S; Sumner, J W; Roumillat, L F

    1984-01-01

    A rapid and sensitive enzyme immunoassay is described for detecting rabies antibody in hybridoma culture fluids. Glass fiber filter disks were used to immobilize gamma-irradiated mouse neuroblastoma cells infected with street or laboratory strains of rabies virus. Bound rabies-specific antibody was detected by reaction with horseradish peroxidase-labeled goat anti-mouse immunoglobulin G. The assay was performed in a 96-well filtration device developed by Cleveland et al. (J. Clin. Microbiol. 15:402-407, 1982) for the typing of herpes simplex viruses. When partially disrupted cells were used, both internal and external viral antigens were available for reaction. The procedure is rapid (less than 4 h for completion) and requires only small amounts of fluid, and the gamma-irradiated antigen is noninfectious. When the procedure was used to screen 145 fluids from rabies-immune spleen-myeloma cell fusions, 132 were positive for rabies antibody. Other commonly used assays for the detection of rabies-specific antibody were less sensitive. Simultaneous analyses of many hybridoma fluids against a battery of street and laboratory strains of rabies virus are possible and allow rapid selection of useful monoclones. PMID:6365963

  17. Probable Rabies Virus Transmission through Organ Transplantation, China, 2015

    PubMed Central

    Zhou, Hang; Zhu, Wuyang; Zeng, Jun; He, Jianfeng; Liu, Kai; Li, Yu; Zhou, Shuwu; Mu, Di; Zhang, Kechun; Yu, Pengcheng; Li, Zhijian; Zhang, Meng; Chen, Xueqiong; Guo, Chun

    2016-01-01

    During July 2015, physicians at a hospital in Beijing, China, diagnosed rabies in 2 patients who had each received a kidney from a common organ donor who had died from acute progressive encephalitis of unknown cause. The patients had rabies incubation periods of 42 and 48 days. Altered mental status developed in both patients and progressively worsened to deep coma within 80 days after transplantation; both patients died. Two other transplant recipients received corneas but remained well after receiving timely rabies prophylaxis. An effective regulatory system for testing donors should be implemented to decrease the occurrence of donor-derived infectious diseases. In addition, health education should be improved to enhance public awareness of transplant-associated infectious diseases. Transplant recipients and other persons with exposure to organs or tissues from donors with rabies must be provided consistent health monitoring and follow-up, including rabies postexposure prophylaxis; any remaining organs and tissues must be quarantined and not transplanted. PMID:27331337

  18. Probable Rabies Virus Transmission through Organ Transplantation, China, 2015.

    PubMed

    Zhou, Hang; Zhu, Wuyang; Zeng, Jun; He, Jianfeng; Liu, Kai; Li, Yu; Zhou, Shuwu; Mu, Di; Zhang, Kechun; Yu, Pengcheng; Li, Zhijian; Zhang, Meng; Chen, Xueqiong; Guo, Chun; Yu, Hongjie

    2016-08-01

    During July 2015, physicians at a hospital in Beijing, China, diagnosed rabies in 2 patients who had each received a kidney from a common organ donor who had died from acute progressive encephalitis of unknown cause. The patients had rabies incubation periods of 42 and 48 days. Altered mental status developed in both patients and progressively worsened to deep coma within 80 days after transplantation; both patients died. Two other transplant recipients received corneas but remained well after receiving timely rabies prophylaxis. An effective regulatory system for testing donors should be implemented to decrease the occurrence of donor-derived infectious diseases. In addition, health education should be improved to enhance public awareness of transplant-associated infectious diseases. Transplant recipients and other persons with exposure to organs or tissues from donors with rabies must be provided consistent health monitoring and follow-up, including rabies postexposure prophylaxis; any remaining organs and tissues must be quarantined and not transplanted. PMID:27331337

  19. Effect of cellular cholesterol depletion on rabies virus infection.

    PubMed

    Hotta, Kozue; Bazartseren, Boldbarrtar; Kaku, Yoshihiro; Noguchi, Akira; Okutani, Akiko; Inoue, Satoshi; Yamada, Akio

    2009-01-01

    Although there are several reports on candidates for rabies virus (RABV) receptor, possible roles played by these receptor candidates in determination of highly neurotropic nature of RABV have not been well understood. Since these candidate receptors for RABV were reported to be frequently associated with cholesterol-rich microdomains characterized by lipid rafts and caveolae structures, we attempted to determine whether the disturbance of microdomains caused by the cholesterol depletion showed any effects on RABV infection. When the cellular cholesterol was depleted by methyl-beta-cyclodextrin (MBCD) treatment, increase in RABV adsorption and infection, but not multiplication rather than suppression was observed in both BHK-21 and HEp-2 cells. These effects exerted by MBCD treatment on RABV infection could be reversed by cholesterol reconstitution. These results suggest that RABV enters BHK-21 or HEp-2 cells through ports of entry other than those located on cholesterol-rich microdomains and raise the possibility that RABV uses different mechanisms to enter the non-neuronal cells. PMID:19010362

  20. An in vivo and in vitro study of rabies virus infection of the rat superior cervical ganglia.

    PubMed

    Tsiang, H; Derer, M; Taxi, J

    1983-01-01

    In the attempt to develop a homogeneous neuronal model to study rabies pathogenesis in vivo and in vitro, the superior cervical ganglia (SCG) were chosen because of their unique features. In vivo infection of the SCG was attempted by inoculation of fixed rabies virus into the anterior eye chamber. However, viral by this route as well as intracerebrally failed to infect this neuronal organ in adult rats whereas the infection was poorly efficient in 24 hours newborn rats. Dissociated cell cultures from the rat embryo SCG were infected in vitro and examined for the presence of rabies specific antigen and release of virus particles in the supernatant. Despite the presence of rabies nucleoprotein in the cytoplasm and the presence of typical Negri bodies, neurons from the rat SCG produced few particles as observed by electron microscopy and no increase in virus yields could be detected by titration of viral infectivity during the infectious cycle. Our observations indicate that although rabies virus is neurotropic as shown in previous studies, all neuronal tissues are not equally susceptible to this viral infection. The resistance of the SCG to rabies virus infection in vivo does not seems to be a lack of accessibility of this organ to infection since other authors had shown that it could be infected by herpes virus. Both in vitro and in vivo experiments show that although neurons from the SCG are susceptible to rabies virus infection, infected cells do not produce rabies infectious virions efficiently.

  1. Genealogical analyses of rabies virus strains from Brazil based on N gene alleles.

    PubMed

    Heinemann, M B; Fernandes-Matioli, F M C; Cortez, A; Soares, R M; Sakamoto, S M; Bernardi, F; Ito, F H; Madeira, A M B N; Richtzenhain, L J

    2002-06-01

    Thirty rabies virus isolates from cows and vampire bats from different regions of São Paulo State, Southeastern Brazil and three rabies vaccines were studied genetically. The analysis was based on direct sequencing of PCR-amplified products of 600 nucleotides coding for the amino terminus of nucleoprotein gene. The sequences were checked to verify their genealogical and evolutionary relationships and possible implication for health programmes. Statistical data indicated that there were no significant genetic differences between samples isolated from distinct hosts, from different geographical regions and between samples collected in the last two decades. According to the HKA test, the variability observed in the sequences is probably due to genetic drift. Since changes in genetic material may produce modifications in the protein responsible for immunogenicity of virus, which may eventually cause vaccine failure in herds, we suggest that continuous efforts in monitoring genetic diversity in rabies virus field strains, in relation to vaccine strains, must be conducted. PMID:12113496

  2. Genealogical analyses of rabies virus strains from Brazil based on N gene alleles.

    PubMed Central

    Heinemann, M. B.; Fernandes-Matioli, F. M. C.; Cortez, A.; Soares, R. M.; Sakamoto, S. M.; Bernardi, F.; Ito, F. H.; Madeira, A. M. B. N.; Richtzenhain, L. J.

    2002-01-01

    Thirty rabies virus isolates from cows and vampire bats from different regions of São Paulo State, Southeastern Brazil and three rabies vaccines were studied genetically. The analysis was based on direct sequencing of PCR-amplified products of 600 nucleotides coding for the amino terminus of nucleoprotein gene. The sequences were checked to verify their genealogical and evolutionary relationships and possible implication for health programmes. Statistical data indicated that there were no significant genetic differences between samples isolated from distinct hosts, from different geographical regions and between samples collected in the last two decades. According to the HKA test, the variability observed in the sequences is probably due to genetic drift. Since changes in genetic material may produce modifications in the protein responsible for immunogenicity of virus, which may eventually cause vaccine failure in herds, we suggest that continuous efforts in monitoring genetic diversity in rabies virus field strains, in relation to vaccine strains, must be conducted. PMID:12113496

  3. Rabies vaccination at a virus-inoculated site as an alternative option to rabies immunoglobulin.

    PubMed

    Morimoto, Kinjiro; Khawplod, Pakamatz; Sato, Yuichiro; Virojanapirom, Phatthamon; Hemachudha, Thiravat

    2016-09-01

    Combined active and passive immunization has been established to be an optimal strategy for rabies post-exposure prophylaxis (PEP). Prompt administration of vaccine and rabies immunoglobulin (RIG) can reliably prevent the disease. However, RIG is unavailable and unaffordable in the majority of cases. On the basis of a model experiment using hamsters, we demonstrated that vaccine injection at the wound site in the same manner as administration of RIG provided protective efficacy that was not inferior to the current optimal PEP, a combination of vaccination and RIG. Further study is needed to determine whether it can replace the use of RIG. PMID:27270361

  4. Molecular characterization of nucleoprotein gene of rabies virus from Maharashtra, India

    PubMed Central

    Mehta, S; Charan, P; Dahake, R; Mukherjee, S; Chowdhary, A

    2016-01-01

    Context: Rabies poses a serious public health concern in developing countries such as India. Aims: The study focuses on molecular diagnosis of street rabies virus (RABV) from human clinical specimens received from Maharashtra, India. Materials and Methods: Nucleoprotein gene from eight (of total 20 suspected samples) rabies cases that tested positive for rabies antigen using reverse transcriptase-polymerase chain reaction (RT-PCR) were sequenced. Results: Sequence analysis using basic local alignment search tool (BLAST) and multiple sequence alignment (MSA) and phylogenetic analysis showed similarity to previously reported sequences from India and those of Arctic lineages. Conclusions: The circulating RABV strains in Maharashtra, India show genetic relatedness to RABV strains reported from Indo-Arctic lineages and India-South and Japan. PMID:26821566

  5. 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.

  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. Rapid discrimination of rabies viruses isolated from various host species in Brazil by multiplex reverse transcription-polymerase chain reaction.

    PubMed

    Sato, Go; Tanabe, Hitomi; Shoji, Youko; Itou, Takuya; Ito, Fumio H; Sato, Tetsuo; Sakai, Takeo

    2005-08-01

    Rabies is carried mainly by mammalian carnivores and vampire bats in Latin America. However, rabies virus (RV) has been isolated in recent years from not only vampire bats in rural areas but also from several non-vampire bat species in urban areas, respectively. Therefore, rapid molecular screening is necessary for efficient epidemiology of these RVs. In this study, we investigated the usefulness of multiplex reverse transcription-polymerase chain reaction (RT-PCR) for determining the origins of 54 RV isolates from various host species in Brazil. And to evaluate the multiplex RT-PCR as a potential diagnostic tool, we investigated the sensitivity of this method. In addition, we compared the results with a phylogenetic tree developed from sequences of the RV glycoprotein (G protein) gene. Multiplex RT-PCR products showed five different sizes of products, whereas the phylogenic tree showed six groups. Of these six groups, four corresponded with the four sizes of the multiplex RT-PCR products. The other two groups showed correspondance with another one size of the multiplex RT-PCR products, indicating that multiplex RT-PCR results reflected the lineage of the 54 isolates. This study also showed that this method can detect trace amounts of RNA. In conclusion, this multiplex RT-PCR method allows the rapid, specific, and simultaneous detection of RVs isolated from various host species in Brazil. PMID:16036175

  8. Phylogeography of rabies virus isolated from herbivores and bats in the Espírito Santo State, Brazil.

    PubMed

    Vieira, Luiz Fernando Pereira; Pereira, Sílvia Regina Ferreira Gonçalves; Carnieli, Pedro; Tavares, Luiz Carlos Barbosa; Kotait, Ivanete

    2013-04-01

    Rabies is enzootic in the State of Espírito Santo, Brazil. Every year, cattle and horses die from rabies that is transmitted by the vampire bat Desmodus rotundus. This paper describes the spread of the rabies virus by the continuous diffusion model using relaxed random walks with BEAST software. Forty-one (41) sequences of gene G from the rabies virus that was isolated from bats and domestic herbivores from several areas of the state between 2006 and 2010 were analyzed. The phylogenetic tree showed three main clusters as well as two sub-clusters under cluster 2. A spatial analysis showed that three strains of the rabies virus spread independently. In general, central Espírito Santo, which is mountainous, was the area where separation of the virus strains occurred. This physical barrier, however, was overcome at some point in time, as samples from different lineages were found in the same microarea.

  9. Phylogeography of rabies virus isolated from herbivores and bats in the Espírito Santo State, Brazil.

    PubMed

    Vieira, Luiz Fernando Pereira; Pereira, Sílvia Regina Ferreira Gonçalves; Carnieli, Pedro; Tavares, Luiz Carlos Barbosa; Kotait, Ivanete

    2013-04-01

    Rabies is enzootic in the State of Espírito Santo, Brazil. Every year, cattle and horses die from rabies that is transmitted by the vampire bat Desmodus rotundus. This paper describes the spread of the rabies virus by the continuous diffusion model using relaxed random walks with BEAST software. Forty-one (41) sequences of gene G from the rabies virus that was isolated from bats and domestic herbivores from several areas of the state between 2006 and 2010 were analyzed. The phylogenetic tree showed three main clusters as well as two sub-clusters under cluster 2. A spatial analysis showed that three strains of the rabies virus spread independently. In general, central Espírito Santo, which is mountainous, was the area where separation of the virus strains occurred. This physical barrier, however, was overcome at some point in time, as samples from different lineages were found in the same microarea. PMID:23264105

  10. Post-exposure Treatment with Anti-rabies VHH and Vaccine Significantly Improves Protection of Mice from Lethal Rabies Infection

    PubMed Central

    Terryn, Sanne; Francart, Aurélie; Rommelaere, Heidi; Stortelers, Catelijne; Van Gucht, Steven

    2016-01-01

    Post-exposure prophylaxis (PEP) against rabies infection consists of a combination of passive immunisation with plasma-derived human or equine immune globulins and active immunisation with vaccine delivered shortly after exposure. Since anti-rabies immune globulins are expensive and scarce, there is a need for cheaper alternatives that can be produced more consistently. Previously, we generated potent virus-neutralising VHH, also called Nanobodies, against the rabies glycoprotein that are effectively preventing lethal disease in an in vivo mouse model. The VHH domain is the smallest antigen-binding functional fragment of camelid heavy chain-only antibodies that can be manufactured in microbial expression systems. In the current study we evaluated the efficacy of half-life extended anti-rabies VHH in combination with vaccine for PEP in an intranasal rabies infection model in mice. The PEP combination therapy of systemic anti-rabies VHH and intramuscular vaccine significantly delayed the onset of disease compared to treatment with anti-rabies VHH alone, prolonged median survival time (35 versus 14 days) and decreased mortality (60% versus 19% survival rate), when treated 24 hours after rabies virus challenge. Vaccine alone was unable to rescue mice from lethal disease. As reported also for immune globulins, some interference of anti-rabies VHH with the antigenicity of the vaccine was observed, but this did not impede the synergistic effect. Post exposure treatment with vaccine and human anti-rabies immune globulins was unable to protect mice from lethal challenge. Anti-rabies VHH and vaccine act synergistically to protect mice after rabies virus exposure, which further validates the possible use of anti-rabies VHH for rabies PEP. PMID:27483431

  11. Post-exposure Treatment with Anti-rabies VHH and Vaccine Significantly Improves Protection of Mice from Lethal Rabies Infection.

    PubMed

    Terryn, Sanne; Francart, Aurélie; Rommelaere, Heidi; Stortelers, Catelijne; Van Gucht, Steven

    2016-08-01

    Post-exposure prophylaxis (PEP) against rabies infection consists of a combination of passive immunisation with plasma-derived human or equine immune globulins and active immunisation with vaccine delivered shortly after exposure. Since anti-rabies immune globulins are expensive and scarce, there is a need for cheaper alternatives that can be produced more consistently. Previously, we generated potent virus-neutralising VHH, also called Nanobodies, against the rabies glycoprotein that are effectively preventing lethal disease in an in vivo mouse model. The VHH domain is the smallest antigen-binding functional fragment of camelid heavy chain-only antibodies that can be manufactured in microbial expression systems. In the current study we evaluated the efficacy of half-life extended anti-rabies VHH in combination with vaccine for PEP in an intranasal rabies infection model in mice. The PEP combination therapy of systemic anti-rabies VHH and intramuscular vaccine significantly delayed the onset of disease compared to treatment with anti-rabies VHH alone, prolonged median survival time (35 versus 14 days) and decreased mortality (60% versus 19% survival rate), when treated 24 hours after rabies virus challenge. Vaccine alone was unable to rescue mice from lethal disease. As reported also for immune globulins, some interference of anti-rabies VHH with the antigenicity of the vaccine was observed, but this did not impede the synergistic effect. Post exposure treatment with vaccine and human anti-rabies immune globulins was unable to protect mice from lethal challenge. Anti-rabies VHH and vaccine act synergistically to protect mice after rabies virus exposure, which further validates the possible use of anti-rabies VHH for rabies PEP. PMID:27483431

  12. In situ apoptosis of adaptive immune cells and the cellular escape of rabies virus in CNS from patients with human rabies transmitted by Desmodus rotundus.

    PubMed

    Fernandes, Elaine Raniero; de Andrade, Heitor Franco; Lancellotti, Carmen Lúcia Penteado; Quaresma, Juarez Antônio Simões; Demachki, Samia; da Costa Vasconcelos, Pedro Fernando; Duarte, Maria Irma Seixas

    2011-03-01

    The aim of the current study was to investigate the apoptosis of neurons, astrocytes and immune cells from human patients that were infected with rabies virus by vampire bats bite. Apoptotic neurons were identified by their morphology and immune cells were identified using double immunostaining. There were very few apoptotic neurons present in infected tissue samples, but there was an increase of apoptotic infiltrating CD4+ and TCD8+ adaptive immune cells in the rabies infected tissue. No apoptosis was present in NK, macrophage and astrocytes. The dissemination of the human rabies virus within an infected host may be mediated by viral escape of the virus from an infected cell and may involve an anti-apoptotic mechanism, which does not kill the neuron or pro-apoptosis of TCD4+ and TCD8+ lymphocytes and which allows for increased proliferation of the virus within the CNS by attenuation of the adaptive immune response. PMID:21255623

  13. Apoptosis induction in brain during the fixed strain of rabies virus infection correlates with onset and severity of illness.

    PubMed

    Theerasurakarn, S; Ubol, S

    1998-08-01

    Viruses such as HIV, influenza, picornavirus and others are known stimulators of apoptosis. This individual cellular elimination is a preferential host defense in regenerative tissues. In contrast, if this death occurred in nonregenerating cells, such as neurons of the central nervous system, may result in disease. The target cell for rabies virus is the neuron. Here we studied the outcome of the interaction between rabies virus (CVS-11) and mouse brain cells. Replication of rabies virus in suckling mouse brain cells resulted in brain cell apoptosis, detected by DNA fragmentation and in situ apoptosis within 25 h after infection and before evidence of intracerebral immune activation. Cell death occurred simultaneously with rabies virus replication. There were clinical signs of illness in infected newborn mice within 24 h after the appearance of DNA fragmentation and before infiltration by lymphocytes. This suggested that onset of illness started independently of the immune function. This conclusion was supported by the occurrence of massive apoptosis followed by paralysis in rabies virus-infected immunosuppressed mice. Direct, viral-induced, neuronal apoptosis was the earliest death mechanism detected in these mice. We propose that pathogenesis of this fixed strain of rabies virus in mice begins with the induction of apoptosis by rabies virus replication. Cerebral damage may then be amplified by immunological mechanisms plus an additional unidentified factor. This is followed by increased permeability of the blood brain barrier.

  14. Genetically modified rabies virus ERA strain is safe and induces long-lasting protective immune response in dogs after oral vaccination.

    PubMed

    Shuai, Lei; Feng, Na; Wang, Xijun; Ge, Jinying; Wen, Zhiyuan; Chen, Weiye; Qin, Lide; Xia, Xianzhu; Bu, Zhigao

    2015-09-01

    Oral immunization in free-roaming dogs is one of the most practical approaches to prevent rabies for developing countries. The safe, efficient and long-lasting protective oral rabies vaccine for dogs is highly sought. In this study, rabies virus (RABV) Evelyn-Rokitnicki-Abelseth (ERA) strain wild-type (rERA) and a genetically modified type (rERAG333E) containing a mutation from arginine to glutamic acid at residue 333 of glycoprotein (G333E) were generated by reverse genetic. The recombinant virus rERAG333E retained growth properties of similar to the parent strain rERA in BHK-21 cell culture. The G333E mutation showed genetic stability during passage into neuroblastoma cells and in the brains of suckling mice and was significantly reduced the virulence of rERA in mice. rERAG333E was immunogenic in dogs by intramuscular inoculation. Mice orally vaccinated with rERAG333E induced strong and one year longer virus neutralizing antibodies (VNA) to RABV, and were completely protected from challenge with lethal street virus at 12months after immunization. Dogs received oral vaccination with rERAG333E induced strong protective RABV VNA response, which lasted for over 3years, and moderate saliva RABV-specific IgA. Moreover, sizeable booster responses to RABV VNA were induced by a second oral dose 1year after the first dose. These results demonstrated that the genetically modified ERA vaccine strain has the potential to serve as a safe and efficient oral live vaccine against rabies in dogs. PMID:26093157

  15. Genetically modified rabies virus ERA strain is safe and induces long-lasting protective immune response in dogs after oral vaccination.

    PubMed

    Shuai, Lei; Feng, Na; Wang, Xijun; Ge, Jinying; Wen, Zhiyuan; Chen, Weiye; Qin, Lide; Xia, Xianzhu; Bu, Zhigao

    2015-09-01

    Oral immunization in free-roaming dogs is one of the most practical approaches to prevent rabies for developing countries. The safe, efficient and long-lasting protective oral rabies vaccine for dogs is highly sought. In this study, rabies virus (RABV) Evelyn-Rokitnicki-Abelseth (ERA) strain wild-type (rERA) and a genetically modified type (rERAG333E) containing a mutation from arginine to glutamic acid at residue 333 of glycoprotein (G333E) were generated by reverse genetic. The recombinant virus rERAG333E retained growth properties of similar to the parent strain rERA in BHK-21 cell culture. The G333E mutation showed genetic stability during passage into neuroblastoma cells and in the brains of suckling mice and was significantly reduced the virulence of rERA in mice. rERAG333E was immunogenic in dogs by intramuscular inoculation. Mice orally vaccinated with rERAG333E induced strong and one year longer virus neutralizing antibodies (VNA) to RABV, and were completely protected from challenge with lethal street virus at 12months after immunization. Dogs received oral vaccination with rERAG333E induced strong protective RABV VNA response, which lasted for over 3years, and moderate saliva RABV-specific IgA. Moreover, sizeable booster responses to RABV VNA were induced by a second oral dose 1year after the first dose. These results demonstrated that the genetically modified ERA vaccine strain has the potential to serve as a safe and efficient oral live vaccine against rabies in dogs.

  16. Molecular and antigenic characterization of rabies viruses from Iran identifies variants with distinct epidemiological origins.

    PubMed

    Nadin-Davis, S A; Simani, S; Armstrong, J; Fayaz, A; Wandeler, A I

    2003-08-01

    A molecular epidemiological study of 48 recent rabies isolates recovered from cases reported throughout Iran identified three distinct viral variants, the evolutionary origins of which were identified by phylogenetic comparison with rabies viruses originating from Europe and Asia. Members of group 1 (15 isolates) were recovered from the northern half of the country only, while those of group 2 (31 isolates) were widely dispersed; both groups clustered within the widely disseminated cosmopolitan lineage. The two isolates of group 3 were detected in the northeastern tip of the country only and belonged to the Arctic strain. Rapid variant discrimination tools, employing restriction fragment length polymorphisms applied to amplified fragments of the viral genome, were devised whilst antigenic characterization of representative viruses identified a small panel of monoclonal antibodies that were also discriminatory. The future application of such methods should provide valuable epidemiological information on rabies incidence in Iran. PMID:12948379

  17. Molecular inferences suggest multiple host shifts of rabies viruses from bats to mesocarnivores in Arizona during 2001-2009.

    PubMed

    Kuzmin, Ivan V; Shi, Mang; Orciari, Lillian A; Yager, Pamela A; Velasco-Villa, Andres; Kuzmina, Natalia A; Streicker, Daniel G; Bergman, David L; Rupprecht, Charles E

    2012-01-01

    In nature, rabies virus (RABV; genus Lyssavirus, family Rhabdoviridae) represents an assemblage of phylogenetic lineages, associated with specific mammalian host species. Although it is generally accepted that RABV evolved originally in bats and further shifted to carnivores, mechanisms of such host shifts are poorly understood, and examples are rarely present in surveillance data. Outbreaks in carnivores caused by a RABV variant, associated with big brown bats, occurred repeatedly during 2001-2009 in the Flagstaff area of Arizona. After each outbreak, extensive control campaigns were undertaken, with no reports of further rabies cases in carnivores for the next several years. However, questions remained whether all outbreaks were caused by a single introduction and further perpetuation of bat RABV in carnivore populations, or each outbreak was caused by an independent introduction of a bat virus. Another question of concern was related to adaptive changes in the RABV genome associated with host shifts. To address these questions, we sequenced and analyzed 66 complete and 20 nearly complete RABV genomes, including those from the Flagstaff area and other similar outbreaks in carnivores, caused by bat RABVs, and representatives of the major RABV lineages circulating in North America and worldwide. Phylogenetic analysis demonstrated that each Flagstaff outbreak was caused by an independent introduction of bat RABV into populations of carnivores. Positive selection analysis confirmed the absence of post-shift changes in RABV genes. In contrast, convergent evolution analysis demonstrated several amino acids in the N, P, G and L proteins, which might be significant for pre-adaptation of bat viruses to cause effective infection in carnivores. The substitution S/T₂₄₂ in the viral glycoprotein is of particular merit, as a similar substitution was suggested for pathogenicity of Nishigahara RABV strain. Roles of the amino acid changes, detected in our study, require

  18. Molecular Inferences Suggest Multiple Host Shifts of Rabies Viruses from Bats to Mesocarnivores in Arizona during 2001–2009

    PubMed Central

    Kuzmin, Ivan V.; Shi, Mang; Orciari, Lillian A.; Yager, Pamela A.; Velasco-Villa, Andres; Kuzmina, Natalia A.; Streicker, Daniel G.; Bergman, David L.; Rupprecht, Charles E.

    2012-01-01

    In nature, rabies virus (RABV; genus Lyssavirus, family Rhabdoviridae) represents an assemblage of phylogenetic lineages, associated with specific mammalian host species. Although it is generally accepted that RABV evolved originally in bats and further shifted to carnivores, mechanisms of such host shifts are poorly understood, and examples are rarely present in surveillance data. Outbreaks in carnivores caused by a RABV variant, associated with big brown bats, occurred repeatedly during 2001–2009 in the Flagstaff area of Arizona. After each outbreak, extensive control campaigns were undertaken, with no reports of further rabies cases in carnivores for the next several years. However, questions remained whether all outbreaks were caused by a single introduction and further perpetuation of bat RABV in carnivore populations, or each outbreak was caused by an independent introduction of a bat virus. Another question of concern was related to adaptive changes in the RABV genome associated with host shifts. To address these questions, we sequenced and analyzed 66 complete and 20 nearly complete RABV genomes, including those from the Flagstaff area and other similar outbreaks in carnivores, caused by bat RABVs, and representatives of the major RABV lineages circulating in North America and worldwide. Phylogenetic analysis demonstrated that each Flagstaff outbreak was caused by an independent introduction of bat RABV into populations of carnivores. Positive selection analysis confirmed the absence of post-shift changes in RABV genes. In contrast, convergent evolution analysis demonstrated several amino acids in the N, P, G and L proteins, which might be significant for pre-adaptation of bat viruses to cause effective infection in carnivores. The substitution S/T242 in the viral glycoprotein is of particular merit, as a similar substitution was suggested for pathogenicity of Nishigahara RABV strain. Roles of the amino acid changes, detected in our study, require

  19. Efficiency of live attenuated and inactivated rabies viruses in prophylactic and post exposure vaccination against the street virus strain.

    PubMed

    Huang, F; Ahmad, W; Duan, M; Liu, Z; Guan, Z; Zhang, M; Qiao, B; Li, Y; Song, Y; Song, Y; Chen, Y; Amjad Ali, M

    2015-06-01

    Rabies remains an enigmatic and widely discussed global infectious disease and causes an increasing number of deaths. The currently used highly effective prophylactic and post exposure (p.e.) vaccination depends solely upon inexpensive, effective and safe vaccines to counteract the spread of the disease. In this study, the potential of an attenuated Chinese rabies vaccine (SRV9) strain in prophylactic and p.e. vaccination against the street strain of rabies virus (RV) was evaluated in mice. Prophylactic vaccination consisting of one intramuscular (i.m.) dose of SRV9 protected 100% of mice from intracerebral (i.c.) challenge with a lethal dose of the street virus. The latter was detected in the brain of mice at day 6 post challenge by RT-PCR. Post exposure vaccination was performed at days 1, 2, 3, 4, 5 and 6 post infection (p.i.) with either SRV9 or inactivated rabies vaccine. The survival rates after i.m. inoculation of SRV9 at the indicated days were 70%, 50%, 30%, 20%, 10%, and 0%, respectively; the corresponding survival rates for the inactivated rabies vaccine were 30%, 20%, 10%, 0%, 0%, and 0%, respectively. However, 100%, 90%, 70%, 50%, 20%, 10%, and 10% of mice survived after i.c. inoculation of SRV9 at the indicated days. The increased permeability of the blood-brain barrier and the infiltration of CD19+ B cells into the central nervous system after i.c. inoculation of SRV9 are regarded as prerequisites for the clearance of the street virus. The obtained data suggest that SRV9 is a promising candidate for prophylactic and p.e. vaccination against rabies infection and that it exhibits a potential for the control of rabies in China.

  20. Pseudotyping of vesicular stomatitis virus with the envelope glycoproteins of highly pathogenic avian influenza viruses.

    PubMed

    Zimmer, Gert; Locher, Samira; Berger Rentsch, Marianne; Halbherr, Stefan J

    2014-08-01

    Pseudotype viruses are useful for studying the envelope proteins of harmful viruses. This work describes the pseudotyping of vesicular stomatitis virus (VSV) with the envelope glycoproteins of highly pathogenic avian influenza viruses. VSV lacking the homotypic glycoprotein (G) gene (VSVΔG) was used to express haemagglutinin (HA), neuraminidase (NA) or the combination of both. Propagation-competent pseudotype viruses were only obtained when HA and NA were expressed from the same vector genome. Pseudotype viruses containing HA from different H5 clades were neutralized specifically by immune sera directed against the corresponding clade. Fast and sensitive reading of test results was achieved by vector-mediated expression of GFP. Pseudotype viruses expressing a mutant VSV matrix protein showed restricted spread in IFN-competent cells. This pseudotype system will facilitate the detection of neutralizing antibodies against virulent influenza viruses, circumventing the need for high-level biosafety containment.

  1. Evidence of rabies virus exposure among humans in the Peruvian Amazon.

    PubMed

    Gilbert, Amy T; Petersen, Brett W; Recuenco, Sergio; Niezgoda, Michael; Gómez, Jorge; Laguna-Torres, V Alberto; Rupprecht, Charles

    2012-08-01

    In May of 2010, two communities (Truenococha and Santa Marta) reported to be at risk of vampire bat depredation were surveyed in the Province Datem del Marañón in the Loreto Department of Perú. Risk factors for bat exposure included age less than or equal to 25 years and owning animals that had been bitten by bats. Rabies virus neutralizing antibodies (rVNAs) were detected in 11% (7 of 63) of human sera tested. Rabies virus ribonucleoprotein (RNP) immunoglobulin G (IgG) antibodies were detected in the sera of three individuals, two of whom were also seropositive for rVNA. Rabies virus RNP IgM antibodies were detected in one respondent with no evidence of rVNA or RNP IgG antibodies. Because one respondent with positive rVNA results reported prior vaccination and 86% (six of seven) of rVNA-positive respondents reported being bitten by bats, these data suggest nonfatal exposure of persons to rabies virus, which is likely associated with vampire bat depredation. PMID:22855749

  2. Evidence of Rabies Virus Exposure among Humans in the Peruvian Amazon

    PubMed Central

    Gilbert, Amy T.; Petersen, Brett W.; Recuenco, Sergio; Niezgoda, Michael; Gómez, Jorge; Laguna-Torres, V. Alberto; Rupprecht, Charles

    2012-01-01

    In May of 2010, two communities (Truenococha and Santa Marta) reported to be at risk of vampire bat depredation were surveyed in the Province Datem del Marañón in the Loreto Department of Perú. Risk factors for bat exposure included age less than or equal to 25 years and owning animals that had been bitten by bats. Rabies virus neutralizing antibodies (rVNAs) were detected in 11% (7 of 63) of human sera tested. Rabies virus ribonucleoprotein (RNP) immunoglobulin G (IgG) antibodies were detected in the sera of three individuals, two of whom were also seropositive for rVNA. Rabies virus RNP IgM antibodies were detected in one respondent with no evidence of rVNA or RNP IgG antibodies. Because one respondent with positive rVNA results reported prior vaccination and 86% (six of seven) of rVNA-positive respondents reported being bitten by bats, these data suggest nonfatal exposure of persons to rabies virus, which is likely associated with vampire bat depredation. PMID:22855749

  3. Generation of recombinant rabies Virus CVS-11 expressing eGFP applied to the rapid virus neutralization test.

    PubMed

    Xue, Xianghong; Zheng, Xuexing; Liang, Hongru; Feng, Na; Zhao, Yongkun; Gao, Yuwei; Wang, Hualei; Yang, Songtao; Xia, Xianzhu

    2014-04-01

    The determination of levels of rabies virus-neutralizing antibody (VNA) provides the foundation for the quantitative evaluation of immunity effects. The traditional fluorescent antibody virus neutralization test (FAVN) using a challenge virus standard (CVS)-11 strain as a detection antigen and staining infected cells with a fluorescein isothiocyanate (FITC)-labeled monoclonal antibody, is expensive and high-quality reagents are often difficult to obtain in developing countries. Indeed, it is essential to establish a rapid, economical, and specific rabies virus neutralization test (VNT). Here, we describe a recombinant virus rCVS-11-eGFP strain that stably expresses enhanced green fluorescent protein (eGFP) based on a reverse genetic system of the CVS-11 strain. Compared to the rCVS-11 strain, the rCVS-11-eGFP strain showed a similar growth property with passaging stability in vitro and pathogenicity in vivo. The rCVS-11-eGFP strain was utilized as a detection antigen to determine the levels of rabies VNAs in 23 human and 29 canine sera; this technique was termed the FAVN-eGFP method. The good reproducibility of FAVN-eGFP was tested with partial serum samples. Neutralization titers obtained from FAVN and FAVN-eGFP were not significantly different. The FAVN-eGFP method allows rapid economical, specific, and high-throughput assessment for the titration of rabies VNAs.

  4. Presence of Virus Neutralizing Antibodies in Cerebral Spinal Fluid Correlates with Non-Lethal Rabies in Dogs

    PubMed Central

    Leyson, Christina M.; Huang, Chien-tsun; Salyards, Gregory; Harvey, Stephen B.; Chen, Zhenhai; He, Biao; Yang, Yang; Hooper, D. C.; Dietzchold, Berhnard; Fu, Zhen F.

    2013-01-01

    Background Rabies is traditionally considered a uniformly fatal disease after onset of clinical manifestations. However, increasing evidence indicates that non-lethal infection as well as recovery from flaccid paralysis and encephalitis occurs in laboratory animals as well as humans. Methodology/Principal Findings Non-lethal rabies infection in dogs experimentally infected with wild type dog rabies virus (RABV, wt DRV-Mexico) correlates with the presence of high level of virus neutralizing antibodies (VNA) in the cerebral spinal fluid (CSF) and mild immune cell accumulation in the central nervous system (CNS). By contrast, dogs that succumbed to rabies showed only little or no VNA in the serum or in the CSF and severe inflammation in the CNS. Dogs vaccinated with a rabies vaccine showed no clinical signs of rabies and survived challenge with a lethal dose of wild-type DRV. VNA was detected in the serum, but not in the CSF of immunized dogs. Thus the presence of VNA is critical for inhibiting virus spread within the CNS and eventually clearing the virus from the CNS. Conclusions/Significance Non-lethal infection with wt RABV correlates with the presence of VNA in the CNS. Therefore production of VNA within the CNS or invasion of VNA from the periphery into the CNS via compromised blood-brain barrier is important for clearing the virus infection from CNS, thereby preventing an otherwise lethal rabies virus infection. PMID:24069466

  5. [Rabies virus study by the plaque forming system. A simplified technique].

    PubMed

    Salaun, J J

    1978-01-01

    The author describes here a simplified and economical plaque forming system for rabies virus study. It is achieved in three steps (cells preparation and infection, apply of overlay, staining). It presents more advantages than previous techniques: time as well as culture media and handling spearing, easy and durable reading. Cells prepared with di-ethyl-amino-ethyl-dextran are added to equal quantities of diluted virus (for titration) or to virus-serum mixing (for seroneutralization) and overlaid after 4 hours with a carboxymethylcellulose medium. Reading is carried out after 6 days incubation and staining with amido black. Plaques obtained are to 2-3 mm in diameter, regularly reproductible, clear and easy to read. This technique enables titration and seroneutralization and even a rabies virus cloning.

  6. A new Ebola virus nonstructural glycoprotein expressed through RNA editing.

    PubMed

    Mehedi, Masfique; Falzarano, Darryl; Seebach, Jochen; Hu, Xiaojie; Carpenter, Michael S; Schnittler, Hans-Joachim; Feldmann, Heinz

    2011-06-01

    Ebola virus (EBOV), an enveloped, single-stranded, negative-sense RNA virus, causes severe hemorrhagic fever in humans and nonhuman primates. The EBOV glycoprotein (GP) gene encodes the nonstructural soluble glycoprotein (sGP) but also produces the transmembrane glycoprotein (GP₁,₂) through transcriptional editing. A third GP gene product, a small soluble glycoprotein (ssGP), has long been postulated to be produced also as a result of transcriptional editing. To identify and characterize the expression of this new EBOV protein, we first analyzed the relative ratio of GP gene-derived transcripts produced during infection in vitro (in Vero E6 cells or Huh7 cells) and in vivo (in mice). The average percentages of transcripts encoding sGP, GP₁,₂, and ssGP were approximately 70, 25, and 5%, respectively, indicating that ssGP transcripts are indeed produced via transcriptional editing. N-terminal sequence similarity with sGP, the absence of distinguishing antibodies, and the abundance of sGP made it difficult to identify ssGP through conventional methodology. Optimized 2-dimensional (2D) gel electrophoresis analyses finally verified the expression and secretion of ssGP in tissue culture during EBOV infection. Biochemical analysis of recombinant ssGP characterized this protein as a disulfide-linked homodimer that was exclusively N glycosylated. In conclusion, we have identified and characterized a new EBOV nonstructural glycoprotein, which is expressed as a result of transcriptional editing of the GP gene. While ssGP appears to share similar structural properties with sGP, it does not appear to have the same anti-inflammatory function on endothelial cells as sGP.

  7. Co-culture: A quick approach for isolation of street rabies virus in murine neuroblastoma cells

    PubMed Central

    Sasikalaveni, A.; Tirumurugaan, K. G.; Manoharan, S.; Raj, G. Dhinakar; Kumanan, K.

    2015-01-01

    Background: Laboratory detection of rabies in most cases is based on detection of the antigen by fluorescent antibody test, however, in weak positive cases confirmative laboratory diagnosis depends on widely accepted mouse inoculation test. Cell lines like neuroblastoma have been used to isolate the virus with greater success not only to target for diagnosis, but also for molecular studies that determine the epidemiology of the circulating street rabies strains and in studies that look at the efficiency of the developed monoclonal antibodies to neutralize the different rabies strains. Due to the recent issues in obtaining ethical permission for mouse experimentation, and also the passages required in the cell lines to isolate the virus, we report herewith a co-culture protocol using the murine neuroblastoma (MNA) cells, which enable quicker isolation of street rabies virus with minimum passages. Objective: This study is not to have an alternative diagnostic assay, but an approach to produce sufficient amount of rabies virus in minimum passages by a co-culture approach in MNA cells. Materials and Methods: The MNA cells are co-cultured by topping the normal cells with infected cells every 48 h and the infectivity was followed up by performing direct fluorescent-antibody test. Results: The co-culture approach results in 100% infectivity and hence the use of live mouse for experimentation could be avoided. Conclusion: Co-culture method provides an alternative for the situations with limited sample volume and for the quicker isolation of virus which warrants the wild type strains without much modification. PMID:27047148

  8. Characterization of M gene-deficient rabies virus with advantages of effective immunization and safety as a vaccine strain.

    PubMed

    Ito, Naoto; Sugiyama, Makoto; Yamada, Kentaro; Shimizu, Kenta; Takayama-Ito, Mutsuyo; Hosokawa, Junji; Minamoto, Nobuyuki

    2005-01-01

    Matrix (M) protein of rabies virus is known to play an important role in assembly and budding of the progeny virus. We generated an M gene-deficient rabies virus, RC-HLDeltaM, using a reverse genetics system of rabies virus RC-HL strain to develop a novel type of vaccine. RC-HLDeltaM infection was confined within a single cell in mouse neuroblastoma cells. This deficient virus failed to generate the progeny virus in the cells. In contrast, RC-HLDeltaM propagated in BHK cells inductively expressing M protein. Suckling and adult mice inoculated intracerebrally with the parental RC-HL strain showed lethal infection and transient body weight loss, respectively, whereas both suckling and adult mice inoculated with RC-HLDeltaM showed no symptoms. The neutralizing antibody against rabies virus was successfully induced by intramuscular immunization with 10(5) focus-forming units of RC-HLDeltaM but not UV-inactivated RC-HL. Intranasal immunization with RC-HLDeltaM resulted in almost the same antibody titer to rabies virus as that in the case of immunization with live RC-HL strain. These findings indicate that RC-HLDeltaM is a candidate for a novel rabies vaccine that is safer and more effective than are current vaccines.

  9. Transmission dynamics of rabies virus in Thailand: Implications for disease control

    PubMed Central

    Denduangboripant, Jessada; Wacharapluesadee, Supaporn; Lumlertdacha, Boonlert; Ruankaew, Nipada; Hoonsuwan, Wirongrong; Puanghat , Apirom; Hemachudha, Thiravat

    2005-01-01

    Background In Thailand, rabies remains a neglected disease with authorities continuing to rely on human death statistics while ignoring the financial burden resulting from an enormous increase in post-exposure prophylaxis. Past attempts to conduct a mass dog vaccination and sterilization program have been limited to Bangkok city and have not been successful. We have used molecular epidemiology to define geographic localization of rabies virus phylogroups and their pattern of spread in Thailand. Methods We analyzed 239 nucleoprotein gene sequences from animal and human brain samples collected from all over Thailand between 1998 and 2002. We then reconstructed a phylogenetic tree correlating these data with geographical information. Results All sequences formed a monophyletic tree of 2 distinct phylogroups, TH1 and TH2. Three subgroups were identified in the TH1 subgroup and were distributed in the middle region of the country. Eight subgroups of TH2 viruses were identified widely distributed throughout the country overlapping the TH1 territory. There was a correlation between human-dependent transportation routes and the distribution of virus. Conclusion Inter-regional migration paths of the viruses might be correlated with translocation of dogs associated with humans. Interconnecting factors between human socioeconomic and population density might determine the transmission dynamics of virus in a rural-to-urban polarity. The presence of 2 or more rabies virus groups in a location might be indicative of a gene flow, reflecting a translocation of dogs within such region and adjacent areas. Different approaches may be required for rabies control based on the homo- or heterogeneity of the virus. Areas containing homogeneous virus populations should be targeted first. Control of dog movement associated with humans is essential. PMID:15985183

  10. Characterization of conformation-specific monoclonal antibodies against rabies virus nucleoprotein

    PubMed Central

    Jiang, Yan; Luo, Yonghuang; Michel, Frank; Hogan, Robert J.; He, Ying

    2010-01-01

    Three anti-rabies virus (RABV) nucleoprotein (N) monoclonal antibodies (Mab) were characterized by immunofluorescence assays, western blotting, and immunohistochemistry. One of these Mabs recognized the antigen by all of the assays, while the other two recognized N only in the native form in the immunofluorescence assay. These data, together with epitope mapping studies, suggest that two anti-N Mabs recognize conformational epitopes located within the N-terminal region of the RABV N protein. The availability of Mabs specific for both linear and epitope-specific antibodies should prove valuable for rabies diagnosis as well as for RABV N protein structure–function studies. PMID:20521069

  11. Government Response to the Discovery of a Rabies Virus Reservoir Species on a Previously Designated Rabies-Free Island, Taiwan, 1999-2014.

    PubMed

    Chang, S-S; Tsai, H-J; Chang, F-Y; Lee, T-S; Huang, K-C; Fang, K-Y; Wallace, R M; Inoue, S; Fei, C-Y

    2016-08-01

    Taiwan had been considered rabies free since 1961. In 2013, Taiwan confirmed the detection of rabies virus in wild Taiwan ferret-badgers. Up to December 2014, there have been 423 rabies-confirmed ferret-badgers and three cases of spillover infection into non-reservoir hosts. Genetic analysis indicates that TFBV is distinct from all other known rabies virus variants. To date, ferret-badger rabies is known to occur only in China and Taiwan. The temporal dynamics of rabid ferret-badgers in Taiwan suggests that the epizootic appears to have subsided to enzootic levels as of December 2014. According to the current epidemiologic data, there is only one TFBV strain in Taiwan. TFBV is still sequestered to the mountainous regions. Humans are at risk mainly through exposure to the virus from infected domestic meso-carnivores, mainly dogs and cats. Dogs and cats should be vaccinated to establish an immunological barrier to stop the spread of the disease from mountainous regions to domestic meso-carnivores. PMID:26542085

  12. Antigenic and genetic characterization of rabies viruses isolated from domestic and wild animals of Brazil identifies the hoary fox as a rabies reservoir.

    PubMed

    Bernardi, F; Nadin-Davis, S A; Wandeler, A I; Armstrong, J; Gomes, A A B; Lima, F S; Nogueira, F R B; Ito, F H

    2005-11-01

    Fifty Brazilian rabies viruses, collected from many different animal species and several regions of the country, were characterized by partial sequencing of the central, variable region of the P gene, a locus useful for sensitive molecular epidemiological studies. Phylogenetic analysis of the sequences, which included comparison with other rabies strains recovered from throughout the Americas, identified three main groups of Brazilian viruses, arbitrarily designated BRL-1 to BRL-3. BRL-1 was found in terrestrial carnivores and clusters with other American strains of the cosmopolitan lineage. BRL-2 comprised two distinct isolates, recovered from two species of non-haematophagous bats, that had evolutionary links to insectivorous-bat-derived strains of North America. BRL-3 consisted of isolates from vampire bats and from livestock species probably infected via contact with vampire bats. The terrestrial group was further subdivided into three subtypes: BRL-1a was associated exclusively with dogs and cats, while BRL-1b and BRL-1c were found exclusively in hoary foxes. These observations strongly support the role of the Brazilian hoary fox as a rabies reservoir. Screening of representative Brazilian rabies viruses against a collection of anti-rabies monoclonal antibodies (mAbs) identified a small panel of mAbs that could be used to discriminate between all Brazilian subgroups as defined by genetic classification in this study. PMID:16227239

  13. Characterization of a pseudorabies virus glycoprotein gene with homology to herpes simplex virus type 1 and type 2 glycoprotein C.

    PubMed Central

    Robbins, A K; Watson, R J; Whealy, M E; Hays, W W; Enquist, L W

    1986-01-01

    A pseudorabies virus (Becker strain) glycoprotein gene was located in the UL region at map position 0.40. The gene was identified by using open reading frame Escherichia coli plasmid expression vectors and specific antibody reagents. A 1.55-kilobase unspliced transcript from the gene was detected in pseudorabies virus-infected tissue culture cells. The DNA sequence revealed a single open reading frame of 1,437 base pairs encoding 479 amino acids. The predicted primary translation product has a molecular weight of 50,860 and contains features of a typical herpesvirus glycoprotein. An E. coli expression plasmid was constructed that contained essentially all of the open reading frame for this gene. Antibodies raised in rabbits against the protein expressed in bacteria by this plasmid immunoprecipitated pseudorabies virus-specific glycoproteins of 92,000 and 74,000 daltons from infected cell extracts. It is likely that these two forms represent different glycosylation states of the protein. Images PMID:3009851

  14. Antigenic typing of Brazilian rabies virus samples isolated from animals and humans, 1989-2000.

    PubMed

    Favoretto, Silvana Regina; Carrieri, Maria Luiza; Cunha, Elenice Maria S; Aguiar, Elizabeth A C; Silva, Luzia Helena Q; Sodre, Miriam M; Souza, Maria Conceição A M; Kotait, Ivanete

    2002-01-01

    Animal and human rabies samples isolated between 1989 and 2000 were typified by means of a monoclonal antibody panel against the viral nucleoprotein. The panel had been previously established to study the molecular epidemiology of rabies virus in the Americas. Samples were isolated in the Diagnostic Laboratory of the Pasteur Institute and in other rabies diagnostic centers in Brazil. In addition to the fixed virus samples CVS-31/96-IP, preserved in mouse brain, and PV-BHK/97, preserved in cell culture, a total of 330 rabies virus samples were isolated from dogs, cats, cattle, horses, bats, sheep, goat, swine, foxes, marmosets, coati and humans. Six antigenic variants that were compatible with the pre-established monoclonal antibodies panel were defined: numbers 2 (dog), 3 (Desmodus rotundus), 4 (Tadarida brasiliensis), 5 (vampire bat from Venezuela), 6 (Lasiurus cinereus) and Lab (reacted to all used antibodies). Six unknown profiles, not compatible with the panel, were also found. Samples isolated from insectivore bats showed the greatest variability and the most commonly isolated variant was variant-3 (Desmodus rotundus). These findings may be related to the existence of multiple independent transmission cycles, involving different bat species. PMID:12048546

  15. The effect of neurotoxin on rabies virus binding to mouse neuroblastoma cells.

    PubMed

    Briggs, D J; Phillips, R M

    1991-08-01

    Mouse neuroblastoma cells were exposed to alpha bungarotoxin, a neurotoxin known to inhibit rabies virus binding to the nicotinic acetylcholine receptor located at the neuromuscular junction in muscle tissue. The total amount of 3H-CVS virus that bound to neurotoxin treated cells was separated into specific and non-specific binding using a cold competition assay. Comparison of untreated and neurotoxin treated cells demonstrated that the majority of cell-associated virus in untreated cells was of a specific nature whereas the majority of the cell-associated virus in neurotoxin treated cells was due to non-specific binding.

  16. Prevalence of Rabies Virus in Foxes Trapped in the Canadian Arctic

    PubMed Central

    Secord, D. C.; Bradley, J. A.; Eaton, R. D.; Mitchell, D.

    1980-01-01

    Brains and salivary glands of 521 trapped arctic foxes (Alopex lagopus) submitted from four different settlement areas in the Northwest Territories were examined for rabies by the standard fluorescent antibody and mouse inoculation tests. Rabies antigen was present in 44 of 201 (21.9%) brains from foxes trapped in the Sachs Harbour area, but submissions from Cambridge Bay (127), Spence Bay (93) and Gjoa Haven (100) were negative. Virus was also present in salivary glands from 43 (97.7%) of these 44 positive foxes. The arctic fox continues to be the main wildlife reservoir of rabies in the Canadian Arctic and foxes in the prodromal stage of the disease pose a particular threat to the trapper. Preexposure vaccination should always be a consideration in this occupational group. PMID:7459793

  17. Virus isolation from saliva and salivary glands of cattle naturally infected with paralytic rabies.

    PubMed

    Delpietro, H A; Larghi, O P; Russo, R G

    2001-02-16

    The infectivity of saliva, salivary and mammary glands, muscle, lung, kidney and liver of 87 cattle infected with paralytic rabies (positive viral isolation from brains) was studied. Fifty percent dilutions of saliva and tissue samples were inoculated intracerebrally into 10- to 15-day-old mice. Viral isolation in mice was confirmed by direct rabies fluorescent-antibody test and the antigenic variant of the isolates characterized by monoclonal antibodies. Rabies virus was isolated from 4.6% of salivary glands and from 1.6% of saliva samples. The rest of the peripheral tissues were negative. Cerebral and peripheral isolates belonged to vampire-bat antigenic variants. These results indicate that cattle infected by vampire bats may be a source of infection for man. The infection risk would depend on the type of contact between rabid cattle and man. PMID:11182465

  18. The importance of immune evasion in the pathogenesis of rabies virus.

    PubMed

    Ito, Naoto; Moseley, Gregory W; Sugiyama, Makoto

    2016-08-01

    Rabies is a zoonotic disease caused by the Lyssavirus rabies virus (RABV) that can infect most mammals, including humans, where it has a case-fatality rate of almost 100%. Although preventable by vaccination, rabies causes c. 59,000 human fatalities every year worldwide. Thus, there exists an urgent need to establish an effective therapy and/or improve dissemination of vaccines for humans and animals. These outcomes require greater understanding of the mechanisms of RABV pathogenesis to identify new molecular targets for the development of therapeutics and/or live vaccines with high levels of safety. Importantly, a number of studies in recent years have indicated that RABV specifically suppresses host immunity through diverse mechanisms and that this is a key process in pathogenicity. Here, we review current understanding of immune modulation by RABV, with an emphasis on its significance to pathogenicity and the potential exploitation of this knowledge to develop new vaccines and antivirals. PMID:27041139

  19. Ineffectiveness and comparative pathogenicity of attenuated rabies virus vaccines for the striped skunk (Mephitis mephitis).

    PubMed

    Rupprecht, C E; Charlton, K M; Artois, M; Casey, G A; Webster, W A; Campbell, J B; Lawson, K F; Schneider, L G

    1990-01-01

    Three attenuated rabies virus vaccines (SAD-B19, ERA/BHK-21, AZA 2) were compared for efficacy and safety in the striped skunk (Mephitis mephitis) by the oral and intranasal routes. The SAD-B19 and ERA/BHK-21 vaccines were given orally; all three vaccines were given intranasally. Oral administration of SAD-B19 and ERA/BHK-21 vaccines induced neither seroconversion nor significant protection against rabies challenge. One skunk which consumed a SAD-B19 vaccine-laden bait succumbed to vaccine-induced rabies. Intranasal instillation of the three vaccines resulted in the deaths of two of six (AZA 2), three of six (ERA/BHK-21) and six of six (SAD-B19) skunks.

  20. The importance of immune evasion in the pathogenesis of rabies virus

    PubMed Central

    ITO, Naoto; MOSELEY, Gregory W.; SUGIYAMA, Makoto

    2016-01-01

    Rabies is a zoonotic disease caused by the Lyssavirus rabies virus (RABV) that can infect most mammals, including humans, where it has a case-fatality rate of almost 100%. Although preventable by vaccination, rabies causes c. 59,000 human fatalities every year worldwide. Thus, there exists an urgent need to establish an effective therapy and/or improve dissemination of vaccines for humans and animals. These outcomes require greater understanding of the mechanisms of RABV pathogenesis to identify new molecular targets for the development of therapeutics and/or live vaccines with high levels of safety. Importantly, a number of studies in recent years have indicated that RABV specifically suppresses host immunity through diverse mechanisms and that this is a key process in pathogenicity. Here, we review current understanding of immune modulation by RABV, with an emphasis on its significance to pathogenicity and the potential exploitation of this knowledge to develop new vaccines and antivirals. PMID:27041139

  1. Evidence of two distinct phylogenetic lineages of dog rabies virus circulating in Cambodia.

    PubMed

    Mey, Channa; Metlin, Artem; Duong, Veasna; Ong, Sivuth; In, Sotheary; Horwood, Paul F; Reynes, Jean-Marc; Bourhy, Hervé; Tarantola, Arnaud; Buchy, Philippe

    2016-03-01

    This first extensive retrospective study of the molecular epidemiology of dog rabies in Cambodia included 149 rabies virus (RABV) entire nucleoprotein sequences obtained from 1998-2011. The sequences were analyzed in conjunction with RABVs from other Asian countries. Phylogenetic reconstruction confirmed the South-East Asian phylogenetic clade comprising viruses from Cambodia, Vietnam, Thailand, Laos and Myanmar. The present study represents the first attempt to classify the phylogenetic lineages inside this clade, resulting in the confirmation that all the Cambodian viruses belonged to the South-East Asian (SEA) clade. Three distinct phylogenetic lineages in the region were established with the majority of viruses from Cambodia closely related to viruses from Thailand, Laos and Vietnam, forming the geographically widespread phylogenetic lineage SEA1. A South-East Asian lineage SEA2 comprised two viruses from Cambodia was identified, which shared a common ancestor with RABVs originating from Laos. Viruses from Myanmar formed separate phylogenetic lineages within the major SEA clade. Bayesian molecular clock analysis suggested that the time to most recent common ancestor (TMRCA) of all Cambodian RABVs dated to around 1950. The TMRCA of the Cambodian SEA1 lineage was around 1964 and that of the SEA2 lineage was around 1953. The results identified three phylogenetically distinct and geographically separated lineages inside the earlier identified major SEA clade, covering at least five countries in the region. A greater understanding of the molecular epidemiology of rabies in South-East Asia is an important step to monitor progress on the efforts to control canine rabies in the region.

  2. Evidence of two distinct phylogenetic lineages of dog rabies virus circulating in Cambodia.

    PubMed

    Mey, Channa; Metlin, Artem; Duong, Veasna; Ong, Sivuth; In, Sotheary; Horwood, Paul F; Reynes, Jean-Marc; Bourhy, Hervé; Tarantola, Arnaud; Buchy, Philippe

    2016-03-01

    This first extensive retrospective study of the molecular epidemiology of dog rabies in Cambodia included 149 rabies virus (RABV) entire nucleoprotein sequences obtained from 1998-2011. The sequences were analyzed in conjunction with RABVs from other Asian countries. Phylogenetic reconstruction confirmed the South-East Asian phylogenetic clade comprising viruses from Cambodia, Vietnam, Thailand, Laos and Myanmar. The present study represents the first attempt to classify the phylogenetic lineages inside this clade, resulting in the confirmation that all the Cambodian viruses belonged to the South-East Asian (SEA) clade. Three distinct phylogenetic lineages in the region were established with the majority of viruses from Cambodia closely related to viruses from Thailand, Laos and Vietnam, forming the geographically widespread phylogenetic lineage SEA1. A South-East Asian lineage SEA2 comprised two viruses from Cambodia was identified, which shared a common ancestor with RABVs originating from Laos. Viruses from Myanmar formed separate phylogenetic lineages within the major SEA clade. Bayesian molecular clock analysis suggested that the time to most recent common ancestor (TMRCA) of all Cambodian RABVs dated to around 1950. The TMRCA of the Cambodian SEA1 lineage was around 1964 and that of the SEA2 lineage was around 1953. The results identified three phylogenetically distinct and geographically separated lineages inside the earlier identified major SEA clade, covering at least five countries in the region. A greater understanding of the molecular epidemiology of rabies in South-East Asia is an important step to monitor progress on the efforts to control canine rabies in the region. PMID:26705238

  3. Ultra-deep sequencing of intra-host rabies virus populations during cross-species transmission.

    PubMed

    Borucki, Monica K; Chen-Harris, Haiyin; Lao, Victoria; Vanier, Gilda; Wadford, Debra A; Messenger, Sharon; Allen, Jonathan E

    2013-11-01

    One of the hurdles to understanding the role of viral quasispecies in RNA virus cross-species transmission (CST) events is the need to analyze a densely sampled outbreak using deep sequencing in order to measure the amount of mutation occurring on a small time scale. In 2009, the California Department of Public Health reported a dramatic increase (350) in the number of gray foxes infected with a rabies virus variant for which striped skunks serve as a reservoir host in Humboldt County. To better understand the evolution of rabies, deep-sequencing was applied to 40 unpassaged rabies virus samples from the Humboldt outbreak. For each sample, approximately 11 kb of the 12 kb genome was amplified and sequenced using the Illumina platform. Average coverage was 17,448 and this allowed characterization of the rabies virus population present in each sample at unprecedented depths. Phylogenetic analysis of the consensus sequence data demonstrated that samples clustered according to date (1995 vs. 2009) and geographic location (northern vs. southern). A single amino acid change in the G protein distinguished a subset of northern foxes from a haplotype present in both foxes and skunks, suggesting this mutation may have played a role in the observed increased transmission among foxes in this region. Deep-sequencing data indicated that many genetic changes associated with the CST event occurred prior to 2009 since several nonsynonymous mutations that were present in the consensus sequences of skunk and fox rabies samples obtained from 20032010 were present at the sub-consensus level (as rare variants in the viral population) in skunk and fox samples from 1995. These results suggest that analysis of rare variants within a viral population may yield clues to ancestral genomes and identify rare variants that have the potential to be selected for if environment conditions change. PMID:24278493

  4. Ultra-Deep Sequencing of Intra-host Rabies Virus Populations during Cross-species Transmission

    PubMed Central

    Borucki, Monica K.; Chen-Harris, Haiyin; Lao, Victoria; Vanier, Gilda; Wadford, Debra A.; Messenger, Sharon; Allen, Jonathan E.

    2013-01-01

    One of the hurdles to understanding the role of viral quasispecies in RNA virus cross-species transmission (CST) events is the need to analyze a densely sampled outbreak using deep sequencing in order to measure the amount of mutation occurring on a small time scale. In 2009, the California Department of Public Health reported a dramatic increase (350) in the number of gray foxes infected with a rabies virus variant for which striped skunks serve as a reservoir host in Humboldt County. To better understand the evolution of rabies, deep-sequencing was applied to 40 unpassaged rabies virus samples from the Humboldt outbreak. For each sample, approximately 11 kb of the 12 kb genome was amplified and sequenced using the Illumina platform. Average coverage was 17,448 and this allowed characterization of the rabies virus population present in each sample at unprecedented depths. Phylogenetic analysis of the consensus sequence data demonstrated that samples clustered according to date (1995 vs. 2009) and geographic location (northern vs. southern). A single amino acid change in the G protein distinguished a subset of northern foxes from a haplotype present in both foxes and skunks, suggesting this mutation may have played a role in the observed increased transmission among foxes in this region. Deep-sequencing data indicated that many genetic changes associated with the CST event occurred prior to 2009 since several nonsynonymous mutations that were present in the consensus sequences of skunk and fox rabies samples obtained from 20032010 were present at the sub-consensus level (as rare variants in the viral population) in skunk and fox samples from 1995. These results suggest that analysis of rare variants within a viral population may yield clues to ancestral genomes and identify rare variants that have the potential to be selected for if environment conditions change. PMID:24278493

  5. Ultra-deep sequencing of intra-host rabies virus populations during cross-species transmission.

    PubMed

    Borucki, Monica K; Chen-Harris, Haiyin; Lao, Victoria; Vanier, Gilda; Wadford, Debra A; Messenger, Sharon; Allen, Jonathan E

    2013-11-01

    One of the hurdles to understanding the role of viral quasispecies in RNA virus cross-species transmission (CST) events is the need to analyze a densely sampled outbreak using deep sequencing in order to measure the amount of mutation occurring on a small time scale. In 2009, the California Department of Public Health reported a dramatic increase (350) in the number of gray foxes infected with a rabies virus variant for which striped skunks serve as a reservoir host in Humboldt County. To better understand the evolution of rabies, deep-sequencing was applied to 40 unpassaged rabies virus samples from the Humboldt outbreak. For each sample, approximately 11 kb of the 12 kb genome was amplified and sequenced using the Illumina platform. Average coverage was 17,448 and this allowed characterization of the rabies virus population present in each sample at unprecedented depths. Phylogenetic analysis of the consensus sequence data demonstrated that samples clustered according to date (1995 vs. 2009) and geographic location (northern vs. southern). A single amino acid change in the G protein distinguished a subset of northern foxes from a haplotype present in both foxes and skunks, suggesting this mutation may have played a role in the observed increased transmission among foxes in this region. Deep-sequencing data indicated that many genetic changes associated with the CST event occurred prior to 2009 since several nonsynonymous mutations that were present in the consensus sequences of skunk and fox rabies samples obtained from 20032010 were present at the sub-consensus level (as rare variants in the viral population) in skunk and fox samples from 1995. These results suggest that analysis of rare variants within a viral population may yield clues to ancestral genomes and identify rare variants that have the potential to be selected for if environment conditions change.

  6. Elucidating the phylodynamics of endemic rabies virus in eastern Africa using whole-genome sequencing

    PubMed Central

    Brunker, Kirstyn; Marston, Denise A; Horton, Daniel L; Cleaveland, Sarah; Fooks, Anthony R; Kazwala, Rudovick; Ngeleja, Chanasa; Lembo, Tiziana; Sambo, Maganga; Mtema, Zacharia J; Sikana, Lwitiko; Wilkie, Gavin; Biek, Roman; Hampson, Katie

    2015-01-01

    Many of the pathogens perceived to pose the greatest risk to humans are viral zoonoses, responsible for a range of emerging and endemic infectious diseases. Phylogeography is a useful tool to understand the processes that give rise to spatial patterns and drive dynamics in virus populations. Increasingly, whole-genome information is being used to uncover these patterns, but the limits of phylogenetic resolution that can be achieved with this are unclear. Here, whole-genome variation was used to uncover fine-scale population structure in endemic canine rabies virus circulating in Tanzania. This is the first whole-genome population study of rabies virus and the first comprehensive phylogenetic analysis of rabies virus in East Africa, providing important insights into rabies transmission in an endemic system. In addition, sub-continental scale patterns of population structure were identified using partial gene data and used to determine population structure at larger spatial scales in Africa. While rabies virus has a defined spatial structure at large scales, increasingly frequent levels of admixture were observed at regional and local levels. Discrete phylogeographic analysis revealed long-distance dispersal within Tanzania, which could be attributed to human-mediated movement, and we found evidence of multiple persistent, co-circulating lineages at a very local scale in a single district, despite on-going mass dog vaccination campaigns. This may reflect the wider endemic circulation of these lineages over several decades alongside increased admixture due to human-mediated introductions. These data indicate that successful rabies control in Tanzania could be established at a national level, since most dispersal appears to be restricted within the confines of country borders but some coordination with neighbouring countries may be required to limit transboundary movements. Evidence of complex patterns of rabies circulation within Tanzania necessitates the use of whole

  7. Skunk rabies.

    PubMed

    Charlton, K M; Webster, W A; Casey, G A; Rupprecht, C E

    1988-01-01

    In North America, the number of cases of rabies diagnosed in skunks generally exceeds that in either raccoons or foxes. Enzootic skunk rabies occurs mainly in four geographic regions: (1) southern Ontario and Quebec and upper New York State; (2) the north central United States and the Canadian provinces of Manitoba, Saskatchewan, and Alberta; (3) California; and (4) south central United States (Texas and several adjacent states). Rabies in these areas (in skunks and, to a large extent, in other terrestrial mammals) is caused mainly by three street virus variants, as determined by monoclonal antibody testing (one variant for areas 2 and 3 and separate variants for each of areas 1 and 4). Experimental studies suggest that the species specificity (e.g., raccoon vs. skunk) of enzootic rabies is due, at least partly, to differences in the pathogenicity of variants of rabies virus.

  8. Characterization of Sri Lanka rabies virus isolates using nucleotide sequence analysis of nucleoprotein gene.

    PubMed

    Arai, Y T; Takahashi, H; Kameoka, Y; Shiino, T; Wimalaratne, O; Lodmell, D L

    2001-01-01

    Thirty-four suspected rabid brain samples from 2 humans, 24 dogs, 4 cats, 2 mongooses, I jackal and I water buffalo were collected in 1995-1996 in Sri Lanka. Total RNA was extracted directly from brain suspensions and examined using a one-step reverse transcription-polymerase chain reaction (RT-PCR) for the rabies virus nucleoprotein (N) gene. Twenty-eight samples were found positive for the virus N gene by RT-PCR and also for the virus antigens by fluorescent antibody (FA) test. Rabies virus isolates obtained from different animal species in different regions of Sri Lanka were genetically homogenous. Sequences of 203 nucleotides (nt)-long RT-PCR products obtained from 16 of 27 samples were found identical. Sequences of 1350 nt of N genes of 14 RT-PCR products were determined. The Sri Lanka isolates under study formed a specific cluster that included also an earlier isolate from India but did not include the known isolates from China, Thailand, Malaysia, Israel, Iran, Oman, Saudi Arabia, Russia, Nepal, Philippines, Japan and from several other countries. These results suggest that one type of rabies virus is circulating among human, dog, cat, mongoose, jackal and water buffalo living near Colombo City and in other five remote regions in Sri Lanka.

  9. A VL-linker-VH Orientation Dependent Single Chain Variable Antibody Fragment Against Rabies Virus G Protein with Enhanced Neutralizing Potency in vivo.

    PubMed

    Cheng, Yue; Li, Zhuang; Xi, Hualong; Gu, Tiejun; Yuan, Ruosen; Chen, Xiaoxu; Jiang, Chunlai; Kong, Wei; Wu, Yongge

    2016-01-01

    Lethal rabies can be prevented effectively by post-exposure prophylactic (PEP) with rabies immunoglobulin (RIG). Single-chain variable fragment (scFv), which is composed of a variable heavy chain (VH) and variable light chain (VL) connected by a peptide linker, may be developed as alternative to RIG for neutralizing rabies virus (RV). However, our previously constructed scFv (FV57S) with the (NH2) VH-linker-VL (COOH) orientation showed a lower neutralizing potency than its parent RIG. This orientation may inhibit FV57S from refolding into an intact and correct conformation. Therefore, the RFV57S protein with a VL-linker-VH orientation was constructed based on FV57S. A HIS tag was incorporated to aid in purification and detection of RFV57S and FV57S. However, abilities of RFV57S and FV57S to bind with the anti-HIS tag mAb were different. Therefore, a novel direct ELISA was established by utilizing a biotin-labeled truncated glycoprotein of RV. Although with similar stability and in vitro neutralizing potency as FV57S, RFV57S showed enhanced binding ability, affinity and in vivo protective efficacy against lethal dose of RV. Our studies support the feasibility of developing a scFv with reversed orientation and provide a novel method for evaluating the binding ability, stability and affinity of engineered antibodies recognizing linear epitope.

  10. Genetic characterization and geographic distribution of rabies virus isolates in Brazil: identification of two reservoirs, dogs and vampire bats.

    PubMed

    Ito, M; Arai, Y T; Itou, T; Sakai, T; Ito, F H; Takasaki, T; Kurane, I

    2001-06-01

    We analyzed 50 rabies virus samples isolated in Brazil from 12 dogs, 11 cats, 5 vampire bats, 15 cattle, 2 horses, 1 pig, 1 sheep, and 3 humans to investigate the molecular epidemiology of rabies viruses. We sequenced 203 nucleotides on the nucleoprotein gene by direct sequencing of the PCR-amplified products. All the isolates belonged to the genotype 1 and homology of the 203 nucleotides was at least 83.7% among isolates. The main reservoirs were estimated based on the homology of nucleotide sequences. Brazilian rabies virus isolates were clustered into two reservoir groups: dogs and vampire bats. All the dog-related rabies virus isolates showed nucleotide homology greater than 99.0%. Vampire bat-related rabies virus isolates showed nucleotide homology greater than 96.6% and could be further divided into subgroups corresponding to areas where viruses were isolated. These data suggest that circulating rabies variants belong to at least two different genotype clusters in Brazil and that these two clusters are maintained independently among vampire bats and dogs.

  11. Genetic characterization and geographic distribution of rabies virus isolates in Brazil: identification of two reservoirs, dogs and vampire bats.

    PubMed

    Ito, M; Arai, Y T; Itou, T; Sakai, T; Ito, F H; Takasaki, T; Kurane, I

    2001-06-01

    We analyzed 50 rabies virus samples isolated in Brazil from 12 dogs, 11 cats, 5 vampire bats, 15 cattle, 2 horses, 1 pig, 1 sheep, and 3 humans to investigate the molecular epidemiology of rabies viruses. We sequenced 203 nucleotides on the nucleoprotein gene by direct sequencing of the PCR-amplified products. All the isolates belonged to the genotype 1 and homology of the 203 nucleotides was at least 83.7% among isolates. The main reservoirs were estimated based on the homology of nucleotide sequences. Brazilian rabies virus isolates were clustered into two reservoir groups: dogs and vampire bats. All the dog-related rabies virus isolates showed nucleotide homology greater than 99.0%. Vampire bat-related rabies virus isolates showed nucleotide homology greater than 96.6% and could be further divided into subgroups corresponding to areas where viruses were isolated. These data suggest that circulating rabies variants belong to at least two different genotype clusters in Brazil and that these two clusters are maintained independently among vampire bats and dogs. PMID:11384221

  12. A comparative study of rabies virus isolates from hematophagous bats in Brazil.

    PubMed

    Castilho, Juliana G; Carnieli, Pedro; Oliveira, Rafael N; Fahl, Willian O; Cavalcante, Rosangela; Santana, Antonio A; Rosa, Wellington L G A; Carrieri, Maria L; Kotait, Ivanete

    2010-10-01

    The Brazilian chiropteran fauna consists of 167 species; of which, three are hematophagous: the common vampire bat (Desmodus rotundus), the white-winged vampire bat (Diaemus youngi), and the hairy-legged vampire bat (Diphylla ecaudata). The aim of this study was to describe the isolation of Rabies virus from common and hairy-legged vampire bats and to report the first comparative antigenic and genetic studies of isolates from these bats. Antigenic and genetic typing of both isolates identified them as antigenic variant 3 (AgV3), the variant frequently isolated from common vampire bats. Phylogenetic analysis showed 99.3% identity between the isolates. This is the first time since 1934 that Rabies virus has been isolated from hairy-legged vampire bats in Brazil. Our analysis provides evidence that the existence of rabies-positive isolates from hairy-legged vampire bats may be the result of an interspecific rabies transmission event from common vampire bats and suggests that roost cohabitation may occur.

  13. Profile of Cytokines and Chemokines Triggered by Wild-Type Strains of Rabies Virus in Mice.

    PubMed

    Appolinário, Camila Michele; Allendorf, Susan Dora; Peres, Marina Gea; Ribeiro, Bruna Devidé; Fonseca, Clóvis R; Vicente, Acácia Ferreira; Antunes, João Marcelo A de Paula; Megid, Jane

    2016-02-01

    Rabies is a lethal infectious disease that causes 55,000 human deaths per year and is transmitted by various mammalian species, such as dogs and bats. The host immune response is essential for avoiding viral progression and promoting viral clearance. Cytokines and chemokines are crucial in the development of an immediate antiviral response; the rabies virus (RABV) attempts to evade this immune response. The virus's capacity for evasion is correlated with its pathogenicity and the host's inflammatory response, with highly pathogenic strains being the most efficient at hijacking the host's defense mechanisms and thereby decreasing inflammation. The purpose of this study was to evaluate the expression of a set of cytokine and chemokine genes that are related to the immune response in the brains of mice inoculated intramuscularly or intracerebrally with two wild-type strains of RABV, one from dog and the other from vampire bat. The results demonstrated that the gene expression profile is intrinsic to the specific rabies variant. The prompt production of cytokines and chemokines seems to be more important than their levels of expression for surviving a rabies infection. PMID:26711511

  14. A comparative study of rabies virus isolates from hematophagous bats in Brazil.

    PubMed

    Castilho, Juliana G; Carnieli, Pedro; Oliveira, Rafael N; Fahl, Willian O; Cavalcante, Rosangela; Santana, Antonio A; Rosa, Wellington L G A; Carrieri, Maria L; Kotait, Ivanete

    2010-10-01

    The Brazilian chiropteran fauna consists of 167 species; of which, three are hematophagous: the common vampire bat (Desmodus rotundus), the white-winged vampire bat (Diaemus youngi), and the hairy-legged vampire bat (Diphylla ecaudata). The aim of this study was to describe the isolation of Rabies virus from common and hairy-legged vampire bats and to report the first comparative antigenic and genetic studies of isolates from these bats. Antigenic and genetic typing of both isolates identified them as antigenic variant 3 (AgV3), the variant frequently isolated from common vampire bats. Phylogenetic analysis showed 99.3% identity between the isolates. This is the first time since 1934 that Rabies virus has been isolated from hairy-legged vampire bats in Brazil. Our analysis provides evidence that the existence of rabies-positive isolates from hairy-legged vampire bats may be the result of an interspecific rabies transmission event from common vampire bats and suggests that roost cohabitation may occur. PMID:20966291

  15. Profile of Cytokines and Chemokines Triggered by Wild-Type Strains of Rabies Virus in Mice.

    PubMed

    Appolinário, Camila Michele; Allendorf, Susan Dora; Peres, Marina Gea; Ribeiro, Bruna Devidé; Fonseca, Clóvis R; Vicente, Acácia Ferreira; Antunes, João Marcelo A de Paula; Megid, Jane

    2016-02-01

    Rabies is a lethal infectious disease that causes 55,000 human deaths per year and is transmitted by various mammalian species, such as dogs and bats. The host immune response is essential for avoiding viral progression and promoting viral clearance. Cytokines and chemokines are crucial in the development of an immediate antiviral response; the rabies virus (RABV) attempts to evade this immune response. The virus's capacity for evasion is correlated with its pathogenicity and the host's inflammatory response, with highly pathogenic strains being the most efficient at hijacking the host's defense mechanisms and thereby decreasing inflammation. The purpose of this study was to evaluate the expression of a set of cytokine and chemokine genes that are related to the immune response in the brains of mice inoculated intramuscularly or intracerebrally with two wild-type strains of RABV, one from dog and the other from vampire bat. The results demonstrated that the gene expression profile is intrinsic to the specific rabies variant. The prompt production of cytokines and chemokines seems to be more important than their levels of expression for surviving a rabies infection.

  16. Rabies Virus CVS-N2c(ΔG) Strain Enhances Retrograde Synaptic Transfer and Neuronal Viability.

    PubMed

    Reardon, Thomas R; Murray, Andrew J; Turi, Gergely F; Wirblich, Christoph; Croce, Katherine R; Schnell, Matthias J; Jessell, Thomas M; Losonczy, Attila

    2016-02-17

    Virally based transsynaptic tracing technologies are powerful experimental tools for neuronal circuit mapping. The glycoprotein-deletion variant of the SAD-B19 vaccine strain rabies virus (RABV) has been the reagent of choice in monosynaptic tracing, since it permits the mapping of synaptic inputs to genetically marked neurons. Since its introduction, new helper viruses and reagents that facilitate complementation have enhanced the efficiency of SAD-B19(ΔG) transsynaptic transfer, but there has been little focus on improvements to the core RABV strain. Here we generate a new deletion mutant strain, CVS-N2c(ΔG), and examine its neuronal toxicity and efficiency in directing retrograde transsynaptic transfer. We find that by comparison with SAD-B19(ΔG), the CVS-N2c(ΔG) strain exhibits a reduction in neuronal toxicity and a marked enhancement in transsynaptic neuronal transfer. We conclude that the CVS-N2c(ΔG) strain provides a more effective means of mapping neuronal circuitry and of monitoring and manipulating neuronal activity in vivo in the mammalian CNS. PMID:26804990

  17. Emerging Technologies for the Detection of Rabies Virus: Challenges and Hopes in the 21st Century

    PubMed Central

    Fooks, Anthony R.; Johnson, Nicholas; Freuling, Conrad M.; Wakeley, Philip R.; Banyard, Ashley C.; McElhinney, Lorraine M.; Marston, Denise A.; Dastjerdi, Akbar; Wright, Edward; Weiss, Robin A.; Müller, Thomas

    2009-01-01

    The diagnosis of rabies is routinely based on clinical and epidemiological information, especially when exposures are reported in rabies-endemic countries. Diagnostic tests using conventional assays that appear to be negative, even when undertaken late in the disease and despite the clinical diagnosis, have a tendency, at times, to be unreliable. These tests are rarely optimal and entirely dependent on the nature and quality of the sample supplied. In the course of the past three decades, the application of molecular biology has aided in the development of tests that result in a more rapid detection of rabies virus. These tests enable viral strain identification from clinical specimens. Currently, there are a number of molecular tests that can be used to complement conventional tests in rabies diagnosis. Indeed the challenges in the 21st century for the development of rabies diagnostics are not of a technical nature; these tests are available now. The challenges in the 21st century for diagnostic test developers are two-fold: firstly, to achieve internationally accepted validation of a test that will then lead to its acceptance by organisations globally. Secondly, the areas of the world where such tests are needed are mainly in developing regions where financial and logistical barriers prevent their implementation. Although developing countries with a poor healthcare infrastructure recognise that molecular-based diagnostic assays will be unaffordable for routine use, the cost/benefit ratio should still be measured. Adoption of rapid and affordable rabies diagnostic tests for use in developing countries highlights the importance of sharing and transferring technology through laboratory twinning between the developed and the developing countries. Importantly for developing countries, the benefit of molecular methods as tools is the capability for a differential diagnosis of human diseases that present with similar clinical symptoms. Antemortem testing for human

  18. Emerging technologies for the detection of rabies virus: challenges and hopes in the 21st century.

    PubMed

    Fooks, Anthony R; Johnson, Nicholas; Freuling, Conrad M; Wakeley, Philip R; Banyard, Ashley C; McElhinney, Lorraine M; Marston, Denise A; Dastjerdi, Akbar; Wright, Edward; Weiss, Robin A; Müller, Thomas

    2009-01-01

    The diagnosis of rabies is routinely based on clinical and epidemiological information, especially when exposures are reported in rabies-endemic countries. Diagnostic tests using conventional assays that appear to be negative, even when undertaken late in the disease and despite the clinical diagnosis, have a tendency, at times, to be unreliable. These tests are rarely optimal and entirely dependent on the nature and quality of the sample supplied. In the course of the past three decades, the application of molecular biology has aided in the development of tests that result in a more rapid detection of rabies virus. These tests enable viral strain identification from clinical specimens. Currently, there are a number of molecular tests that can be used to complement conventional tests in rabies diagnosis. Indeed the challenges in the 21st century for the development of rabies diagnostics are not of a technical nature; these tests are available now. The challenges in the 21st century for diagnostic test developers are two-fold: firstly, to achieve internationally accepted validation of a test that will then lead to its acceptance by organisations globally. Secondly, the areas of the world where such tests are needed are mainly in developing regions where financial and logistical barriers prevent their implementation. Although developing countries with a poor healthcare infrastructure recognise that molecular-based diagnostic assays will be unaffordable for routine use, the cost/benefit ratio should still be measured. Adoption of rapid and affordable rabies diagnostic tests for use in developing countries highlights the importance of sharing and transferring technology through laboratory twinning between the developed and the developing countries. Importantly for developing countries, the benefit of molecular methods as tools is the capability for a differential diagnosis of human diseases that present with similar clinical symptoms. Antemortem testing for human

  19. Rabies virus inactivation by binary ethylenimine: new method for inactivated vaccine production.

    PubMed Central

    Larghi, O P; Nebel, A E

    1980-01-01

    The inactivation dynamics of rabies virus (PV strain) by binary ethylenimine, and the immunogenic properites and the stability of the vaccines prepared using this agent, were studied. Binary ethylenimine at a final concentration of 0.01 M was prepared wtih 2-bromoethylamine hydrobromide in alkaline solutions, either separately from or in suspensions of rabies virus propagated in BHK cells. The infectivity of virus suspensions containing more than 108 plaque-forming units per 0.1 ml was inactivated in 2 h when the inactivating agent was prepared before its addition to the suspensions, and in3 h when prepared directly in the suspensions. Liquid vaccines prepared in this manner and stored at different temperatures maintained potency for 1 month at 37 degrees C and for 6 months at 4 degrees C and 22 to 25 degrees C. Lyophilized vaccine maintained its potency for 6 months at the three temperatures. The inactivated vaccine mixed with aluminum or oil adjuvant at high dilutions protected guinea pigs against challenge. This safer procedure for rabies virus inactivation offers promise for the production of effective vaccines for the immunization of dogs and cattle. PMID:7358836

  20. Studies on antigenic and genomic properties of Brazilian rabies virus isolates.

    PubMed

    Schaefer, R; Batista, H B R; Franco, A C; Rijsewijk, F A M; Roehe, P M

    2005-05-20

    Despite the recognized stability of rabies virus, differences among isolates from different species have been found. This work was carried out with the aim to identify antigenic and genomic differences in Brazilian rabies virus isolates and to verify whether such alterations would bear any relationship with the different hosts for the virus in nature. For that, 79 Brazilian rabies viruses isolated from different host species and from distinct regions within Brazil were submitted to antigenic characterization with a panel of 11 monoclonal antibodies (Mabs) directed to lyssavirus antigens and to genomic analyses by the reverse transcriptase-polymerase chain reaction (RT-PCR) amplification of the N gene followed by restriction endonuclease analysis (REA). In addition, the nucleotide sequences of part of the N gene (225 bp) of seven isolates, taken as representative of the majority of the viruses under study, were determined. The analyses with the Mabs and RT-PCR/REA allowed the identification of two major groups of variants, the first formed by most isolates of cattle and bats and the second formed by viruses of dog origin. Partial sequencing of the N gene confirmed the similarity among isolates from cattle origin and those of vampire bats. However, viruses from non-haematophagous bats exhibited consistent differences from those of vampire bat isolates. Such findings suggest that the variants have evolved fairly stable modifications, which are not altered after passage in a dead-end host of a distinct species. No association could be established between antigenic or genomic alterations and geographic distribution of the isolates, which suggests that evolution of the virus has been directed to adaptation to the host species. PMID:15863275

  1. High Diversity of Rabies Viruses Associated with Insectivorous Bats in Argentina: Presence of Several Independent Enzootics

    PubMed Central

    Piñero, Carolina; Gury Dohmen, Federico; Beltran, Fernando; Martinez, Leila; Novaro, Laura; Russo, Susana; Palacios, Gustavo; Cisterna, Daniel M.

    2012-01-01

    Background Rabies is a fatal infection of the central nervous system primarily transmitted by rabid animal bites. Rabies virus (RABV) circulates through two different epidemiological cycles: terrestrial and aerial, where dogs, foxes or skunks and bats, respectively, act as the most relevant reservoirs and/or vectors. It is widely accepted that insectivorous bats are not important vectors of RABV in Argentina despite the great diversity of bat species and the extensive Argentinean territory. Methods We studied the positivity rate of RABV detection in different areas of the country, and the antigenic and genetic diversity of 99 rabies virus (RABV) strains obtained from 14 species of insectivorous bats collected in Argentina between 1991 and 2008. Results Based on the analysis of bats received for RABV analysis by the National Rabies system of surveillance, the positivity rate of RABV in insectivorous bats ranged from 3.1 to 5.4%, depending on the geographic location. The findings were distributed among an extensive area of the Argentinean territory. The 99 strains of insectivorous bat-related sequences were divided into six distinct lineages associated with Tadarida brasiliensis, Myotis spp, Eptesicus spp, Histiotus montanus, Lasiurus blosseviilli and Lasiurus cinereus. Comparison with RABV sequences obtained from insectivorous bats of the Americas revealed co-circulation of similar genetic variants in several countries. Finally, inter-species transmission, mostly related with Lasiurus species, was demonstrated in 11.8% of the samples. Conclusions This study demonstrates the presence of several independent enzootics of rabies in insectivorous bats of Argentina. This information is relevant to identify potential areas at risk for human and animal infection. PMID:22590657

  2. Evaluation of In vitro Antiviral Activity of Datura metel Linn. Against Rabies Virus

    PubMed Central

    Roy, Soumen; Mukherjee, Sandeepan; Pawar, Sandip; Chowdhary, Abhay

    2016-01-01

    Objective: The soxhlet and cold extracts of Datura metel Linn. were evaluated for in vitro antirabies activity. Materials and Methods: Soxhlet and cold extraction method were used to extract Datura (fruit and seed) extracts. In vitro cytotoxicity assay was performed by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide assay. Based on the CC50 range, the in vitro antirabies activity of the extracts was screened by rapid fluorescent focus inhibition test and molecular method. Results: The Datura (fruit and seed) extracts were not cytotoxic below 5 mg/ml (CC50). Titer of 10−4 rabies virus challenge virus standard (RV CVS) (1 50% tissue culture infective dose [1 TCID50]) was obtained by RFFT method and the challenge dose of 10 TCID50 was used for antirabies assay. Datura fruit and seed (soxhlet and cold) extracts showed 50% inhibition of RV CVS at 2.5 mg/ml and 1.25 mg/ml (inhibitory concentration 50% [IC50]), respectively. The tested extracts showed selectivity index (CC50/IC50) ranging from 2 to 4. The viral RNA was extracted and real-time reverse transcription-polymerase chain reaction was performed which also revealed a 2-fold reduction of viral load at 1.25 mg/ml of the Datura seed (soxhlet methanolic and cold aqueous) extracts. Conclusion: To the best of our knowledge, this is the first study of in vitro antiviral activity of D. metel Linn. against rabies virus. Datura seed extracts have a potential in vitro antirabies activity and, in future, can be further screened for in vivo activity against rabies virus in murine model. SUMMARY In the present study, Datura metel. Linn showed and in-vitro anti rabies activity in Vero cell line which was determined by RFFIT method and PCR method

  3. Evaluation of In vitro Antiviral Activity of Datura metel Linn. Against Rabies Virus

    PubMed Central

    Roy, Soumen; Mukherjee, Sandeepan; Pawar, Sandip; Chowdhary, Abhay

    2016-01-01

    Objective: The soxhlet and cold extracts of Datura metel Linn. were evaluated for in vitro antirabies activity. Materials and Methods: Soxhlet and cold extraction method were used to extract Datura (fruit and seed) extracts. In vitro cytotoxicity assay was performed by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide assay. Based on the CC50 range, the in vitro antirabies activity of the extracts was screened by rapid fluorescent focus inhibition test and molecular method. Results: The Datura (fruit and seed) extracts were not cytotoxic below 5 mg/ml (CC50). Titer of 10−4 rabies virus challenge virus standard (RV CVS) (1 50% tissue culture infective dose [1 TCID50]) was obtained by RFFT method and the challenge dose of 10 TCID50 was used for antirabies assay. Datura fruit and seed (soxhlet and cold) extracts showed 50% inhibition of RV CVS at 2.5 mg/ml and 1.25 mg/ml (inhibitory concentration 50% [IC50]), respectively. The tested extracts showed selectivity index (CC50/IC50) ranging from 2 to 4. The viral RNA was extracted and real-time reverse transcription-polymerase chain reaction was performed which also revealed a 2-fold reduction of viral load at 1.25 mg/ml of the Datura seed (soxhlet methanolic and cold aqueous) extracts. Conclusion: To the best of our knowledge, this is the first study of in vitro antiviral activity of D. metel Linn. against rabies virus. Datura seed extracts have a potential in vitro antirabies activity and, in future, can be further screened for in vivo activity against rabies virus in murine model. SUMMARY In the present study, Datura metel. Linn showed and in-vitro anti rabies activity in Vero cell line which was determined by RFFIT method and PCR method PMID:27695266

  4. Interferon-inducible GTPase: a novel viral response protein involved in rabies virus infection.

    PubMed

    Li, Ling; Wang, Hualei; Jin, Hongli; Cao, Zengguo; Feng, Na; Zhao, Yongkun; Zheng, Xuexing; Wang, Jianzhong; Li, Qian; Zhao, Guoxing; Yan, Feihu; Wang, Lina; Wang, Tiecheng; Gao, Yuwei; Tu, Changchun; Yang, Songtao; Xia, Xianzhu

    2016-05-01

    Rabies virus infection is a major public health concern because of its wide host-interference spectrum and nearly 100 % lethality. However, the interactions between host and virus remain unclear. To decipher the authentic response in the central nervous system after rabies virus infection, a dynamic analysis of brain proteome alteration was performed. In this study, 104 significantly differentially expressed proteins were identified, and intermediate filament, interferon-inducible GTPases, and leucine-rich repeat-containing protein 16C were the three outstanding groups among these proteins. Interferon-inducible GTPases were prominent because of their strong upregulation. Moreover, quantitative real-time PCR showed distinct upregulation of interferon-inducible GTPases at the level of transcription. Several studies have shown that interferon-inducible GTPases are involved in many biological processes, such as viral infection, endoplasmic reticulum stress response, and autophagy. These findings indicate that interferon-inducible GTPases are likely to be a potential target involved in rabies pathogenesis or the antiviral process.

  5. Real-time Imaging of Rabies Virus Entry into Living Vero cells

    PubMed Central

    Xu, Haijiao; Hao, Xian; Wang, Shaowen; Wang, Zhiyong; Cai, Mingjun; Jiang, Junguang; Qin, Qiwei; Zhang, Maolin; Wang, Hongda

    2015-01-01

    Understanding the mechanism of rabies virus (RABV) infection is vital for prevention and therapy of virulent rabies. However, the infection mechanism remains largely uncharacterized due to the limited methods and viral models. Herein, we utilized a powerful single-virus tracking technique to dynamically and globally visualize the infection process of the live attenuated rabies vaccine strain-SRV9 in living Vero cells. Firstly, it was found that the actin-enriched filopodia is in favor of virus reaching to the cell body. Furthermore, by carrying out drug perturbation experiments, we confirmed that RABV internalization into Vero cells proceeds via classical dynamin-dependent clathrin-mediated endocytosis with requirement for intact actin, but caveolae-dependent endocytosis is not involved. Then, our real-time imaging results unambiguously uncover the characteristics of viral internalization and cellular transport dynamics. In addition, our results directly and quantitatively reveal that the intracellular motility of internalized RABV particles is largely microtubule-dependent. Collectively, our work is crucial for understanding the initial steps of RABV infection, and elucidating the mechanisms of post-infection. Significantly, the results provide profound insight into development of novel and effective antiviral targets. PMID:26148807

  6. Interferon-inducible GTPase: a novel viral response protein involved in rabies virus infection.

    PubMed

    Li, Ling; Wang, Hualei; Jin, Hongli; Cao, Zengguo; Feng, Na; Zhao, Yongkun; Zheng, Xuexing; Wang, Jianzhong; Li, Qian; Zhao, Guoxing; Yan, Feihu; Wang, Lina; Wang, Tiecheng; Gao, Yuwei; Tu, Changchun; Yang, Songtao; Xia, Xianzhu

    2016-05-01

    Rabies virus infection is a major public health concern because of its wide host-interference spectrum and nearly 100 % lethality. However, the interactions between host and virus remain unclear. To decipher the authentic response in the central nervous system after rabies virus infection, a dynamic analysis of brain proteome alteration was performed. In this study, 104 significantly differentially expressed proteins were identified, and intermediate filament, interferon-inducible GTPases, and leucine-rich repeat-containing protein 16C were the three outstanding groups among these proteins. Interferon-inducible GTPases were prominent because of their strong upregulation. Moreover, quantitative real-time PCR showed distinct upregulation of interferon-inducible GTPases at the level of transcription. Several studies have shown that interferon-inducible GTPases are involved in many biological processes, such as viral infection, endoplasmic reticulum stress response, and autophagy. These findings indicate that interferon-inducible GTPases are likely to be a potential target involved in rabies pathogenesis or the antiviral process. PMID:26906695

  7. A recombinant rabies virus (ERAGS) for use in a bait vaccine for swine

    PubMed Central

    2016-01-01

    Purpose Rabies viruses (RABV) circulating worldwide in various carnivores occasionally cause fatal encephalitis in swine. In this study, the safety and immunogenicity of a recombinant rabies virus, the ERAGS strain constructed with a reverse genetics system, was evaluated in domestic pigs. Materials and Methods Growing pigs were administered 1 mL (108.0 FAID50/mL) of the ERAGS strain via intramuscular (IM) or oral routes and were observed for 4 weeks' post-inoculation. Three sows were also inoculated with 1 mL of the ERAGS strain via the IM route. The safety and immunogenicity in swine were evaluated using daily observation and a virus-neutralizing assay (VNA). Fluorescent antibody tests (FAT) for the RABV antigen and reverse transcriptase-polymerase chain reaction (RT-PCR) assays for the detection of the nucleocapsid (N) gene of RABV were conducted with brain tissues from the sows after necropsy. Results The growing pigs and sows administered the ERAGS strain did not exhibit any clinical sign of rabies during the test period test and did develop VNA titers. The growing pigs inoculated with the ERAGS strain via the IM route showed higher VNA titers than did those receiving oral administration. FAT and RT-PCR assays were unable to detect RABV in several tissues, including brain samples from the sows. Conclusion Our results suggest that the ERAGS strain was safe in growing pigs and sows and induced moderate VNA titers in pigs. PMID:27489807

  8. Real-time Imaging of Rabies Virus Entry into Living Vero cells.

    PubMed

    Xu, Haijiao; Hao, Xian; Wang, Shaowen; Wang, Zhiyong; Cai, Mingjun; Jiang, Junguang; Qin, Qiwei; Zhang, Maolin; Wang, Hongda

    2015-01-01

    Understanding the mechanism of rabies virus (RABV) infection is vital for prevention and therapy of virulent rabies. However, the infection mechanism remains largely uncharacterized due to the limited methods and viral models. Herein, we utilized a powerful single-virus tracking technique to dynamically and globally visualize the infection process of the live attenuated rabies vaccine strain-SRV9 in living Vero cells. Firstly, it was found that the actin-enriched filopodia is in favor of virus reaching to the cell body. Furthermore, by carrying out drug perturbation experiments, we confirmed that RABV internalization into Vero cells proceeds via classical dynamin-dependent clathrin-mediated endocytosis with requirement for intact actin, but caveolae-dependent endocytosis is not involved. Then, our real-time imaging results unambiguously uncover the characteristics of viral internalization and cellular transport dynamics. In addition, our results directly and quantitatively reveal that the intracellular motility of internalized RABV particles is largely microtubule-dependent. Collectively, our work is crucial for understanding the initial steps of RABV infection, and elucidating the mechanisms of post-infection. Significantly, the results provide profound insight into development of novel and effective antiviral targets.

  9. Integrating the landscape epidemiology and genetics of RNA viruses: rabies in domestic dogs as a model.

    PubMed

    Brunker, K; Hampson, K; Horton, D L; Biek, R

    2012-12-01

    Landscape epidemiology and landscape genetics combine advances in molecular techniques, spatial analyses and epidemiological models to generate a more real-world understanding of infectious disease dynamics and provide powerful new tools for the study of RNA viruses. Using dog rabies as a model we have identified how key questions regarding viral spread and persistence can be addressed using a combination of these techniques. In contrast to wildlife rabies, investigations into the landscape epidemiology of domestic dog rabies requires more detailed assessment of the role of humans in disease spread, including the incorporation of anthropogenic landscape features, human movements and socio-cultural factors into spatial models. In particular, identifying and quantifying the influence of anthropogenic features on pathogen spread and measuring the permeability of dispersal barriers are important considerations for planning control strategies, and may differ according to cultural, social and geographical variation across countries or continents. Challenges for dog rabies research include the development of metapopulation models and transmission networks using genetic information to uncover potential source/sink dynamics and identify the main routes of viral dissemination. Information generated from a landscape genetics approach will facilitate spatially strategic control programmes that accommodate for heterogeneities in the landscape and therefore utilise resources in the most cost-effective way. This can include the efficient placement of vaccine barriers, surveillance points and adaptive management for large-scale control programmes.

  10. The effect of interferon on the receptor sites to rabies virus on mouse neuroblastoma cells

    SciTech Connect

    Briggs, D.J.

    1989-01-01

    The binding of rabies virus to mouse neuroblastoma cells (MNA) primed with alpha interferon (IFN-{alpha}), beta interferon (IFN-{beta}), or alpha bungarotoxin (BTX) was examined. A saturable number of receptor sites to rabies virus was calculated by increasing the amount of {sup 3}H-CVS added to a constant number of untreated MNA cells. MNA cells were then exposed to 20 I.U. of IFN-{alpha}, IFN-{beta}, or 1 {mu}g of BTX and assayed to determine if these treatments had an effect on the number of receptor sites to rabies virus. Total amount of {sup 3}H-CVS bound to MNA cells was determined during a three hour incubation period. Cold competition assays using 1,000 fold excess unlabeled CVS were used to determine non-specific binding for each treatment. Specific binding was then calculated by subtracting non-specific binding from the total amount of CVS bound to MNA cells. A similar amount of total viral protein bound to untreated and IFN-{beta}, and BTX treated cells after 180 minutes of incubation. The bound protein varied by only 0.07 {mu}g. However, the amount of specific and non-specific binding varied a great deal between treatments. BTX caused an increase in non-specific and a decrease in specific binding of rabies virus. IFN-{beta} produced variable results in non-specific and specific binding while IFN-{alpha} caused mainly specific binding to occur. The most significant change brought about by IFN-{alpha} was an increase in the rate of viral attachment. At 30 minutes post-infection, IFN-{alpha} treated cells had bound 90% of the total amount of virus bound to untreated cells after 180 minutes. The increased binding rate did not cause a productive infection of rabies virus. No viral production was evident after an incubation period of 48 hours in either IFN-{alpha} or IFN-{beta} treated cells.

  11. Stability of attenuated live virus rabies vaccine in baits targeted to wild foxes under operational conditions.

    PubMed Central

    Lawson, K F; Bachmann, P

    2001-01-01

    The viability of an attenuated live virus rabies vaccine in a bait targeted to red foxes was examined under various operational conditions in a series of experiments in Ontario. The virus was relatively stable over a 28-day period in the field, losing a mean 0.5, s = 0.2 log10 of virus titer. The micro-environment into which the bait was placed (open cultivated field, grassy meadow, wooded grove, sun or shade) did not make an appreciable difference in the viability of the virus. There was no significant difference (P < or = 0.05) between mean ambient temperatures and the temperature of fluids in blister packs of baits placed in sun or shade. Sixty-three percent of foxes fed baits exposed to sun and shade conditions for 21 days (titer 10(6.2) tissue culture infective doses per 1 mL) developed rabies virus-neutralizing antibodies. Storage of vaccine baits at -30 degrees C prior to bait distribution was important in maintaining virus viability. PMID:11360859

  12. Antibodies to rabies virus in terrestrial wild mammals in native rainforest on the north coast of São Paulo State, Brazil.

    PubMed

    Araujo, Danielle B; Martorelli, Luzia A; Kataoka, Ana Paula G A; Campos, Angélica C A; Rodrigues, Camila S; Sanfilippo, Luiz F; Cunha, Elenice S; Durigon, Edison L; Favoretto, Silvana R

    2014-07-01

    Rabies causes thousands of human and animal deaths worldwide each year. The emergent importance of rabies in wild animals demonstrates the necessity of epidemiologic studies of infection in these species toward the development of better strategies for prevention and control of rabies. We analyzed the circulation of rabies virus among wildlife species from a native rainforest in São Paulo State, Brazil. We used the rapid fluorescent focus inhibition test (RFFIT) to test for rabies virus-neutralizing antibodies in 139 captured terrestrial mammals and the fluorescent antibody test (FAT), mouse inoculation test (MIT), and reverse-transcriptase (RT)-PCR to test for virus in samples from the central nervous system of 53 animals found dead. The percentage of samples positive by RFFIT was 10.8%. All samples tested by FAT, MIT, and RT-PCR were negative. Research should be continued to obtain a better understanding of the role of wildlife in the circulation and transmission of rabies virus.

  13. Detection of multiple strains of rabies virus RNA using primers designed to target Mexican vampire bat variants.

    PubMed Central

    Loza-Rubio, E.; Rojas-Anaya, E.; Banda-Ruíz, V. M.; Nadin-Davis, S. A.; Cortez-García, B.

    2005-01-01

    A reverse transcription-polymerase chain reaction (RT-PCR), that uses primers specifically designed to amplify a portion of the N gene of vampire bat strains of rabies that circulate in Mexico, but also recognizing most of the rabies variants circulating in endemic areas, was established. This standardized PCR assay was able to detect viral RNA in tenfold serial dilutions up to a 10(7) dilution using stock virus at an original titre of 10(7.5) LD50. The assay was highly specific for rabies virus. Forty different rabies isolates recovered from different species and geographical regions in the country were diagnosed as positive and negative by the fluorescent antibody test (FAT). These same samples were re-examined by both PCR and the mouse inoculation test (MIT). Compared with MIT the PCR exhibited an epidemiological sensitivity of 86% and a specificity of 91% while its positive predictive value was 96%. PMID:16181515

  14. Detection of multiple strains of rabies virus RNA using primers designed to target Mexican vampire bat variants.

    PubMed

    Loza-Rubio, E; Rojas-Anaya, E; Banda-Ruíz, V M; Nadin-Davis, S A; Cortez-García, B

    2005-10-01

    A reverse transcription-polymerase chain reaction (RT-PCR), that uses primers specifically designed to amplify a portion of the N gene of vampire bat strains of rabies that circulate in Mexico, but also recognizing most of the rabies variants circulating in endemic areas, was established. This standardized PCR assay was able to detect viral RNA in tenfold serial dilutions up to a 10(7) dilution using stock virus at an original titre of 10(7.5) LD50. The assay was highly specific for rabies virus. Forty different rabies isolates recovered from different species and geographical regions in the country were diagnosed as positive and negative by the fluorescent antibody test (FAT). These same samples were re-examined by both PCR and the mouse inoculation test (MIT). Compared with MIT the PCR exhibited an epidemiological sensitivity of 86% and a specificity of 91% while its positive predictive value was 96%. PMID:16181515

  15. The anterograde transport of rabies virus in rat sensory dorsal root ganglia neurons.

    PubMed

    Tsiang, H; Lycke, E; Ceccaldi, P E; Ermine, A; Hirardot, X

    1989-08-01

    We have previously described the capacity of neurites extending from cultured rat sensory dorsal root ganglia (DRG) neurons to transport rabies virus through axoplasm in the retrograde direction. Here we report the infection of cultured neurons derived from the DRG and the subsequent anterograde transport of rabies virus from the infected cell somas through the extending neurites to its release into the culture supernatant. Viral transport was monitored by titration of the virus yield in the external compartment. Both early and late transport mechanisms of rabies virions were identified. The first one occurred a few hours post-infection and was undetectable 6 h later, before the initiation of viral replication. The velocity of this first wave of infective virions was in the range of 100 to 400 mm/day. The early viral transport was probably the result of a direct translocation of infective virions from the somatic site of entry to the neuritic extensions and subsequent release into the culture medium without replication in the cellular perikaryon. The second virus transport peak was detected 48 h post-infection. In this case, the virions detected in the neuritic compartment were presumably the progeny of the inoculated virus which had replicated in the perikaryon before the viral transport occurs. Using a four-compartment culture device we were able to demonstrate, simultaneously, retrograde and anterograde transport of the virus. The presence of antirabies serum in contact with the exposed neurites did not inhibit either the retrograde or the anterograde transport mechanisms. The viral release from the neuritic extensions after the fast anterograde transport was evaluated to be in the range of 150 to 300 infectious virions per bundle of neurites per day.

  16. 9 CFR 113.312 - Rabies Vaccine, Live Virus.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... calculations, five replicate virus titrations shall be conducted on a sample of the vaccine virus dilution used... rehydrated vaccine which, on the basis of previous titrations, has been diluted to the proposed minimum... disrupted and undiluted cell fluids from each lot shall be tested. (2) Virus titrations. Final...

  17. 9 CFR 113.312 - Rabies Vaccine, Live Virus.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... calculations, five replicate virus titrations shall be conducted on a sample of the vaccine virus dilution used... rehydrated vaccine which, on the basis of previous titrations, has been diluted to the proposed minimum... disrupted and undiluted cell fluids from each lot shall be tested. (2) Virus titrations. Final...

  18. 9 CFR 113.312 - Rabies Vaccine, Live Virus.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... calculations, five replicate virus titrations shall be conducted on a sample of the vaccine virus dilution used... rehydrated vaccine which, on the basis of previous titrations, has been diluted to the proposed minimum... disrupted and undiluted cell fluids from each lot shall be tested. (2) Virus titrations. Final...

  19. 9 CFR 113.312 - Rabies Vaccine, Live Virus.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... calculations, five replicate virus titrations shall be conducted on a sample of the vaccine virus dilution used... rehydrated vaccine which, on the basis of previous titrations, has been diluted to the proposed minimum... disrupted and undiluted cell fluids from each lot shall be tested. (2) Virus titrations. Final...

  20. 9 CFR 113.312 - Rabies Vaccine, Live Virus.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... calculations, five replicate virus titrations shall be conducted on a sample of the vaccine virus dilution used... rehydrated vaccine which, on the basis of previous titrations, has been diluted to the proposed minimum... disrupted and undiluted cell fluids from each lot shall be tested. (2) Virus titrations. Final...

  1. [The Infectious and Pathogenic Characteristics of Rabies Virus Strain CTNCEC25].

    PubMed

    Wang, Chunhua; Luo, Shan; Rong, Weihua; Liu, Yongdi; Li, Hui; Zhu, Shimao; Tian, Hua; Zhou, Wei; Guo, Caiping

    2015-09-01

    To investigate the phenotypic characteristics of the strain of the rabies virus CTNCEC25, the strain of the China rabies virus CTN-1 adapted to primary chicken embryo cells (CECs), Vero cells, and mouse neuroblastoma N2a cells was inoculated with CTNCEC25 and parental CTN-1 strains to explore the cytopathic effect (CPE) and growth kinetics of CTNCEC25 on cultured cells. To determine the pathogenicity of CTNCEC25, suckling mice, adult mice, guinea pigs and rabbits were inoculated with CTNCEC25 via the intracerebral route and their survival monitored every day. Furthermore, the CTNCEC25 strain was passed serially in CECs for 20 passages and then 3 passages in the brains of suckling mice to determine phenotypic stability. CTNCEC25 achieved similar growth kinetics in Vero cells and N2a cells compared with parental CTN-1, but CTNCEC25 replicated more efficiently in CECs than the CTN-1 strain with a titer 72 h after infection reaching 10(7.5-7.6) FFU/mL, which was significantly higher than the 10(5.8) FFU/mL achieved by its parental strain, CTN-1. Moreover, CTNCEC25 induced apparent CPE in Vero cells, CECs and N2a cells. Analyses of intracerebral inoculation demonstrated that CTNCEC25 was attenuated profoundly in adult mice and was completely apathogenic to guinea pigs and rabbits, though it caused death in suckling mice. The CTNCEC25 strain proliferated steadily after serial passage in CECs and the brains of suckling mice, and remained avirulent in adult mice. These results suggest that CTNCEC25 is a highly attenuated and genetically stable strain of the rabies virus. CTNCEC25 replicated stably and efficiently in cultured cells and achieved high titers, so it could be a promising and safe vaccine strain for rabies prevention in China. PMID:26738284

  2. Rabies virus infection in Eptesicus fuscus bats born in captivity (naïve bats).

    PubMed

    Davis, April D; Jarvis, Jodie A; Pouliott, Craig; Rudd, Robert J

    2013-01-01

    The study of rabies virus infection in bats can be challenging due to quarantine requirements, husbandry concerns, genetic differences among animals, and lack of medical history. To date, all rabies virus (RABV) studies in bats have been performed in wild caught animals. Determining the RABV exposure history of a wild caught bat based on the presence or absence of viral neutralizing antibodies (VNA) may be misleading. Previous studies have demonstrated that the presence of VNA following natural or experimental inoculation is often ephemeral. With this knowledge, it is difficult to determine if a seronegative, wild caught bat has been previously exposed to RABV. The influence of prior rabies exposure in healthy, wild caught bats is unknown. To investigate the pathogenesis of RABV infection in bats born in captivity (naïve bats), naïve bats were inoculated intramuscularly with one of two Eptesicus fuscus rabies virus variants, EfV1 or EfV2. To determine the host response to a heterologous RABV, a separate group of naïve bats were inoculated with a Lasionycteris noctivagans RABV (LnV1). Six months following the first inoculation, all bats were challenged with EfV2. Our results indicate that naïve bats may have some level of innate resistance to intramuscular RABV inoculation. Additionally, naïve bats inoculated with the LnV demonstrated the lowest clinical infection rate of all groups. However, primary inoculation with EfV1 or LnV did not appear to be protective against a challenge with the more pathogenic EfV2.

  3. [The Infectious and Pathogenic Characteristics of Rabies Virus Strain CTNCEC25].

    PubMed

    Wang, Chunhua; Luo, Shan; Rong, Weihua; Liu, Yongdi; Li, Hui; Zhu, Shimao; Tian, Hua; Zhou, Wei; Guo, Caiping

    2015-09-01

    To investigate the phenotypic characteristics of the strain of the rabies virus CTNCEC25, the strain of the China rabies virus CTN-1 adapted to primary chicken embryo cells (CECs), Vero cells, and mouse neuroblastoma N2a cells was inoculated with CTNCEC25 and parental CTN-1 strains to explore the cytopathic effect (CPE) and growth kinetics of CTNCEC25 on cultured cells. To determine the pathogenicity of CTNCEC25, suckling mice, adult mice, guinea pigs and rabbits were inoculated with CTNCEC25 via the intracerebral route and their survival monitored every day. Furthermore, the CTNCEC25 strain was passed serially in CECs for 20 passages and then 3 passages in the brains of suckling mice to determine phenotypic stability. CTNCEC25 achieved similar growth kinetics in Vero cells and N2a cells compared with parental CTN-1, but CTNCEC25 replicated more efficiently in CECs than the CTN-1 strain with a titer 72 h after infection reaching 10(7.5-7.6) FFU/mL, which was significantly higher than the 10(5.8) FFU/mL achieved by its parental strain, CTN-1. Moreover, CTNCEC25 induced apparent CPE in Vero cells, CECs and N2a cells. Analyses of intracerebral inoculation demonstrated that CTNCEC25 was attenuated profoundly in adult mice and was completely apathogenic to guinea pigs and rabbits, though it caused death in suckling mice. The CTNCEC25 strain proliferated steadily after serial passage in CECs and the brains of suckling mice, and remained avirulent in adult mice. These results suggest that CTNCEC25 is a highly attenuated and genetically stable strain of the rabies virus. CTNCEC25 replicated stably and efficiently in cultured cells and achieved high titers, so it could be a promising and safe vaccine strain for rabies prevention in China.

  4. Carbohydrate Structure of Sindbis Virus Glycoprotein E2 from Virus Grown in Hamster and Chicken Cells

    PubMed Central

    Burke, David; Keegstra, Kenneth

    1979-01-01

    Sindbis virus was used as a probe to examine glycosylation processes in two different species of cultured cells. Parallel studies were carried out analyzing the carbohydrate added to Sindbis glycoprotein E2 when the virus was grown in chicken embryo cells and BHK cells. The Pronase glycopeptides of Sindbis glycoprotein E2 were purified by a combination of ion-exchange and gel filtration chromatography. Four glycopeptides were resolved, ranging in molecular weight from 1,800 to 2,700. Structures are proposed for each of the four glycopeptides, based on data obtained by quantitative composition analyses, methylation analyses, and degradation of the glycopeptides using purified exo- and endoglycosidases. The largest three glycopeptides (S1, S2, and S3) have similar structures but differ in the extent of sialylation. All three contain N-acetylglucosamine, mannose, galactose, and fucose, in a structure similar to oligosaccharides found on other glycoproteins. Glycopeptide S1 has two residues of sialic acid, whereas glycopeptides S2 and S3 contain 1 and 0 residues of sialic acid, respectively. The smallest glycopeptide, S4, contains only N-acetyglucosamine and mannose, and is also similar to mannose-rich oligosaccharides found on other glycoproteins. Each of the complex glycopeptides (S1, S2, or S3) from virus grown in BHK cells is indistinguishable from the corresponding glycopeptides derived from virus grown in chicken cells. Glycopeptide S4 is also very similar in size, composition, and sugar linkages from virus derived from the two hosts. These results suggest that chicken cells and BHK cells have similar glycosylation mechanisms and glycosylate Sindbis glycoprotein E2 in nearly identical ways. PMID:430605

  5. Exposure to rabies virus in a population of free-ranging capuchin monkeys (Cebus apella nigritus) in a fragmented, environmentally protected area in southeastern Brazil.

    PubMed

    Machado, Gustavo Puglia; Antunes, João Marcelo Azevedo de Paula; Uieda, Wilson; Biondo, Alexander Welker; Cruvinel, Tatiana Morosini de Andrade; Kataoka, Ana Paula; Martorelli, Luzia Fátima Alves; de Jong, David; Amaral, Jeanne Margareth Gimenes; Hoppe, Estevam Guilherme Lux; Guerra Neto, Guilherme; Megid, Jane

    2012-07-01

    The aim of this study is to assess the frequency of rabies antibodies in free-ranging capuchin monkeys (Cebus apella nigritus) in a fragmented, environmentally protected, rural area of southeastern Brazil. Thirty-six free-ranging monkeys were tested by the rapid fluorescent focus inhibition test for detection of antibodies against rabies virus. Four individuals (11.11 %) had neutralizing antibody titers ≥ 0.25 IU/mL, demonstrating rabies virus exposure. PMID:22430558

  6. [Rabies virus in Nyctinomops laticaudatus bats in the City of Rio de Janeiro: isolation, titration and epidemiology].

    PubMed

    da Silva, Marlon Vicente; Xavier, Sheila de Matos; Moreira, Wildeberg Cal; dos Santos, Beatriz Cristina Pereira; Esbérard, Carlos E L

    2007-01-01

    The first case report of rabies in bats of the species Nyctinomops laticaudatus, in the city of Rio de Janeiro City, is presented. Virus isolation and titration were performed in different tissues, and high titers were found in the brain and salivary glands. Rabies occurrence in such an infrequent species in this state suggests that the disease may be more prevalent than it appears to be.

  7. Immunogenicity Studies in Carnivores Using a Rabies Virus Construct with a Site-Directed Deletion in the Phosphoprotein

    PubMed Central

    Vos, Ad; Conzelmann, Karl-Klaus; Finke, Stefan; Müller, Thomas; Teifke, Jens; Fooks, Anthony R.; Neubert, Andreas

    2011-01-01

    Different approaches have been applied to develop highly attenuated rabies virus vaccines for oral vaccination of mesocarnivores. One prototype vaccine construct is SAD dIND1, which contains a deletion in the P-gene severely limiting the inhibition of type-1 interferon induction. Immunogenicity studies in foxes and skunks were undertaken to investigate whether this highly attenuated vaccine would be more immunogenic than the parental SAD B19 vaccine strain. In foxes, it was demonstrated that SAD dIND1 protected the animals against a rabies infection after a single oral dose, although virus neutralizing antibody titres were lower than in foxes orally vaccinated with the SAD B19 virus as observed in previous experiments. In contrast, skunks receiving 107.5 FFU SAD dIND1 did not develop virus neutralizing antibodies and were not protected against a subsequent rabies infection. PMID:21991446

  8. Importance of rabies virus nucleoprotein in viral evasion of interferon response in the brain.

    PubMed

    Masatani, Tatsunori; Ito, Naoto; Ito, Yuki; Nakagawa, Keisuke; Abe, Masako; Yamaoka, Satoko; Okadera, Kota; Sugiyama, Makoto

    2013-07-01

    By using a cultured neuroblastoma cell line, the present authors recently showed that the N protein of virulent rabies virus fixed strain Nishigahara (Ni), but not that of the attenuated derivative Ni-CE, mediates evasion of induction of type I interferon (IFN). In this study, to determine whether Ni N protein indeed fulfills this function in vivo, the abilities to suppress IFN responses in the mouse brain of Ni-CE and the virulent chimeric virus CE(NiN), which has the N gene from Ni in the genetic background of Ni-CE, were compared. It was demonstrated that CE(NiN) propagates and spreads more efficiently than does Ni-CE in the brain and that IFN response in brains infected with CE(NiN) is weaker than in those infected with Ni-CE. It was also shown that amino acids at positions 273 and 394 in the N protein, which are known as pathogenic determinants, affect the ability of the viruses to suppress IFN response in the brain. These findings strongly suggest that, in the brain, rabies virus N protein plays important roles in evasion of innate immune responses and thereby in efficient propagation and spread of virus leading to lethal outcomes of infection.

  9. Immune response and protection in raccoons (Procyon lotor) following consumption of baits containing ONRAB®, a human adenovirus rabies glycoprotein recombinant vaccine.

    PubMed

    Brown, L J; Rosatte, R C; Fehlner-Gardiner, C; Taylor, J S; Davies, J C; Donovan, D

    2012-10-01

    We investigated the immune response and protection conferred in raccoons (Procyon lotor) following consumption of ONRAB(®) oral rabies vaccine baits. Forty-two wild-caught, captive raccoons were each offered an ONRAB vaccine bait; 21 controls received no vaccine baits. Blood samples collected from all raccoons before treatment, and each week posttreatment for 16 wk, were assessed for the presence of rabies virus antibody. In the bait group, an individual was considered to have responded to vaccination if serum samples from three or more consecutive weeks were antibody-positive. Using this criterion, 77% (20/26) of raccoons that consumed ONRAB baits with no observed vaccine spillage (full dose) demonstrated a humoral immune response. In the group that received a partial dose (0.05-0.90 mL vaccine recovered), 50% (8/16) of raccoons responded to vaccination. Regardless of the vaccine dose received, among the 28 raccoons that responded to vaccination 18 had antibody initially detectable at week 2 and 22 remained antibody-positive for at least 10 consecutive weeks. Kinetics of the humoral immune response suggest that the best time to conduct postbaiting surveillance for evidence of vaccination would be 6-13 wk following bait deployment, with the highest antibody prevalence expected between weeks 8-10. A sub-sample of 29 raccoons (20 ONRAB, 9 controls) was challenged with raccoon rabies virus variant 350 days posttreatment. Eight of nine controls (89%) developed rabies whereas 15/20 vaccinates (75%) survived. Survival following rabies challenge was significantly higher in raccoons presented ONRAB vaccine baits.

  10. Desmodus rotundus and Artibeus spp. bats might present distinct rabies virus lineages.

    PubMed

    Fahl, Willian Oliveira; Carnieli, Pedro; Castilho, Juliana Galera; Carrieri, Maria Luiza; Kotait, Ivanete; Iamamoto, Keila; Oliveira, Rafael Novaes; Brandão, Paulo Eduardo

    2012-01-01

    In Brazil, bats have been assigned an increasing importance in public health as they are important rabies reservoirs. Phylogenetic studies have shown that rabies virus (RABV) strains from frugivorous bats Artibeus spp. are closely associated to those from the vampire bat Desmodus rotundus, but little is known about the molecular diversity of RABV in Artibeus spp. The N and G genes of RABV isolated from Artibeus spp. and cattle infected by D. rotundus were sequenced, and phylogenetic trees were constructed. The N gene nucleotides tree showed three clusters: one for D. rotundus and two for Artibeus spp. Regarding putative N amino acid-trees, two clusters were formed, one for D. rotundus and another for Artibeus spp. RABV G gene phylogeny supported the distinction between D. rotundus and Artibeus spp. strains. These results show the intricate host relationship of RABV's evolutionary history, and are invaluable for the determination of RABV infection sources.

  11. Desmodus rotundus and Artibeus spp. bats might present distinct rabies virus lineages.

    PubMed

    Fahl, Willian Oliveira; Carnieli, Pedro; Castilho, Juliana Galera; Carrieri, Maria Luiza; Kotait, Ivanete; Iamamoto, Keila; Oliveira, Rafael Novaes; Brandão, Paulo Eduardo

    2012-01-01

    In Brazil, bats have been assigned an increasing importance in public health as they are important rabies reservoirs. Phylogenetic studies have shown that rabies virus (RABV) strains from frugivorous bats Artibeus spp. are closely associated to those from the vampire bat Desmodus rotundus, but little is known about the molecular diversity of RABV in Artibeus spp. The N and G genes of RABV isolated from Artibeus spp. and cattle infected by D. rotundus were sequenced, and phylogenetic trees were constructed. The N gene nucleotides tree showed three clusters: one for D. rotundus and two for Artibeus spp. Regarding putative N amino acid-trees, two clusters were formed, one for D. rotundus and another for Artibeus spp. RABV G gene phylogeny supported the distinction between D. rotundus and Artibeus spp. strains. These results show the intricate host relationship of RABV's evolutionary history, and are invaluable for the determination of RABV infection sources. PMID:23146155

  12. Characterization of a virulent dog-originated rabies virus affecting more than twenty fallow deer (Dama dama) in Inner Mongolia, China.

    PubMed

    Zhu, Hongwei; Chen, Xiaoyun; Shao, Xiqun; Ba, Hengxing; Wang, Fengxue; Wang, Hualei; Yang, Yong; Sun, Na; Ren, Jingqiang; Cheng, Shipeng; Wen, Yongjun

    2015-04-01

    Rabies has emerged as a serious problem in the most recent years in northern China. A rabies virus (RABV) isolate, IMDRV-13, was recovered from brain samples of dog-bitten rabid fallow deer (Dama dama) in a farm in Hohhot, Inner Mongolia. We tested the susceptibility of mouse neuroblastoma (MNA) cells and BSR cells as well as that of adult mice to IMDRV-13. The isolate was found to be a virulent isolate with an equivalent pathogenicity index (0.12) and a slight lower neurotropism index (1.07) compared with those of challenge virus standard, CVS-24, which was 0.13 and 1.23, respectively. The complete genome of IMDRV-13 was determined subsequently and found to be 11,924 nucleotides (nt) in length with the same genomic organization as other RABVs. Phylogenetic tree based on complete genome sequences of 43 RABV isolates and strains indicated that IMDRV-13, along with other two isolates in Inner Mongolia, CNM1101C and CNM1104D, clustered within the dog-associated China I clade, which is also the dominant lineage in the current rabies epidemic in China. In addition, sequence analysis of the glycoprotein G identified an amino acid substitution (I338→T338) unique to the IMDRV-13 within antigenic sites III (330-338), this mutation also leads to an additional potential N-glycosylation site (N336), which may represent a useful model to study relationship of N-glycosylation in G protein and specific properties such as pathogenicity or host adaption of RABV. PMID:25614955

  13. Characterization of a virulent dog-originated rabies virus affecting more than twenty fallow deer (Dama dama) in Inner Mongolia, China.

    PubMed

    Zhu, Hongwei; Chen, Xiaoyun; Shao, Xiqun; Ba, Hengxing; Wang, Fengxue; Wang, Hualei; Yang, Yong; Sun, Na; Ren, Jingqiang; Cheng, Shipeng; Wen, Yongjun

    2015-04-01

    Rabies has emerged as a serious problem in the most recent years in northern China. A rabies virus (RABV) isolate, IMDRV-13, was recovered from brain samples of dog-bitten rabid fallow deer (Dama dama) in a farm in Hohhot, Inner Mongolia. We tested the susceptibility of mouse neuroblastoma (MNA) cells and BSR cells as well as that of adult mice to IMDRV-13. The isolate was found to be a virulent isolate with an equivalent pathogenicity index (0.12) and a slight lower neurotropism index (1.07) compared with those of challenge virus standard, CVS-24, which was 0.13 and 1.23, respectively. The complete genome of IMDRV-13 was determined subsequently and found to be 11,924 nucleotides (nt) in length with the same genomic organization as other RABVs. Phylogenetic tree based on complete genome sequences of 43 RABV isolates and strains indicated that IMDRV-13, along with other two isolates in Inner Mongolia, CNM1101C and CNM1104D, clustered within the dog-associated China I clade, which is also the dominant lineage in the current rabies epidemic in China. In addition, sequence analysis of the glycoprotein G identified an amino acid substitution (I338→T338) unique to the IMDRV-13 within antigenic sites III (330-338), this mutation also leads to an additional potential N-glycosylation site (N336), which may represent a useful model to study relationship of N-glycosylation in G protein and specific properties such as pathogenicity or host adaption of RABV.

  14. 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.

  15. 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.

  16. Enhanced growth and plaquing of rabies virus in static chick embryo cell culture.

    PubMed

    Sekine, N; Yoshino, K

    1976-08-01

    The 7-day egg passage line of HEP Flury strain of rabies virus was inoculated to primary chick embyro (CE) cells prepared in different ways to compared efficiencies of viral growth and plaquing. Special care to minimize cellular damage due to trypsin at the step of monodispersion and sowing a comparatively large number of cells for monolayer preparation were required for rabies plaquing, whereas such cares were not necessary for plaquing of vesicular stomatitis virus. Plaque number and size were increased by incorporation of a high concentration of thymidine into cell growth medium. Various other means to produce a static state of CE cells were tested, and a maximal plaquing efficiency was obtained when dishes receiving a massive number of dispersed cells in MEM plus 1% calf serum were incubated at 37 C for 1 day without any buffering for monolayer preparation and postinfection incubation was done at 32 C in a CO2-incubator. Bottle cultures of CE cells prepared in a similar manner, when infected with HEP Flury virus, yielded a markedly higher titer of virus that CE cells prepared by our previous standard method. PMID:185442

  17. Axonal transport of rabies virus in the central nervous system of the rat.

    PubMed

    Gillet, J P; Derer, P; Tsiang, H

    1986-11-01

    Stereotaxic inoculation of rabies virus into specific nuclei in the central nervous system has been used for the investigation of the central neural transport mechanisms of viral information. The infection was monitored by specific fluorescence and peroxidase studies and the titration of viral infectivity in dissected brain areas. Twenty-four hours after inoculation into the striatum, cortex, or substantia nigra, infected neurons were detected only in cells from areas and nuclei which were related to the site of inoculation. The distribution of infected neurons showed that retrograde axoplasmic flow plays a determining role in the transport of rabies virus 24 hours after delivery of virus to specific target nuclei. Local destruction of neurons by kainic acid at the site of viral inoculation did not prevent the uptake and subsequent retrograde axonal transport of virus. There was an overall correlation between the major neural connections of the inoculated areas (e.g. the striatum) and the infected areas 24 hours later (e.g. the substantia nigra).

  18. 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

  19. Experimental rabies virus infection in Artibeus jamaicensis bats with CVS-24 variants.

    PubMed

    Reid, J E; Jackson, A C

    2001-12-01

    An experimental model of rabies was established in the fruit-eating bat species Artibeus jamaicensis. The infections caused by CVS-N2c and CVS-B2c, which are both stable variants of CVS-24, were compared after inoculation of adult bats in the right masseter muscle. CVS-N2c produced neurologic signs of rabies with paresis, ataxia, and inability to fly, while CVS-B2c did not produce neurologic signs. Bats were sacrificed and the distribution of rabies virus antigen was assessed in tissue sections with immunoperoxidase staining. Both viruses spread to the brain stem and bilaterally to the trigeminal ganglia by days 2 to 3. CVS-N2c had disseminated widely in the central nervous system (CNS) by day 4 and had involved the spinal cord, thalamus, cerebellum, and cerebral cortex. CVS-B2c had infected neurons in the spinal cord on day 5 and in the cerebellum, thalamus, and cerebral cortex on day 6. Infected pyramidal neurons of the hippocampus were observed on day 5 in CVS-N2c infection, but infected neurons were never noted in the hippocampus in CVS-B2c infection. CVS-N2c infected many more neurons and more prominently involved neuronal processes than CVS-B2c. CVS-N2c spread more efficiently in the CNS than CVS-B2c. Morphologic changes of apoptosis or biochemical evidence of DNA fragmentation were not observed in neurons with either virus after this route of inoculation. The different neurovirulent properties of these CVS variants in this model were not related to their in vivo ability to induce apoptosis.

  20. Applications of pox virus vectors to vaccination: an update.

    PubMed

    Paoletti, E

    1996-10-15

    Recombinant pox viruses have been generated for vaccination against heterologous pathogens. Amongst these, the following are notable examples. (i) The engineering of the Copenhagen strain of vaccinia virus to express the rabies virus glycoprotein. When applied in baits, this recombinant has been shown to vaccinate the red fox in Europe and raccoons in the United States, stemming the spread of rabies virus infection in the wild. (ii) A fowlpox-based recombinant expressing the Newcastle disease virus fusion and hemagglutinin glycoproteins has been shown to protect commercial broiler chickens for their lifetime when the vaccine was administered at 1 day of age, even in the presence of maternal immunity against either the Newcastle disease virus or the pox vector. (iii) Recombinants of canarypox virus, which is restricted for replication to avian species, have provided protection against rabies virus challenge in cats and dogs, against canine distemper virus, feline leukemia virus, and equine influenza virus disease. In humans, canarypox virus-based recombinants expressing antigens from rabies virus, Japanese encephalitis virus, and HIV have been shown to be safe and immunogenic. (iv) A highly attenuated vaccinia derivative, NYVAC, has been engineered to express antigens from both animal and human pathogens. Safety and immunogenicity of NYVAC-based recombinants expressing the rabies virus glycoprotein, a polyprotein from Japanese encephalitis virus, and seven antigens from Plasmodium falciparum have been demonstrated to be safe and immunogenic in early human vaccine studies. PMID:8876138

  1. [Comparison of 4 technics for serologic titration of antibodies against rabies virus in dogs].

    PubMed

    Blancou, J; Aubert, M F; Cain, E

    1983-10-01

    Four different serological techniques were used for the determination of antibody levels against rabies virus in 55 vaccinated street dogs: mouse neutralization test (reference), rapid fluorescent focus inhibition test, plaque reduction test and immunoenzymatic test (protein A). The results obtained with each of the last three methods were compared with those obtained with the reference test: correlation coefficients were, respectively, 0.810, 0.812 and 0.682, each being significantly correlated with the others. Therefore the three techniques may each be recommended as an alternative to the mouse neutralization test for routine titrations; the immunoenzymatic method, which titrates not only neutralizing antibodies being, nevertheless, better used as a screening test.

  2. [Prokaryotic expression and immunogenicity analysis of glycoprotein from infectious hematopoietic necrosis virus].

    PubMed

    Xu, Li-ming; Liu, Hong-bai; Yin, Jia-sheng; Lu, Tong-yan

    2013-09-01

    In order to detect Infectious hematopoietic necrosis virus with immunological methods, the surface glycoprotein of a recent IHNV-Sn isolated from farmed rainbow trout ( Oncorhynchus mykiss ) in China was amplified and cloned into pET27b(+) vector (designated as pET27b-G ). The expression of recombinant plasmid pET27b-G in E. coli BL21(DE3) was induced and determined by SDS-PAGE analysis. The predicted molecular weight of glycoprotein protein was approximately 55 kD and was confirmed in this study. The inclusion body of glycoprotein was treated with urea at different urea concentrations, and dialyzed into PBS buffer. Purified glycoprotein with high concentration was obtained after dialyzed in the PBS buffer. Antisera against glycoprotein were produced from immunized rabbits. The prepared antisera could react specifically with both the recombinant glycoprotein and natural glycoprotein of the IHNV-Sn isolated in the test of indirect ELISA, and the titer against the recombinant glycoprotein was 1:20,000. IFA showed that the antisera can recognize the glycoprotein located on the surface of IHNV-Sn and IHNV reference strain. These results indicated that the expressed glycoprotein was immunogenical and antigenical and could be functional as the natural IHNV glycoprotein. These results established a foundation for further study on vaccine and rapid diagnosis of IHNV.

  3. Complex Epidemiology of a Zoonotic Disease in a Culturally Diverse Region: Phylogeography of Rabies Virus in the Middle East

    PubMed Central

    Horton, Daniel L.; McElhinney, Lorraine M.; Freuling, Conrad M.; Marston, Denise A.; Banyard, Ashley C.; Goharrriz, Hooman; Wise, Emma; Breed, Andrew C.; Saturday, Greg; Kolodziejek, Jolanta; Zilahi, Erika; Al-Kobaisi, Muhannad F.; Nowotny, Norbert; Mueller, Thomas; Fooks, Anthony R.

    2015-01-01

    The Middle East is a culturally and politically diverse region at the gateway between Europe, Africa and Asia. Spatial dynamics of the fatal zoonotic disease rabies among countries of the Middle East and surrounding regions is poorly understood. An improved understanding of virus distribution is necessary to direct control methods. Previous studies have suggested regular trans-boundary movement, but have been unable to infer direction. Here we address these issues, by investigating the evolution of 183 rabies virus isolates collected from over 20 countries between 1972 and 2014. We have undertaken a discrete phylogeographic analysis on a subset of 139 samples to infer where and when movements of rabies have occurred. We provide evidence for four genetically distinct clades with separate origins currently circulating in the Middle East and surrounding countries. Introductions of these viruses have been followed by regular and multidirectional trans-boundary movements in some parts of the region, but relative isolation in others. There is evidence for minimal regular incursion of rabies from Central and Eastern Asia. These data support current initiatives for regional collaboration that are essential for rabies elimination. PMID:25811659

  4. Human rabies transmitted by vampire bats: antigenic and genetic characterization of rabies virus isolates from the Amazon region (Brazil and Ecuador).

    PubMed

    Castilho, Juliana Galera; Carnieli, Pedro; Durymanova, Ekaterina A; Fahl, Willian de Oliveira; Oliveira, Rafael de Novaes; Macedo, Carla Isabel; da Rosa, Elizabeth Salbe Travassos; Mantilla, Anibal; Carrieri, Maria Luiza; Kotait, Ivanete

    2010-10-01

    Since 2004, the main transmitter of human rabies in Latin America has been the vampire bat (Desmodus rotundus). Based on the nucleoprotein of the rabies virus (RV), we analyzed antigenic and genetic profiles of isolates from 29 samples taken from humans living in different areas of the Amazon region. Two isolates were from Ecuador and 27 from the Northern and Northeastern regions of Brazil, which were obtained during outbreaks in various municipalities in the states of Pará and Maranhão in the years 2004 and 2005. The partial N gene (nt 104-1477) of the 29 isolates was sequenced, and the sequences were used to build a neighbor-joining tree with the Kimura-2 parameter model. All 29 human RV isolates were identified as belonging to antigenic variant 3 (AgV3) and were genetically grouped into the D. rotundus cluster, which was divided into two subclusters (A and B), subcluster A in turn being divided into four genetic groups (A1, A2, A3 and A4). Genetic and molecular markers characterizing these genetic lineages were also identified. The results of this study show that the isolates belong to the same rabies cycle as that of the vampire bat D. rotundus. However, the division of clusters within the lineage associated with D. rotundus shows that different genetic sublineages of the virus were circulating in the Amazon region during the study period. Our findings suggest that there are phylogeographic differences between isolates obtained over a short period.

  5. Retro-transduction by virus pseudotyped with glycoprotein of vesicular stomatitis virus

    SciTech Connect

    Ohishi, Masahisa; Shioda, Tatsuo; Sakuragi, Jun-ichi . E-mail: sakuragi@biken.osaka-u.ac.jp

    2007-05-25

    A virus pseudotyped with glycoprotein of vesicular stomatitis virus (VSV-G) can enter various cell types at a relatively high titer. We observed that the amount of viral antigen from VSV-G pseudotyped human immunodeficiency virus type 1 (HIV-1) producing cells was much higher than that from their non-pseudotyped counterparts. This enhanced viral antigen production was not observed when we used HIV-1 pol mutant, viral enzyme inhibitors, HIV Env protein, or VSV-G fusion defective mutants. The transfection experiment using GFP-expressing virus showed time-dependent expansion of GFP-positive cells and viral DNA integration. These results suggested that the increase in viral antigen yield was caused by the release of a progeny virus following retro-transduction by the pseudotyped virus of the cells within the transfected cell culture. The infectivity as well as the amount of VSV-G on virus particles per unit of viral antigen was significantly different before and after the onset of the yield enhancement. This suggests that results of infection assays of the virus pseudotyped with VSV-G may be affected by the occurrence of such enhancement. This means that, while pseudotyping with VSV-G is a simple and effective method, this procedure should be carefully considered when the virus is produced for infectivity assays.

  6. Adeno-associated viruses serotype 2-mediated RNA interference efficiently inhibits rabies virus replication in vitro and in vivo.

    PubMed

    Wu, Hong-Xia; Wang, Hua-Lei; Guo, Xiao-Feng; Yang, Yu-Jiao; Ma, Jin-Zhu; Wang, Tie-Cheng; Gao, Yu-Wei; Zhao, Yong-Kun; Yang, Song-Tao; Xia, Xian-Zhu

    2013-10-01

    To investigate the potential of adeno-associated viruses serotype 2 (AAV2)-mediated RNA interference (RNAi) as an antiviral agent against rabies, recombinant AAV2 vectors expressing siRNA targeting the nucleoprotein (N) gene of rabies virus (RABV) (rAAV-N796) were constructed and evaluated. When NA cells pretreated with rAAV-N796 were challenged with RABV, there was a 37.8 ± 3.4% to 55.1 ± 5.3% reduction in RABV virus titer. When cells pre-challenged with RABV were treated with rAAV-N796, there was a 4.4 ± 1.4 to 28.8 ± 3.2% reduction in RABV virus titer. Relative quantification of RABV transcripts using real-time PCR and Western blot revealed that the knockdown of RABV-N gene transcripts was based on the rAAV-N796 inoculation titer. When any NA cells were treated with rAAV-N796 before or after challenged with RABV, significant reduction in virus titer was observed in both administrations. Mice treated intracerebrally with rAAV-N796 exhibited 50 ± 5.3 and 62.5 ± 4.7% protection when challenged intracerebrally or intramuscally, respectively, with lethal RABV. When mice treated intramuscularly with rAAV-N796 were challenged intramuscularly with lethal RABV, they exhibited 37.5 ± 3.7% protection. When mice were intracerebrally and intramuscularly with rAAV-N796 24 hr after exposure to RABV infection, they exhibited 25 ± 4.1% protection The N gene mRNA levels in the brains of challenged mice with three different administrations were reduced (55, 68, 32 and 25%, respectively). These results indicated that AAV2 vector-mediated siRNA delivery in vitro in NA cells inhibited RABV multiplication, inhibited RABV multiplication in vivo in the mice brain and imparted partial protection against lethal rabies. So, it may have a potential to be used as an alternative antiviral approach against rabies.

  7. Vaccinating the vampire bat Desmodus rotundus against rabies.

    PubMed

    Almeida, M F; Martorelli, L F A; Aires, C C; Barros, R F; Massad, E

    2008-11-01

    The objective of this study was to extend the previous work of indirect oral rabies immunization of vampire bats (Desmodus rotundus) maintained in captivity, which demonstrated the immunogenicity of the V-RG vaccine (Vaccinia-Rabies Glycoprotein) and indicated that although the results had been encouraging, a new method for concentrating the vaccine should be tested in order to avoid vaccine loss and increase the survival proportion of bats after rabies challenge. In this study, three groups of seven bats each were tested with vaccine concentrated by ultrafiltration through a cellulose membrane. The vaccine was homogenized in Vaseline paste and applied to the back of one vector bat, which was then reintroduced into its group. A dose of 10(5.0) MICLD(50) rabies virus was used by intramuscular route to challenge the bats postvaccination. The survival proportion in the three groups after the challenge was 71.4%, 71.4% and 100%.

  8. Vaccinating the vampire bat Desmodus rotundus against rabies.

    PubMed

    Almeida, M F; Martorelli, L F A; Aires, C C; Barros, R F; Massad, E

    2008-11-01

    The objective of this study was to extend the previous work of indirect oral rabies immunization of vampire bats (Desmodus rotundus) maintained in captivity, which demonstrated the immunogenicity of the V-RG vaccine (Vaccinia-Rabies Glycoprotein) and indicated that although the results had been encouraging, a new method for concentrating the vaccine should be tested in order to avoid vaccine loss and increase the survival proportion of bats after rabies challenge. In this study, three groups of seven bats each were tested with vaccine concentrated by ultrafiltration through a cellulose membrane. The vaccine was homogenized in Vaseline paste and applied to the back of one vector bat, which was then reintroduced into its group. A dose of 10(5.0) MICLD(50) rabies virus was used by intramuscular route to challenge the bats postvaccination. The survival proportion in the three groups after the challenge was 71.4%, 71.4% and 100%. PMID:18761044

  9. 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.

  10. Generation of a recombinant rabies Flury LEP virus carrying an additional G gene creates an improved seed virus for inactivated vaccine production.

    PubMed

    Tao, Lihong; Ge, Jinying; Wang, Xijun; Wen, Zhiyuan; Zhai, Hongyue; Hua, Tao; Zhao, Bolin; Kong, Dongni; Yang, Chinglai; Bu, Zhigao

    2011-01-01

    The rabies Flury Low Egg Passage virus (LEP) has been widely used as a seed virus to generate inactive vaccine. Here, we established a reverse genetic system for LEP and generated a recombinant LEP virus (rLEP-G) that carries two identical G genes. This recombinant virus showed similar properties to those of LEP with respect to in vitro growth, neurotropism index, and virulence in mice. rLEP-G produced 4.3-fold more G protein than did LEP in BHK-21 cells. The inactivated vaccine generated from rLEP-G induced significantly higher virus neutralization titers in mice and dogs than those produced in response to LEP-derived vaccine. Our results suggest that rLEP-G is an improved seed virus candidate for inactivated rabies virus vaccine manufacture.

  11. Development and evaluation of a new immunohistochemistry-based test for the detection of rabies virus neutralizing antibodies

    PubMed Central

    Madhusudana, Shampur N; Malavalli, Bhavana V; Thankappan, Ullas P; Sundramoorthy, Subha; Belludi, Ashwin Y; Pulagumbaly, Srinivasa B; Sanyal, Sampada

    2014-01-01

    Rabies claims about 55 000 human lives and many hundreds of thousands of livestock every year, worldwide. Despite a heavy disease burden, laboratory facilities to diagnose the infection remain scarce in most countries of the developing world where the disease is endemic. Rapid Fluorescent Focus Inhibition Test (RFFIT) and Fluorescent Antibody Virus Neutralization Test (FAVN) are the common tests done in the rabies diagnostic laboratories to detect and quantitate Rabies Virus Neutralizing Antibodies (RVNA). RFFIT is most often employed in confirming seroconversion following prophylactic vaccination, and to aid ante-mortem diagnosis in suspected cases of rabies. Though this remains one of the most sought-after diagnostic services in rabies laboratories, the requirements for expensive anti-rabies fluorochrome antibody conjugate and a fluorescent microscope restrict its performance to only a few reference laboratories. Cost-effective laboratory diagnostic methods employing affordable technology are a need of the hour in the rabies-endemic countries. In this study we have developed a new immunohistochemistry-based neutralization test and extensively evaluated it along with RFFIT. One hundred and 20 human serum samples collected after post-exposure vaccination were subjected to both the tests for determining RVNA titers. The results obtained with the new test correlated significantly with those of RFFIT. Further validation of the inter- and intra- assay precision, lower limit of quantification (LLOQ) and specificity was also performed. The best correlation between the 2 methods, however, was observed only when the RVNA concentrations in the samples were >20 IU/mL. Overall, the immunohistrochemistry-based neutralization test yielded satisfactory results. We suggest that it might serve as a cost-effective alternative to RFFIT in low-resource settings in the developing countries. PMID:24583787

  12. Mason-Pfizer monkey virus: analysis and localization of virion proteins and glycoproteins.

    PubMed Central

    Schochetman, G; Kortright, K; Schlom, J

    1975-01-01

    The polypeptide composition of Mason-Pfizer monkey virus was determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Six major polypeptides of molecular weights 68,000, 27,000, 20,000, 14,000, 12,000, and 10,000 were resolved regardless of the cell type (i.e., two human and two rhesus) in which the virus was grown. Protein gp68 (68,000) represented the major virus glycoprotein and protein gp20 (20,000) represented a minor glycoprotein of the virion, again regardless of the cell type of origin of the virus. Protein gp68 appears to be located on the outer surface of the viral envelope, as demonstrated by lactoperoxidase catalyzed iodination of intact virions. Additional glycoproteins were shown to be virion associated; their presence depended, however, on the cell type in which the virus was propagated. PMID:810603

  13. 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

  14. 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.

  15. 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

  16. Respiratory syncytial virus envelope glycoprotein (G) has a novel structure.

    PubMed Central

    Satake, M; Coligan, J E; Elango, N; Norrby, E; Venkatesan, S

    1985-01-01

    Amino acid sequence of human respiratory syncytial virus envelope glycoprotein (G) was deduced from the DNA sequence of a recombinant plasmid and confirmed by limited amino acid microsequencing of purified 90K G protein. The calculated molecular mass of the protein encoded by the only long open reading frame of 298 amino acids was 32,588 daltons and was somewhat smaller than the 36K polypeptide translated in vitro from mRNA selected by this plasmid. Inspection of the sequence revealed a single hydrophobic domain of 23 amino acids capable of membrane insertion at 41 residues from the N-terminus. There was no N-terminal signal sequence and the hydrophilic N-terminal 20 residues probably represent the cytoplasmic tail of the protein. The N-terminally oriented membrane insertion was somewhat analogous to paramyxovirus hemagglutinin-neuraminidase (HN) and influenza neuraminidase (NA). The protein was moderately hydrophilic and rich in hydroxy-amino acids. It was both N- and O-glycosylated with the latter contributing significantly to the net molecular mass 90K. Images PMID:4069997

  17. Disulfide Bonds in Hepatitis C Virus Glycoprotein E1 Control the Assembly and Entry Functions of E2 Glycoprotein

    PubMed Central

    Wahid, Ahmed; Helle, François; Descamps, Véronique; Duverlie, Gilles; Penin, François

    2013-01-01

    Class II membrane fusion proteins have been described in viruses in which the envelope proteins are derived from a precursor polyprotein containing two transmembrane glycoproteins arranged in tandem. Although the second protein, which carries the membrane fusion function, is in general well characterized, the companion protein, which is a protein chaperone for the folding of the fusion protein, is less well characterized for some viruses, like hepatitis C virus (HCV). To investigate the role of the class II companion glycoprotein E1 of HCV, we chose to target conserved cysteine residues in the protein, and we systematically mutated them in a full-length infectious HCV clone by reverse genetics. All the mutants were infectious, albeit with lower titers than the wild-type virus. The reduced infectivity was in part due to a decrease in viral assembly, as revealed by measurement of intracellular infectivity and by quantification of core protein released from cells transfected with mutant genomes. Analyses of mutated proteins did not show any major defect in folding. However, the mutations reduced virus stability, and they could also affect the density of infectious viral particles. Mutant viruses also showed a defect in cell-to-cell transmission. Finally, our data indicate that HCV glycoprotein E1 can also affect the fusion protein E2 by modulating its recognition by the cellular coreceptor CD81. Therefore, in the context of HCV, our data identify an additional function of a class II companion protein as a molecule that can control the binding capacity of the fusion protein. PMID:23175356

  18. Titration and neutralization of rabies virus (ERA strain) following its replication in a pig fallopian tube cell line.

    PubMed

    Bouillant, A M; Tabel, H; Greig, A S

    1974-04-01

    The ERA strain of rabies virus was found to replicate and produce a constant cytopathic effect in a cell line derived from a gilt's Fallopian tube. Titration and neutralization tests were made by a microtest technique in which one TCID(50) corresponded approximately to one mouse LD(50).

  19. Recombinant rabies virus expressing the H protein of canine distemper virus protects dogs from the lethal distemper challenge.

    PubMed

    Wang, Feng-Xue; Zhang, Shu-Qin; Zhu, Hong-Wei; Yang, Yong; Sun, Na; Tan, Bin; Li, Zhen-Guang; Cheng, Shi-Peng; Fu, Zhen F; Wen, Yong-Jun

    2014-12-01

    The rabies virus (RV) vector LBNSE expressing foreign antigens have shown considerable promise as vaccines against viral and bacteria diseases, which is effective and safe. We produced a new RV-based vaccine vehicle expressing 1.824 kb hemagglutinin (H) gene of the canine distemper virus (CDV) by reverse genetics technology. The recombinant virus LBNSE-CDV-H retained growth properties similar to those of vector LBNSE both in BSR and mNA cell culture. The H gene of CDV was expressed and detected by immunostaining. To compare the immunogenicity of LBNSE-CDV-H, dogs were immunized with each of these recombinant viruses by intramuscular (i.m.). The dogs were bled at third weeks after the immunization for the measurement of virus neutralizing antibody (VNA) and then challenged with virulent virus (ZJ 7) at fourth weeks. The parent virus (LBNSE) without expression of any foreign molecules was included for comparison. Dogs inoculated with LBNSE-CDV-H showed no any signs of disease and exhibited seroconversion against both RV and CDV H protein. The LBNSE-CDV-H did not cause disease in dogs and conferred protection from challenge with a lethal wild type CDV strain, demonstrating its potential value for wildlife conservation efforts. Together, these studies suggest that recombinant RV expressing H protein from CDV stimulated high levels of adaptive immune responses (VNA), and protected all dogs challenge infection. PMID:25465178

  20. Focal Adhesion Kinase Is Involved in Rabies Virus Infection through Its Interaction with Viral Phosphoprotein P

    PubMed Central

    Fouquet, Baptiste; Nikolic, Jovan; Larrous, Florence; Bourhy, Hervé; Wirblich, Christoph

    2014-01-01

    ABSTRACT The rabies virus (RABV) phosphoprotein P is a multifunctional protein: it plays an essential role in viral transcription and replication, and in addition, RABV P has been identified as an interferon antagonist. Here, a yeast two-hybrid screen revealed that RABV P interacts with the focal adhesion kinase (FAK). The binding involved the 106-to-131 domain, corresponding to the dimerization domain of P and the C-terminal domain of FAK containing the proline-rich domains PRR2 and PRR3. The P-FAK interaction was confirmed in infected cells by coimmunoprecipitation and colocalization of FAK with P in Negri bodies. By alanine scanning, we identified a single mutation in the P protein that abolishes this interaction. The mutant virus containing a substitution of Ala for Arg in position 109 in P (P.R109A), which did not interact with FAK, is affected at a posttranscriptional step involving protein synthesis and viral RNA replication. Furthermore, FAK depletion inhibited viral protein expression in infected cells. This provides the first evidence of an interaction of RABV with FAK that positively regulates infection. IMPORTANCE Rabies virus exhibits a small genome that encodes a limited number of viral proteins. To maintain efficient virus replication, some of them are multifunctional, such as the phosphoprotein P. We and others have shown that P establishes complex networks of interactions with host cell components. These interactions have revealed much about the role of P and about host-pathogen interactions in infected cells. Here, we identified another cellular partner of P, the focal adhesion kinase (FAK). Our data shed light on the implication of FAK in RABV infection and provide evidence that P-FAK interaction has a proviral function. PMID:25410852

  1. Rabies virus inactivates cofilin to facilitate viral budding and release.

    PubMed

    Zan, Jie; An, Shu-Ting; Mo, Kai-Kun; Zhou, Jian-Wei; Liu, Juan; Wang, Hai-Long; Yan, Yan; Liao, Min; Zhou, Ji-Yong

    2016-09-01

    Cytoplasmic actin and actin-associated proteins have been identified in RABV particles. Although actin is involved in RABV entry into cells, the specific role of actin in RABV budding and release remains unknown. Our study found that RABV M protein-mediated virion budding depends on intact actin filaments. Confocal microscopy demonstrated a block to virions budding, with a number of M protein-mediated budding vesicles detained in the cell cytoplasm. Furthermore, RABV infection resulted in inactivation of cofilin and upregulation of phosphorylated cofilin. Knockdown of cofilin reduced RABV release. These results for the first time indicate that RABV infection resulted in upregulation of phosphorylated cofilin to facililtate actin polymerization for virus budding. PMID:27396619

  2. Uukuniemi virus glycoproteins accumulate in and cause morphological changes of the Golgi complex in the absence of virus maturation.

    PubMed Central

    Gahmberg, N; Kuismanen, E; Keränen, S; Pettersson, R F

    1986-01-01

    We have studied the transport of the Uukuniemi virus membrane glycoproteins in baby hamster kidney and chick embryo cells by using a temperature-sensitive mutant (ts12). Uukuniemi virus assembles in the Golgi complex, where both glycoproteins G1 and G2 and nucleocapsid protein N accumulate (E. Kuismanen, B. Bång, M. Hurme, and R. F. Pettersson, J. Virol. 51:137-146, 1984). At the restrictive temperature (39 degrees C), the glycoproteins of ts12 were transported to the Golgi complex as in wild-type, virus-infected cells, whereas the nucleocapsid protein failed to accumulate there. Pulse-chase labeling followed by immunoprecipitation and treatment with endo-beta-N-acetylglucosaminidase H showed that G1 synthesized at 39 degrees C in ts12-infected cells had an altered mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting a lack of terminal glycosylation. The typical Uukuniemi virus-induced vacuolization and expansion of the Golgi complex could be seen also in ts12-infected cells at 39 degrees C, although no virus particles were formed. This suggests that the morphological changes were induced by the Uukuniemi virus glycoproteins. In wild-type virus- or ts12-infected cells, G1 and G2 could not be chased out from the Golgi complex even after 6 h of treatment with cycloheximide. The glycoproteins were thus retained in the Golgi even under conditions when no virus maturation took place and when nucleocapsids did not accumulate in the Golgi region. Accordingly, the glycoproteins of Uukuniemi virus were found to have properties resembling those of Golgi-specific proteins. This virus model system may be useful in studying the synthesis and transport of membrane proteins that are transported to and retained in the Golgi. Images PMID:3512854

  3. In vivo differential susceptibility of sensory neurons to rabies virus infection.

    PubMed

    Velandia-Romero, Myriam L; Castellanos, Jaime E; Martínez-Gutiérrez, Marlén

    2013-08-20

    There is controversy with regard to the entry pathway of the rabies virus (RABV) into the central nervous system (CNS). Some authors have suggested that the virus inoculated at the periphery is captured and transported to CNS only by motor neurons; however, it has been reported that dorsal root ganglia (DRG) sensory neurons capture and transport the virus to the spinal cord (SC) and then to the brain. It is probable that preferences for one pathway or another depend on the site of inoculation and the post-infection time. Therefore, in the present study, we evaluated different vertebral segments and post-infection times, along with the location, number, and subpopulation of sensory neurons susceptible to infection after inoculating RABV in the footpads of adult mice. It was noted that the virus inoculated in the footpad preferentially entered the CNS through the large-sized DRG sensory neurons, while infection of the motor neurons occurred later. Further, it was found that the virus was dispersed in spinal cord trans-synaptically through the interneurons, arriving at both sensory neurons and contralateral motor neurons. In conclusion, we observed that RABV inoculated in the plantar footpad is captured preferentially by large sensory neurons and is transported to the DRG, where it replicates and is spread to the SC using transynaptic jumps, infecting sensory and motor neurons at the same level before ascending to the brain.

  4. Increased pathogenicity of rabies virus due to modification of a non-coding region.

    PubMed

    Virojanapirom, Phatthamon; Yamada, Kentaro; Khawplod, Pakamatz; Nishizono, Akira; Hemachudha, Thiravat

    2016-11-01

    Sub-passaging of QS-05, a street rabies virus (RABV) isolate, in non-neuronal cells resulted in a virus with higher pathogenicity, QS-BHK-P7. Four full-length cDNA plasmids were constructed and the corresponding recombinant viruses were recovered: rQS-05, rQS-BHK-P7 and rQS05-2475G/rQS-BHK-P7-2475A (made by switching of intergenic P-M between these two backbones). rQS-BHK-P7-2475 A virus had eight instead of seven adenosines in its poly(A) sequence. Interestingly, mutant viruses with 6 or 8 adenosines infected more neuroblastoma cells than their parental ones. Mice that were infected intracerebrally and intramuscularly with rQS05-2475G and rQS-BHK-P7 exhibited highest mortality. However, mice infected with rQS-BHK-P7-2475AA had the shortest survival time. This study demonstrates that modifications in the non-coding region may play a role in determining the virulence of RABV. PMID:27558122

  5. Identification of a human immunodeficiency virus type 1 envelope glycoprotein variant resistant to cold inactivation.

    PubMed

    Kassa, Aemro; Finzi, Andrés; Pancera, Marie; Courter, Joel R; Smith, Amos B; Sodroski, Joseph

    2009-05-01

    The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein trimer consists of gp120 and gp41 subunits and undergoes a series of conformational changes upon binding to the receptors, CD4 and CCR5/CXCR4, that promote virus entry. Surprisingly, we found that the envelope glycoproteins of some HIV-1 strains are functionally inactivated by prolonged incubation on ice. Serial exposure of HIV-1 to extremes of temperature, followed by expansion of replication-competent viruses, allowed selection of a temperature-resistant virus. The envelope glycoproteins of this virus resisted cold inactivation due to a single passage-associated change, H66N, in the gp120 exterior envelope glycoprotein. Histidine 66 is located within the gp41-interactive inner domain of gp120 and, in other studies, has been shown to decrease the sampling of the CD4-bound conformation by unliganded gp120. Substituting asparagine or other amino acid residues for histidine 66 in cold-sensitive HIV-1 envelope glycoproteins resulted in cold-stable phenotypes. Cold inactivation of the HIV-1 envelope glycoproteins occurred even at high pH, indicating that protonation of histidine 66 is not necessary for this process. Increased exposure of epitopes in the ectodomain of the gp41 transmembrane envelope glycoprotein accompanied cold inactivation, but shedding of gp120 did not. An amino acid change in gp120 (S375W) that promotes the CD4-bound state or treatment with soluble CD4 or a small-molecule CD4 mimic resulted in increased cold sensitivity. These results indicate that the CD4-bound intermediate of the HIV-1 envelope glycoproteins is cold labile; avoiding the CD4-bound state increases temperature stability.

  6. A method for simultaneous detection and identification of Brazilian dog- and vampire bat-related rabies virus by reverse transcription loop-mediated isothermal amplification assay.

    PubMed

    Saitou, Yasumasa; Kobayashi, Yuki; Hirano, Shinji; Mochizuki, Nobuyuki; Itou, Takuya; Ito, Fumio H; Sakai, Takeo

    2010-09-01

    At present, the sporadic occurrence of human rabies in Brazil can be attributed primarily to dog- and vampire bat-related rabies viruses. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) was employed as a simultaneous detection method for both rabies field variants within 60 min. Vampire bat-related rabies viruses could be distinguished from dog variants by digesting amplicons of the RT-LAMP reaction using the restriction enzyme AlwI. Amplification and digestion could both be completed within 120 min after RNA extraction. In addition, the RT-LAMP assay also detected rabies virus in isolates from Brazilian frugivorous bats and Ugandan dog, bovine and goat samples. In contrast, there were false negative results from several Brazilian insectivorous bats and all of Chinese dog, pig, and bovine samples using the RT-LAMP assay. This study showed that the RT-LAMP assay is effective for the rapid detection of rabies virus isolates from the primary reservoir in Brazil. Further improvements are necessary so that the RT-LAMP assay can be employed for the universal detection of genetic variants of rabies virus in the field.

  7. A method for simultaneous detection and identification of Brazilian dog- and vampire bat-related rabies virus by reverse transcription loop-mediated isothermal amplification assay.

    PubMed

    Saitou, Yasumasa; Kobayashi, Yuki; Hirano, Shinji; Mochizuki, Nobuyuki; Itou, Takuya; Ito, Fumio H; Sakai, Takeo

    2010-09-01

    At present, the sporadic occurrence of human rabies in Brazil can be attributed primarily to dog- and vampire bat-related rabies viruses. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) was employed as a simultaneous detection method for both rabies field variants within 60 min. Vampire bat-related rabies viruses could be distinguished from dog variants by digesting amplicons of the RT-LAMP reaction using the restriction enzyme AlwI. Amplification and digestion could both be completed within 120 min after RNA extraction. In addition, the RT-LAMP assay also detected rabies virus in isolates from Brazilian frugivorous bats and Ugandan dog, bovine and goat samples. In contrast, there were false negative results from several Brazilian insectivorous bats and all of Chinese dog, pig, and bovine samples using the RT-LAMP assay. This study showed that the RT-LAMP assay is effective for the rapid detection of rabies virus isolates from the primary reservoir in Brazil. Further improvements are necessary so that the RT-LAMP assay can be employed for the universal detection of genetic variants of rabies virus in the field. PMID:20403387

  8. Spatial and temporal dynamics of rabies virus variants in big brown bat populations across Canada: footprints of an emerging zoonosis.

    PubMed

    Nadin-Davis, Susan A; Feng, Yuqin; Mousse, Delphine; Wandeler, Alexander I; Aris-Brosou, Stéphane

    2010-05-01

    Phylogenetic analysis of a collection of rabies viruses that currently circulate in Canadian big brown bats (Eptesicus fuscus) identified five distinct lineages which have emerged from a common ancestor that existed over 400 years ago. Four of these lineages are regionally restricted in their range while the fifth lineage, comprising two-thirds of all specimens, has emerged in recent times and exhibits a recent demographic expansion with rapid spread across the Canadian range of its host. Four of these viral lineages are shown to circulate in the US. To explore the role of the big brown bat host in dissemination of these viral variants, the population structure of this species was explored using both mitochondrial DNA and nuclear microsatellite markers. These data suggest the existence of three subpopulations distributed in British Columbia, mid-western Canada (Alberta and Saskatchewan) and eastern Canada (Quebec and Ontario), respectively. We suggest that these three bat subpopulations may differ by their level of female phylopatry, which in turn affects the spread of rabies viruses. We discuss how this bat population structure has affected the historical spread of rabies virus variants across the country and the potential impact of these events on public health concerns regarding rabies.

  9. Validation and standardization of virus neutralizing test using indirect immunoperoxidase technique for the quantification of antibodies to rabies virus.

    PubMed

    Ogawa, T; Gamoh, K; Aoki, H; Kobayashi, R; Etoh, M; Senda, M; Hirayama, N; Nishimura, M; Shiraishi, R; Servat, A; Cliquet, F

    2008-08-01

    A virus neutralizing test using an indirect immunoperoxidase technique (VNT-IIP) for rabies has been developed for the titration of dog and cat serum samples in Japan. The VNT-IIP has the advantage that results obtained can be viewed by the naked eye. The purpose of this study was to validate the VNT-IIP and compare it with one of the international standard methods, the fluorescent antibody virus neutralization test (FAVNT). The VNT-IIP showed satisfactory repeatability, high analytical specificity and good accuracy. Regarding the comparison between the VNT-IIP and the FAVNT, the VNT-IIP showed good agreement (91.9%), high sensitivity (92.8%) as well as specificity (87.0%) and good correlation (r = 0.92). As described above, the validation of the VNT-IIP was satisfactory and the performances of the test proved to be equivalent to those of an international standard method.

  10. Immune Clearance of Attenuated Rabies Virus Results in Neuronal Survival with Altered Gene Expression

    PubMed Central

    Gomme, Emily A.; Wirblich, Christoph; Addya, Sankar; Rall, Glenn F.; Schnell, Matthias J.

    2012-01-01

    Rabies virus (RABV) is a highly neurotropic pathogen that typically leads to mortality of infected animals and humans. The precise etiology of rabies neuropathogenesis is unknown, though it is hypothesized to be due either to neuronal death or dysfunction. Analysis of human brains post-mortem reveals surprisingly little tissue damage and neuropathology considering the dramatic clinical symptomology, supporting the neuronal dysfunction model. However, whether or not neurons survive infection and clearance and, provided they do, whether they are functionally restored to their pre-infection phenotype has not been determined in vivo for RABV, or any neurotropic virus. This is due, in part, to the absence of a permanent “mark” on once-infected cells that allow their identification long after viral clearance. Our approach to study the survival and integrity of RABV-infected neurons was to infect Cre reporter mice with recombinant RABV expressing Cre-recombinase (RABV-Cre) to switch neurons constitutively expressing tdTomato (red) to expression of a Cre-inducible EGFP (green), permanently marking neurons that had been infected in vivo. We used fluorescence microscopy and quantitative real-time PCR to measure the survival of neurons after viral clearance; we found that the vast majority of RABV-infected neurons survive both infection and immunological clearance. We were able to isolate these previously infected neurons by flow cytometry and assay their gene expression profiles compared to uninfected cells. We observed transcriptional changes in these “cured” neurons, predictive of decreased neurite growth and dysregulated microtubule dynamics. This suggests that viral clearance, though allowing for survival of neurons, may not restore them to their pre-infection functionality. Our data provide a proof-of-principle foundation to re-evaluate the etiology of human central nervous system diseases of unknown etiology: viruses may trigger permanent neuronal damage that

  11. [Sequencing and analysis of complete genome of rabies viruses isolated from Chinese Ferret-Badger and dog in Zhejiang province].

    PubMed

    Lei, Yong-Liang; Wang, Xiao-Guang; Tao, Xiao-Yan; Li, Hao; Meng, Sheng-Li; Chen, Xiu-Ying; Liu, Fu-Ming; Ye, Bi-Feng; Tang, Qing

    2010-01-01

    Based on sequencing the full-length genomes of four Chinese Ferret-Badger and dog, we analyze the properties of rabies viruses genetic variation in molecular level, get the information about rabies viruses prevalence and variation in Zhejiang, and enrich the genome database of rabies viruses street strains isolated from China. Rabies viruses in suckling mice were isolated, overlapped fragments were amplified by RT-PCR and full-length genomes were assembled to analyze the nucleotide and deduced protein similarities and phylogenetic analyses from Chinese Ferret-Badger, dog, sika deer, vole, used vaccine strain were determined. The four full-length genomes were sequenced completely and had the same genetic structure with the length of 11, 923 nts or 11, 925 nts including 58 nts-Leader, 1353 nts-NP, 894 nts-PP, 609 nts-MP, 1575 nts-GP, 6386 nts-LP, and 2, 5, 5 nts- intergenic regions(IGRs), 423 nts-Pseudogene-like sequence (psi), 70 nts-Trailer. The four full-length genomes were in accordance with the properties of Rhabdoviridae Lyssa virus by BLAST and multi-sequence alignment. The nucleotide and amino acid sequences among Chinese strains had the highest similarity, especially among animals of the same species. Of the four full-length genomes, the similarity in amino acid level was dramatically higher than that in nucleotide level, so the nucleotide mutations happened in these four genomes were most synonymous mutations. Compared with the reference rabies viruses, the lengths of the five protein coding regions had no change, no recombination, only with a few point mutations. It was evident that the five proteins appeared to be stable. The variation sites and types of the four genomes were similar to the reference vaccine or street strains. And the four strains were genotype 1 according to the multi-sequence and phylogenetic analyses, which possessed the distinct district characteristics of China. Therefore, these four rabies viruses are likely to be street viruses

  12. Genetic and phylogenetic characterization of rabies virus isolates from wildlife and livestock in Paraiba, Brazil.

    PubMed

    Shoji, Y; Kobayashi, Y; Sato, G; Gomes, A A B; Itou, T; Ito, F H; Sakai, T

    2006-01-01

    Thirty-four rabies virus (RV) isolates from foxes (8), insectivore bats (9), cattle (14), sheep (1), a goat (1) and a donkey (1) from Paraiba state, northeastern Brazil, were genetically characterized. Sequences of 890 nts of nucleoprotein (N) genes of these isolates were analyzed and compared with those of other Brazilian isolates characterized earlier. Phylogenetic analysis revealed three genetical lineages of RV co-existing in this region. Each lineage was found to be associated with particular host species and to circulate independently of each other. The first lineage was found in foxes (Dusicyon sp.) and could be discriminated from domestic carnivore isolates from Sao Paulo, Goias and Minas Gerais in the southern and central Brazil. The second lineage was associated with insectivorous bats (Molossus spp.) and differed from vampire bat-associated RV isolates. The third lineage was found in livestock and clustered with vampire bat-associated RV isolates from Sao Paulo, Tocantins, Goias and Matto Grosso. These results indicate that RV of these genetic lineages are cocirculating in the Paraiba state and that livestock in this region are infected with vampire bat-associated RV, suggesting that the vampire bat is the main reservoir of livestock rabies in this region. PMID:16599183

  13. 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.

  14. Human rabies transmitted by vampire bats: antigenic and genetic characterization of rabies virus isolates from the Amazon region (Brazil and Ecuador).

    PubMed

    Castilho, Juliana Galera; Carnieli, Pedro; Durymanova, Ekaterina A; Fahl, Willian de Oliveira; Oliveira, Rafael de Novaes; Macedo, Carla Isabel; da Rosa, Elizabeth Salbe Travassos; Mantilla, Anibal; Carrieri, Maria Luiza; Kotait, Ivanete

    2010-10-01

    Since 2004, the main transmitter of human rabies in Latin America has been the vampire bat (Desmodus rotundus). Based on the nucleoprotein of the rabies virus (RV), we analyzed antigenic and genetic profiles of isolates from 29 samples taken from humans living in different areas of the Amazon region. Two isolates were from Ecuador and 27 from the Northern and Northeastern regions of Brazil, which were obtained during outbreaks in various municipalities in the states of Pará and Maranhão in the years 2004 and 2005. The partial N gene (nt 104-1477) of the 29 isolates was sequenced, and the sequences were used to build a neighbor-joining tree with the Kimura-2 parameter model. All 29 human RV isolates were identified as belonging to antigenic variant 3 (AgV3) and were genetically grouped into the D. rotundus cluster, which was divided into two subclusters (A and B), subcluster A in turn being divided into four genetic groups (A1, A2, A3 and A4). Genetic and molecular markers characterizing these genetic lineages were also identified. The results of this study show that the isolates belong to the same rabies cycle as that of the vampire bat D. rotundus. However, the division of clusters within the lineage associated with D. rotundus shows that different genetic sublineages of the virus were circulating in the Amazon region during the study period. Our findings suggest that there are phylogeographic differences between isolates obtained over a short period. PMID:20637811

  15. Recombinant rabies virus expressing IFNα1 enhanced immune responses resulting in its attenuation and stronger immunogenicity.

    PubMed

    Wang, Yifei; Tian, Qin; Xu, Xiaojuan; Yang, Xianfeng; Luo, Jun; Mo, Weiyu; Peng, Jiaojiao; Niu, Xuefeng; Luo, Yongwen; Guo, Xiaofeng

    2014-11-01

    Several studies have shown that type 1 interferons (IFNs) exert multiple biological effects on both innate and adaptive immune responses. Here, we investigated the pathogenicity and immunogenicity of recombinant rabies virus (RABV) expressing canine interferon α1 (rHEP-CaIFNα1). It was shown that Kun Ming (KM) mice that received a single intramuscular immunization with rHEP-CaIFNα1 had an earlier increase and a higher level of virus-neutralizing antibody titers compared with immunization of the parent HEP-Flury. A challenge experiment further confirmed that more mice that were immunized with rHEP-CaIFNα1 survived compared with mice immunized with the parent virus. Quantitative real-time PCR indicated that rHEP-CaIFNα1 induced a stronger innate immune response, especially the type 1 IFN response. Flow cytometry was conducted to show that rHEP-CaIFNα1 recruited more activated B cells in lymph nodes and CD8 T cells in the peripheral blood, which is beneficial to achieve virus clearance in the early infective stage. PMID:25310498

  16. Antigenic and genetic characterization of the first rabies virus isolated from the bat Eumops perotis in Brazil.

    PubMed

    Castilho, Juliana Galera; Canello, Flávia Marchizeli; Scheffer, Karin Corrêa; Achkar, Samira Maria; Carrieri, Maria Luiza; Kotait, Ivanete

    2008-01-01

    Although the main transmitters of rabies in Brazil are dogs and vampire bats, the role of other species such as insectivorous and frugivorous bats deserves special attention, as the rabies virus has been isolated from 36 bat species. This study describes the first isolation of the rabies virus from the insectivorous bat Eumops perotis. The infected animal was found in the city of Ribeirão Preto, São Paulo. The virus was identified by immunofluorescence antibody test (FAT) in central nervous system (CNS) samples, and the isolation was carried out in N2A cell culture and adult mice. The sample was submitted to antigenic typing using a panel of monoclonal antibodies (CDC/Atlanta/USA). The DNA sequence of the nucleoprotein gene located between nucleotides 102 and 1385 was aligned with homologous sequences from GenBank using the CLUSTAL/W method, and the alignment was used to build a neighbor-joining distance-based phylogenetic tree with the K-2-P model. CNS was negative by FAT, and only one mouse died after inoculation with a suspension from the bat's CNS. Antigenic typing gave a result that was not compatible with the patterns defined by the panel. Phylogenetic analysis showed that the virus isolated segregated into the same cluster related to other viruses isolated from insectivorous bats belonging to genus Nyctinomops ssp. (98.8% nucleotide identity with each other). PMID:18488088

  17. Cloning, sequence, and expression of the glycoprotein gene of infectious hematopoietic necrosis virus, a fish rhabdovirus

    SciTech Connect

    Feyereisen-Koener, J.M.

    1987-01-01

    Double-stranded cDNA was prepared from infectious hematopoietic necrosis virus mRNA and cloned into the plasmid vector pUC8. A coprotein (G-protein) of infectious hematopoietic necrosis virus was selected by hybridization to a /sup 32/P-labeled probe. The restriction map and nucleotide sequence of the mRNA encoding the glycoprotein of infectious hematopoietic necrosis virus was determined using this full-length cDNA clone.

  18. 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.

  19. Development of Primer Pairs from Molecular Typing of Rabies Virus Variants Present in Mexico.

    PubMed

    Bastida-González, Fernando; Ramírez-Hernández, Dolores G; Chavira-Suárez, Erika; Lara-Padilla, Eleazar; Zárate-Segura, Paola

    2016-01-01

    Nucleoprotein (N) gene from rabies virus (RABV) is a useful sequence target for variant studies. Several specific RABV variants have been characterized in different mammalian hosts such as skunk, dog, and bats by using anti-nucleocapsid monoclonal antibodies (MAbs) via indirect fluorescent antibody (IFA) test, a technique not available in many laboratories in Mexico. In the present study, a total of 158 sequences of N gene from RABV were used to design eight pairs of primers (four external and four internal primers), for typing four different RABV variants (dog, skunk, vampire bat, and nonhematophagous bat) which are most common in Mexico. The results indicate that the primer and the typing variant from the brain samples, submitted to nested and/or real-time PCR, are in agreement in all four singleplex reactions, and the designed primer pairs are an alternative for use in specific variant RABV typing.

  20. Development of Primer Pairs from Molecular Typing of Rabies Virus Variants Present in Mexico

    PubMed Central

    Ramírez-Hernández, Dolores G.; Lara-Padilla, Eleazar; Zárate-Segura, Paola

    2016-01-01

    Nucleoprotein (N) gene from rabies virus (RABV) is a useful sequence target for variant studies. Several specific RABV variants have been characterized in different mammalian hosts such as skunk, dog, and bats by using anti-nucleocapsid monoclonal antibodies (MAbs) via indirect fluorescent antibody (IFA) test, a technique not available in many laboratories in Mexico. In the present study, a total of 158 sequences of N gene from RABV were used to design eight pairs of primers (four external and four internal primers), for typing four different RABV variants (dog, skunk, vampire bat, and nonhematophagous bat) which are most common in Mexico. The results indicate that the primer and the typing variant from the brain samples, submitted to nested and/or real-time PCR, are in agreement in all four singleplex reactions, and the designed primer pairs are an alternative for use in specific variant RABV typing. PMID:27563666

  1. Genetic analysis of phosphoprotein and matrix protein of rabies viruses isolated in Brazil.

    PubMed

    Kobayashi, Yuki; Okuda, Hiromi; Nakamura, Kana; Sato, Go; Itou, Takuya; Carvalho, Adolorata A B; Silva, Marlon V; Mota, Carla S; Ito, Fumio H; Sakai, Takeo

    2007-11-01

    To investigate the genetic characteristics of phosphoprotein (P) and matrix protein (M) genes of variable rabies virus (RV) prevalent in Brazil, the authors genetically characterized the P and M genes from 30 Brazilian RV field isolates. Phylogenetic analysis based on the P and M genes revealed the presence of six RV variants that consisted primarily of three insectivorous bats, the vampire bat, dog and fox in Brazil. Specific amino acid substitutions corresponding to these phylogenetic lineages were observed, with Asp(42) and Glu(62) in the P protein found to be characteristic of Brazilian chiroptera- and carnivora-related RVs, respectively. Amino acid sequence motifs predicted to associate with a viral function in the P and M proteins were conserved among Brazilian RV variants. PMID:18057829

  2. Development of Primer Pairs from Molecular Typing of Rabies Virus Variants Present in Mexico.

    PubMed

    Bastida-González, Fernando; Ramírez-Hernández, Dolores G; Chavira-Suárez, Erika; Lara-Padilla, Eleazar; Zárate-Segura, Paola

    2016-01-01

    Nucleoprotein (N) gene from rabies virus (RABV) is a useful sequence target for variant studies. Several specific RABV variants have been characterized in different mammalian hosts such as skunk, dog, and bats by using anti-nucleocapsid monoclonal antibodies (MAbs) via indirect fluorescent antibody (IFA) test, a technique not available in many laboratories in Mexico. In the present study, a total of 158 sequences of N gene from RABV were used to design eight pairs of primers (four external and four internal primers), for typing four different RABV variants (dog, skunk, vampire bat, and nonhematophagous bat) which are most common in Mexico. The results indicate that the primer and the typing variant from the brain samples, submitted to nested and/or real-time PCR, are in agreement in all four singleplex reactions, and the designed primer pairs are an alternative for use in specific variant RABV typing. PMID:27563666

  3. Evolutionary History and Phylogeography of Rabies Viruses Associated with Outbreaks in Trinidad

    PubMed Central

    Seetahal, Janine F. R.; Velasco-Villa, Andres; Allicock, Orchid M.; Adesiyun, Abiodun A.; Bissessar, Joseph; Amour, Kirk; Phillip-Hosein, Annmarie; Marston, Denise A.; McElhinney, Lorraine M.; Shi, Mang; Wharwood, Cheryl-Ann; Fooks, Anthony R.; Carrington, Christine V. F.

    2013-01-01

    Bat rabies is an emerging disease of public health significance in the Americas. The Caribbean island of Trinidad experiences periodic outbreaks within the livestock population. We performed molecular characterisation of Trinidad rabies virus (RABV) and used a Bayesian phylogeographic approach to investigate the extent to which outbreaks are a result of in situ evolution versus importation of virus from the nearby South American mainland. Trinidadian RABV sequences were confirmed as bat variant and clustered with Desmodus rotundus (vampire bat) related sequences. They fell into two largely temporally defined lineages designated Trinidad I and II. The Trinidad I lineage which included sequences from 1997–2000 (all but two of which were from the northeast of the island) was most closely related to RABV from Ecuador (2005, 2007), French Guiana (1990) and Venezuela (1993, 1994). Trinidad II comprised sequences from the southwest of the island, which clustered into two groups: Trinidad IIa, which included one sequence each from 2000 and 2007, and Trinidad IIb including all 2010 sequences. The Trinidad II sequences were most closely related to sequences from Brazil (1999, 2004) and Uruguay (2007, 2008). Phylogeographic analyses support three separate RABV introductions from the mainland from which each of the three Trinidadian lineages arose. The estimated dates for the introductions and subsequent lineage expansions suggest periods of in situ evolution within Trinidad following each introduction. These data also indicate co-circulation of Trinidad lineage I and IIa during 2000. In light of these findings and the likely vampire bat origin of Trinidadian RABV, further studies should be conducted to investigate the relationship between RABV spatiotemporal dynamics and vampire bat population ecology, in particular any movement between the mainland and Trinidad. PMID:23991230

  4. Evolutionary history and phylogeography of rabies viruses associated with outbreaks in Trinidad.

    PubMed

    Seetahal, Janine F R; Velasco-Villa, Andres; Allicock, Orchid M; Adesiyun, Abiodun A; Bissessar, Joseph; Amour, Kirk; Phillip-Hosein, Annmarie; Marston, Denise A; McElhinney, Lorraine M; Shi, Mang; Wharwood, Cheryl-Ann; Fooks, Anthony R; Carrington, Christine V F

    2013-01-01

    Bat rabies is an emerging disease of public health significance in the Americas. The Caribbean island of Trinidad experiences periodic outbreaks within the livestock population. We performed molecular characterisation of Trinidad rabies virus (RABV) and used a Bayesian phylogeographic approach to investigate the extent to which outbreaks are a result of in situ evolution versus importation of virus from the nearby South American mainland. Trinidadian RABV sequences were confirmed as bat variant and clustered with Desmodus rotundus (vampire bat) related sequences. They fell into two largely temporally defined lineages designated Trinidad I and II. The Trinidad I lineage which included sequences from 1997-2000 (all but two of which were from the northeast of the island) was most closely related to RABV from Ecuador (2005, 2007), French Guiana (1990) and Venezuela (1993, 1994). Trinidad II comprised sequences from the southwest of the island, which clustered into two groups: Trinidad IIa, which included one sequence each from 2000 and 2007, and Trinidad IIb including all 2010 sequences. The Trinidad II sequences were most closely related to sequences from Brazil (1999, 2004) and Uruguay (2007, 2008). Phylogeographic analyses support three separate RABV introductions from the mainland from which each of the three Trinidadian lineages arose. The estimated dates for the introductions and subsequent lineage expansions suggest periods of in situ evolution within Trinidad following each introduction. These data also indicate co-circulation of Trinidad lineage I and IIa during 2000. In light of these findings and the likely vampire bat origin of Trinidadian RABV, further studies should be conducted to investigate the relationship between RABV spatiotemporal dynamics and vampire bat population ecology, in particular any movement between the mainland and Trinidad.

  5. Detection of rabies virus RNA isolated from several species of animals in Brazil by RT-PCR.

    PubMed

    Ito, M; Itou, T; Sakai, T; Santos, M F; Arai, Y T; Takasaki, T; Kurane, I; Ito, F H

    2001-12-01

    Brain samples from different animal species including humans: five vampire bats, 14 cattle, 12 dogs, 11 cats, two horses, one pig, one sheep and three humans collected from various geographical regions of Brazil were found to be positive for rabies by means of the fluorescent antibody test (FAT) and the mouse inoculation test (MIT). The brain samples were retested for rabies by means of the reverse transcription and polymerase chain reaction (RT-PCR) with 2 primer sets (P1/P2 and RHNI/RHNS3), which amplified full or partial regions on the nucleoprotein (N) gene of the rabies virus, respectively. Brain samples from five vampire bats, 13 cattle, one horse and one sheep failed to yield PCR products when the RHN1/RHNS3 primer pair was used, but all brain samples successfully yielded the products when the P1/P2 primer pair was used. These results suggest that Brazilian rabies virus isolates could be principally divided into two populations according to genetic difference. PMID:11789609

  6. 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

  7. 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

  8. [Effect of rabies virus infection on the expression of parvalbumin, calbindin and calretinin in mouse cerebral cortex].

    PubMed

    Torres-Fernández, Orlando; Yepes, Gloria E; Gómez, Javier E; Pimienta, Hernán J

    2004-03-01

    Some clinical features of rabies and experimental evidence from cell culture and laboratory animals suggest impairment of gabaergic neurotransmission. Several types of gabaergic neurons occur in the cerebral cortex. They can be identified by three neuronal markers: the calcium binding proteins (CaBPs) parvalbumin (PV), calbindin (CB) and calretinin (CR). Rabies virus spreads throughout the cerebral cortex; however, rabies cytopathic effects on gabaergic neurons are unknown. The expression of calcium-binding proteins (CaBPs) parvalbumin (PV), calbindin (CB) and calretinin (CR) was studied in the frontal cortex of mice. The effect of gabaergic neurons was evaluated immunohistochemically. The distribution patterns of CaBPs in normal mice and in mice infected with 'fixed' or 'street' rabies virus were compared. PV was found in multipolar neurons located in all cortical layers except layer I, and in pericellular clusters of terminal knobs surrounding the soma of pyramidal neurons. CB-immunoreactivity was distributed in two cortical bands. One was composed of round neurons enclosed by a heavily labeled neuropil; this band corresponds to supragranular layers II and III. The other was a weakly stained band of neuropil which contained scattered multipolar CB-ir neurons; this corresponds to infragranular layers V and VI. The CR-ir neurons were bipolar fusiform cells located in all layers of cortex, but concentrated in layers II and III. A feature common to samples infected with both types of viruses was a more intense immunoreactivity to PV in contrast to normal samples. The infection with 'street' virus did not cause additional changes in the expression of CaBPs. However, the infection with 'fixed' virus produced a remarkable reduction of CB-immunoreactivity demonstrated by the loss of CB-ir neurons and low neuropil stain in the frontal cortex. In addition, the size of CR-ir neurons in the cingulate cortex was decreased.

  9. Identification and genetic characterization of rabies virus from Egyptian water buffaloes (Bubalus bubalis) bitten by a fox.

    PubMed

    El-Tholoth, Mohamed; El-Beskawy, Mohamed; Hamed, Mohamed F

    2015-09-01

    Rabies is caused by negative strand RNA-virus classified in the genus Lyssavirus, family Rhabdoviridae of the order Mononegavirales. The aim of the present study was to identify and analyze nucleotides sequence of nucleoprotein (N) gene of rabies virus (RABV) from two cases of water buffaloes (Bubalus bubalis) bitten by a fox in Egypt, 2013. The diseased buffaloes showed nervous manifestations with fever. Specimens from brains of the buffaloes with suspected rabies were collected. RABV in collected samples was identified using direct fluorescent antibody (dFA) technique, histopathological examination and reverse transcription-polymerase chain reaction (RT-PCR). Also, nucleotides sequence of partially amplified nucleoprotein (N) gene was compared with the other street strains of RABV available on GenBank. The results revealed that RABV antigen was identified in the brains of diseased buffaloes by dFA technique and the characteristic intracytoplasmic inclusions (Negri bodies) and RABV nucleic acid were detected by histopathology and RT-PCR, respectively. The identified virus showed close genetic relationship with street strains identified previously from dogs in different Governorates in Egypt and with strains identified in Israel and Jordan indicating transmission of the virus between Egyptian Governorates with a potential transmission from and/or to our neighboring countries. PMID:26396980

  10. Identification and genetic characterization of rabies virus from Egyptian water buffaloes (Bubalus bubalis) bitten by a fox.

    PubMed

    El-Tholoth, Mohamed; El-Beskawy, Mohamed; Hamed, Mohamed F

    2015-09-01

    Rabies is caused by negative strand RNA-virus classified in the genus Lyssavirus, family Rhabdoviridae of the order Mononegavirales. The aim of the present study was to identify and analyze nucleotides sequence of nucleoprotein (N) gene of rabies virus (RABV) from two cases of water buffaloes (Bubalus bubalis) bitten by a fox in Egypt, 2013. The diseased buffaloes showed nervous manifestations with fever. Specimens from brains of the buffaloes with suspected rabies were collected. RABV in collected samples was identified using direct fluorescent antibody (dFA) technique, histopathological examination and reverse transcription-polymerase chain reaction (RT-PCR). Also, nucleotides sequence of partially amplified nucleoprotein (N) gene was compared with the other street strains of RABV available on GenBank. The results revealed that RABV antigen was identified in the brains of diseased buffaloes by dFA technique and the characteristic intracytoplasmic inclusions (Negri bodies) and RABV nucleic acid were detected by histopathology and RT-PCR, respectively. The identified virus showed close genetic relationship with street strains identified previously from dogs in different Governorates in Egypt and with strains identified in Israel and Jordan indicating transmission of the virus between Egyptian Governorates with a potential transmission from and/or to our neighboring countries.

  11. Immunostained plaque assay for detection and titration of rabies virus infectivity.

    PubMed

    Park, Jun-Sun; Um, Jihye; Choi, Young-Ki; Lee, Yeong Seon; Ju, Young Ran; Kim, Su Yeon

    2016-02-01

    The fluorescent antibody test (FAT) is the most commonly used method for detection of the rabies virus (RV). The plaque assay can only be applied to fixed RVs, and cannot be used for street RVs. In this study, plaque formation allowing the determination of both fixed and street RVs was achieved using the immune plaque assay. The immune plaque assay carried out using both fixed and street RVs showed the formation of clear and countable plaques after immunostaining with anti-RV P monoclonal antibody and HRP-conjugated anti-mouse IgG. Plaque size increased with incubation time, and the plaque morphology differed according to viral strain. Fixed RVs had the dot-shaped regular plaque morphology and street RVs had the small irregular-shape plaque morphology. In addition, no significant differences were observed between the growth kinetics of the KGH strain when the virus was titrated using the FAT and the immune plaque assay. It allowed the successful detection and quantification of both street and fixed RVs through the production of clear, countable plaques, making it easy to obtain objective results. The assay is an applicable tool for the detection of RVs in various investigations, including virus neutralizing antibody testing, cell-to-cell spread, and viral drug sensitivity testing.

  12. 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

  13. Animal-associated exposure to rabies virus among travelers, 1997-2012.

    PubMed

    Gautret, Philippe; Harvey, Kira; Pandey, Prativa; Lim, Poh Lian; Leder, Karin; Piyaphanee, Watcharapong; Shaw, Marc; McDonald, Susan C; Schwartz, Eli; Esposito, Douglas H; Parola, Philippe

    2015-04-01

    Among travelers, rabies cases are rare, but animal bites are relatively common. To determine which travelers are at highest risk for rabies, we studied 2,697 travelers receiving care for animal-related exposures and requiring rabies postexposure prophylaxis at GeoSentinel clinics during 1997-2012. No specific demographic characteristics differentiated these travelers from other travelers seeking medical care, making it challenging to identify travelers who might benefit from reinforced pretravel rabies prevention counseling. Median travel duration was short for these travelers: 15 days for those seeking care after completion of travel and 20 days for those seeking care during travel. This finding contradicts the view that preexposure rabies vaccine recommendations should be partly based on longer travel durations. Over half of exposures occurred in Thailand, Indonesia, Nepal, China, and India. International travelers to rabies-endemic regions, particularly Asia, should be informed about potential rabies exposure and benefits of pretravel vaccination, regardless of demographics or length of stay.

  14. Protection against Marek's disease by a fowlpox virus recombinant expressing the glycoprotein B of Marek's disease virus.

    PubMed

    Nazerian, K; Lee, L F; Yanagida, N; Ogawa, R

    1992-03-01

    Fowlpox virus (FPV) recombinants expressing the glycoprotein B and the phosphorylated protein (pp38) of the GA strain of Marek's disease virus (MDV) were assayed for their ability to protect chickens against challenge with virulent MDV. The recombinant FPV expressing the glycoprotein B gene elicited neutralizing antibodies against MDV, significantly reduced the level of cell-associated viremia, and, similar to the conventional herpesvirus of turkeys, protected chickens against challenge with the GA strain and the highly virulent RB1B and Md5 strains of MDV. The recombinant FPV expressing the pp38 gene failed to either elicit neutralizing antibodies against MDV or protect the vaccinated chickens against challenge with MDV.

  15. Involvement of extraneural tissues and upregulation of inducible nitric oxide synthase after experimental infection with rabies virus in BALB/c mice and LEW/SsN rats.

    PubMed

    Liao, Pi-Hung; Hsu, Yung-Hsiang; Yang, Hui-Hua; Wang, Ming-Hseng; Chen, Li-Kuang

    2012-09-01

    Rabies virus can cause fatal encephalomyelitis, but the involvement of extraneural organs has not been well characterized. In this study, we investigated the histopathological changes and the distribution of viral antigens in extraneural organs after pathogenic rabies virus infection in mouse and rat models. In histopathological examination, classical viral encephalitis and rabies-specific Negri body were observed in the brain. In addition to the central nervous system (CNS), inflammatory responses were found in other organs, such as the heart, kidney, liver, and lung. Similarly, immunohistochemical staining and reverse transcription-polymerase chain reaction revealed the presence of rabies virus in the CNS and extraneural tissues. Moreover, macrophages, especially in the lung and heart, were involved in the infection. Transcriptional analyses of the expression of inducible nitric oxide synthase (iNOS) demonstrated that rabies virus potentiated the gene expression of iNOS in the brain, lung, and heart. The immunoreactive iNOS-positive macrophages were detected adjacent to the infection. These results suggest that macrophages are involved in the extraneural infection and the expression of iNOS in macrophages contributes to the formation of tissue inflammation. Our study indicates the involvement of extraneural organs following rabies virus infection, which may aggravate the progression of this deadly disease.

  16. Factors affecting recombinant Western equine encephalitis virus glycoprotein production in the baculovirus system.

    PubMed

    Toth, Ann M; Geisler, Christoph; Aumiller, Jared J; Jarvis, Donald L

    2011-12-01

    In an effort to produce processed, soluble Western equine encephalitis virus (WEEV) glycoproteins for subunit therapeutic vaccine studies, we isolated twelve recombinant baculoviruses designed to express four different WEEV glycoprotein constructs under the transcriptional control of three temporally distinct baculovirus promoters. The WEEV glycoprotein constructs encoded full-length E1, the E1 ectodomain, an E26KE1 polyprotein precursor, and an artificial, secretable E2E1 chimera. The three different promoters induced gene expression during the immediate early (ie1), late (p6.9), and very late (polh) phases of baculovirus infection. Protein expression studies showed that the nature of the WEEV construct and the timing of expression both influenced the quantity and quality of recombinant glycoprotein produced. The full-length E1 product was insoluble, irrespective of the timing of expression. Each of the other three constructs yielded soluble products and, in these cases, the timing of expression was important, as higher protein processing efficiencies were generally obtained at earlier times of infection. However, immediate early expression did not yield detectable levels of every WEEV product, and expression during the late (p6.9) or very late (polh) phases of infection provided equal or higher amounts of processed, soluble product. Thus, while earlier foreign gene expression can provide higher recombinant glycoprotein processing efficiencies in the baculovirus system, in the case of the WEEV glycoproteins, earlier expression did not provide larger amounts of high quality, soluble recombinant glycoprotein product.

  17. Epitope mapping of the infectious hematopoietic necrosis virus glycoprotein by flow cytometry.

    PubMed

    Xu, Li-Ming; Liu, Miao; Zhao, Jing-Zhuang; Cao, Yong-Sheng; Yin, Jia-Sheng; Liu, Hong-Bai; Lu, Tongyan

    2014-10-01

    The glycoprotein of infectious hematopoietic necrosis virus was truncated to ten overlapping fragments. All fragments were displayed on the inner membrane of the Escherichia coli periplasm. After disruption of the outer membrane, spheroplasts that had anchored with the glycoprotein fragment were incubated with an anti-glycoprotein polyclonal antibody. Prey pairs were detected and quantitated by flow cytometry with all fragments but one, G2, reacting with the polyclonal antibody. The antigenicity of all ten fragments was analyzed using conventional methods, and epitopes were localized in all fragments, except for G2 and were consistent with FCM analysis. Antigenicity of purified glycoprotein fusion proteins was confirmed by western blotting and ELISA. This method provides a rapid, quantitative and simple strategy for identifying linear B cell epitopes of a given protein.

  18. 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

  19. 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

  20. 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.

  1. MHC class II DRB diversity in raccoons (Procyon lotor) reveals associations with raccoon rabies virus (Lyssavirus).

    PubMed

    Srithayakumar, Vythegi; Castillo, Sarrah; Rosatte, Rick C; Kyle, Christopher J

    2011-02-01

    In North America, the raccoon rabies virus (RRV) is an endemic wildlife disease which causes acute encephalopathies and is a strong selective force on raccoons (Procyon lotor), with estimates of ∼85% of the population succumbing to the disease when epizootic. RRV is regarded as a lethal disease if untreated; therefore, no evolutionary response would be expected of raccoon populations. However, variable immune responses to RRV have been observed in raccoons indicating a potential for evolutionary adaptation. Studies of variation within the immunologically important major histocompatibility complex (MHC) have revealed relationships between MHC alleles and diseases in humans and other wildlife species. This enhances our understanding of how hosts and pathogens adapt and co-evolve. In this study, we used RRV as a model system to study host-pathogen interaction in raccoons from a challenge study and from four wild populations that differ in exposure times and viral lineages. We investigated the potential role of Prlo-DRB polymorphism in relation to susceptibility/resistance to RRV in 113 RRV positive and 143 RRV negative raccoons. Six alleles were found to be associated with RRV negative status and five alleles with RRV positive animals. We found variable patterns of MHC associations given the relative number of selective RRV sweeps in the studied regions and correlations between MHC diversity and RRV lineages. The allelic associations established provide insight into how the genetic variation of raccoons may affect the disease outcome and this can be used to examine similar associations between other rabies variants and their hosts.

  2. [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

  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.

  4. Experimental oral immunization of ferret badgers (Melogale moschata) with a recombinant canine adenovirus vaccine CAV-2-E3Δ-RGP and an attenuated rabies virus SRV9.

    PubMed

    Zhao, Jinghui; Liu, Ye; Zhang, Shoufeng; Fang, Lijun; Zhang, Fei; Hu, Rongliang

    2014-04-01

    Ferret badgers (Melogale moschata) are a major reservoir of rabies virus in southeastern China. Oral immunization has been shown to be a practical method for wildlife rabies management in Europe and North America. Two groups of 20 ferret badgers were given a single oral dose of a recombinant canine adenovirus-rabies vaccine, CAV-2-E3Δ-RGP, or an experimental attenuated rabies virus vaccine, SRV9. At 21 days, all ferret badgers had seroconverted, with serum virus-neutralizing antibodies ranging from 0.1 to 4.5 IU/mL. Titers were >0.50 IU/mL (an acceptable level) in 17/20 and 16/20 animals receiving CAV-2-E3Δ-RGP or SRV9, respectively. The serologic results indicate that the recombinant CAV-2-E3Δ-RGP is at least as effective as the attenuated rabies virus vaccine. Both may be considered for additional research as oral rabies vaccine candidates for ferret badgers.

  5. 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

  6. Role of chemokines in the enhancement of BBB permeability and inflammatory infiltration after rabies virus infection.

    PubMed

    Kuang, Yi; Lackay, Sarah N; Zhao, Ling; Fu, Zhen F

    2009-09-01

    Induction of innate immunity, particularly through the induction of interferon and chemokines, by rabies virus (RABV) infection has been reported to be inversely correlated with pathogenicity. To further investigate the association between the expression of chemokines and RABV infection, laboratory-attenuated RABV (B2C) and wild-type (wt) RABV (DRV) were administered to Balb/c mice intramuscularly. Chemokine expression, inflammatory cell infiltration, and blood-brain barrier (BBB) permeability were evaluated at various time points after infection. At day 3 post-infection (p.i.) there was very little inflammation in the central nervous system (CNS) and BBB permeability did not change in mice infected with either virus when compared with mock-infected mice. At 6 day p.i., infection with B2C induced the expression of inflammatory chemokines and infiltration of inflammatory cells into the CNS, while these changes were minimal in DRV-infected mice. Furthermore, infection with B2C significantly enhanced BBB permeability comparing to infection with DRV. Among the upregulated chemokines, the expression of IP-10 was best correlated with infiltration of inflammatory cells into the CNS and enhancement of BBB permeability. These data indicate that laboratory-attenuated RABV induces expression of chemokines and infiltration of inflammatory cells into the CNS. Upregulation of chemokines by B2C may have triggered the change in BBB permeability, which helps infiltration of inflammatory cells into the CNS, and thus attenuation of RABV.

  7. 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.

  8. Increased interleukin-10 associated with low IL-6 concentration correlated with greater survival rates in mice infected by rabies virus vaccinated against it and immunomodulated with P. acnes.

    PubMed

    Megid, J; Kaneno, R; Nozaki, C N; Brito, C J C; Almeida, M F

    2004-11-01

    Macrophage activity, cytokines serum concentration, serum neutralizing antibodies and lethality by rabies were evaluated in swiss mice experimentally infected with street rabies virus and submitted or not to antirabies vaccination and immunomodulation with P. acnes. Animals were killed at different times and serum was collected in order to evaluate cytokines concentration; peritonial and splenic macrophages were collected for macrophage activity evaluation. Greater survival rates higher IL-10 and low IL-6 serum concentration were observed in vaccinated animals treated using P. acnes.

  9. Immunological responses to envelope glycoprotein 120 from subtypes of human immunodeficiency virus type 1.

    PubMed

    Gilljam, G; Svensson, A; Ekström, A; Wahren, B

    1999-07-01

    The outer envelope glycoprotein (gp120) from subtypes A-E of HIV-1 was purified using a specific high mannose-binding lectin, Galanthus nivalis agglutinin. All isolates were grown in peripheral blood lymphocyte cells in order to avoid selection in cell lines. A comparison of the reactivities of the envelope proteins was made using sera from patients infected with the different subtypes. In this study, the B and C subtype envelope glycoproteins showed the strongest immunological reactivity, when reacted with sera from patients infected with the same subtype of virus. On the other hand, sera of patients infected with subtype A or C virus had the strongest and broadest reactivities, to envelope glycoproteins of many subtypes. The purified gp120 proteins from all five subtypes stimulated mononuclear cells from HIV-1 (subtype B)-infected patients, indicating conserved T cell-activating epitopes. The immunological reactivities indicate that strong antigenicity does not always predict the broadest immunogenicity of an envelope glycoprotein. Glycoprotein 120 from foreign subtypes may serve to induce strong cross-reactive immune responses.

  10. 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.

  11. Black-backed jackal exposure to rabies virus, canine distemper virus, and Bacillus anthracis in Etosha National Park, Namibia.

    PubMed

    Bellan, Steve E; Cizauskas, Carrie A; Miyen, Jacobeth; Ebersohn, Karen; Küsters, Martina; Prager, K C; Van Vuuren, Moritz; Sabeta, Claude; Getz, Wayne M

    2012-04-01

    Canine distemper virus (CDV) and rabies virus (RABV) occur worldwide in wild carnivore and domestic dog populations and pose threats to wildlife conservation and public health. In Etosha National Park (ENP), Namibia, anthrax is endemic and generates carcasses frequently fed on by an unusually dense population of black-backed jackals (Canis mesomelas). Using serology, phylogenetic analyses (on samples obtained from February 2009-July 2010), and historical mortality records (1975-2011), we assessed jackal exposure to Bacillus anthracis (BA; the causal bacterial agent of anthrax), CDV, and RABV. Prevalence of antibodies against BA (95%, n = 86) and CDV (71%, n = 80) was relatively high, while that of antibodies against RABV was low (9%, n = 81). Exposure to BA increased significantly with age, and all animals >6 mo old were antibody-positive. As with BA, prevalence of antibodies against CDV increased significantly with age, with similar age-specific trends during both years of the study. No significant effect of age was found on the prevalence of antibodies against RABV. Three of the seven animals with antibodies against RABV were monitored for more than 1 yr after sampling and showed no signs of active infection. Mortality records revealed that rabid animals are destroyed nearly every year inside the ENP tourist camps. Phylogenetic analyses demonstrated that jackal RABV in ENP is part of the same transmission cycle as other dog-jackal RABV cycles in Namibia. PMID:22493112

  12. Black-backed jackal exposure to rabies virus, canine distemper virus, and Bacillus anthracis in Etosha National Park, Namibia.

    PubMed

    Bellan, Steve E; Cizauskas, Carrie A; Miyen, Jacobeth; Ebersohn, Karen; Küsters, Martina; Prager, K C; Van Vuuren, Moritz; Sabeta, Claude; Getz, Wayne M

    2012-04-01

    Canine distemper virus (CDV) and rabies virus (RABV) occur worldwide in wild carnivore and domestic dog populations and pose threats to wildlife conservation and public health. In Etosha National Park (ENP), Namibia, anthrax is endemic and generates carcasses frequently fed on by an unusually dense population of black-backed jackals (Canis mesomelas). Using serology, phylogenetic analyses (on samples obtained from February 2009-July 2010), and historical mortality records (1975-2011), we assessed jackal exposure to Bacillus anthracis (BA; the causal bacterial agent of anthrax), CDV, and RABV. Prevalence of antibodies against BA (95%, n = 86) and CDV (71%, n = 80) was relatively high, while that of antibodies against RABV was low (9%, n = 81). Exposure to BA increased significantly with age, and all animals >6 mo old were antibody-positive. As with BA, prevalence of antibodies against CDV increased significantly with age, with similar age-specific trends during both years of the study. No significant effect of age was found on the prevalence of antibodies against RABV. Three of the seven animals with antibodies against RABV were monitored for more than 1 yr after sampling and showed no signs of active infection. Mortality records revealed that rabid animals are destroyed nearly every year inside the ENP tourist camps. Phylogenetic analyses demonstrated that jackal RABV in ENP is part of the same transmission cycle as other dog-jackal RABV cycles in Namibia.

  13. Persistent infections of a field strain of rabies virus in murine neuroblastoma (NA-C1300) cell cultures.

    PubMed

    Webster, W A; Charlton, K M; Casey, G A

    1989-10-01

    Rabies virus from the brain of a striped skunk (Mephitis mephitis) from Ontario was inoculated into murine neuroblastoma (NA-C1300) cell cultures. These cultures were incubated and the cells were subcultured every three to four days. The presence of viral antigen in the cell cultures was monitored by direct immunofluorescent staining and in the culture fluids by titration in either baby hamster kidney (BHK/C13) or NA cells or in experimental mice. The virus-infected NA cultures evolved from an initial high viral concentration in supernatant fluid through a period of decreasing titers of infectious virus in the supernatant fluids to a final phase where no infectious virus has been found following cell culture and animal inoculation methods attempted although the persistently infected cells remained 95-100% viral nucleocapsid antigen-positive. Possible mechanisms involved in the perpetuation of this infection are discussed. This is the first report of a persistent infection of cell cultures by a field strain of rabies virus.

  14. Persistent infections of a field strain of rabies virus in murine neuroblastoma (NA-C1300) cell cultures.

    PubMed Central

    Webster, W A; Charlton, K M; Casey, G A

    1989-01-01

    Rabies virus from the brain of a striped skunk (Mephitis mephitis) from Ontario was inoculated into murine neuroblastoma (NA-C1300) cell cultures. These cultures were incubated and the cells were subcultured every three to four days. The presence of viral antigen in the cell cultures was monitored by direct immunofluorescent staining and in the culture fluids by titration in either baby hamster kidney (BHK/C13) or NA cells or in experimental mice. The virus-infected NA cultures evolved from an initial high viral concentration in supernatant fluid through a period of decreasing titers of infectious virus in the supernatant fluids to a final phase where no infectious virus has been found following cell culture and animal inoculation methods attempted although the persistently infected cells remained 95-100% viral nucleocapsid antigen-positive. Possible mechanisms involved in the perpetuation of this infection are discussed. This is the first report of a persistent infection of cell cultures by a field strain of rabies virus. PMID:2590871

  15. Field use of a vaccinia-rabies recombinant vaccine for the control of sylvatic rabies in Europe and North America.

    PubMed

    Brochier, B; Aubert, M F; Pastoret, P P; Masson, E; Schon, J; Lombard, M; Chappuis, G; Languet, B; Desmettre, P

    1996-09-01

    During recent years, most research on the control of sylvatic rabies has concentrated on developing methods of oral vaccination of wild rabies vectors. To improve both the safety and the stability of the vaccine used, a recombinant vaccinia virus, which expresses the immunising glycoprotein of rabies virus (VRG), has been developed and tested extensively in the laboratory as well as in the field. From 1989 to 1995, approximately 8.5 million VRG vaccine doses were dispersed in Western Europe to vaccinate red foxes (Vulpes vulpes), and in the United States of America (USA) to vaccinate raccoons (Procyon lotor) and coyotes (Canis latrans). In Europe, the use of VRG has led to the elimination of sylvatic rabies from large areas of land, which have consequently been freed from the need for vaccination. Nevertheless, despite very good examples of cross-border cooperation, reinfections have occurred in some regions, due to the difficulty of co-ordinating vaccination plans among neighbouring countries. In the USA, preliminary data from field trails indicate a significant reduction in the incidence of rabies in vaccinated areas. PMID:9025144

  16. 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

  17. Rabies Virus Infection in Ferret Badgers (Melogale moschata subaurantiaca) in Taiwan: A Retrospective Study.

    PubMed

    Chang, Jen-Chieh; Tsai, Kuo-Jung; Hsu, Wei-Cheng; Tu, Yang-Chang; Chuang, Wei-Chieh; Chang, Chia-Yi; Chang, Shih-Wei; Lin, Te-En; Fang, Kuo-Yun; Chang, Yung-Fu; Tsai, Hsiang-Jung; Lee, Shu-Hwae

    2015-10-01

    Fifteen ferret badgers (Melogale moschata subaurantiaca), collected 2010-13 and stored frozen, were submitted for rabies diagnosis by direct fluorescent antibody test and reverse transcription PCR. We detected seven positive animal samples, including some from 2010, which indicated that the ferret badger population in Taiwan had been affected by rabies prior to 2010.

  18. 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.

  19. 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.

  20. Genome-Wide Transcriptional Profiling Reveals Two Distinct Outcomes in Central Nervous System Infections of Rabies Virus

    PubMed Central

    Zhang, Daiting; He, Feilong; Bi, Shuilian; Guo, Huixia; Zhang, Baoshi; Wu, Fan; Liang, Jiaqi; Yang, Youtian; Tian, Qin; Ju, Chunmei; Fan, Huiying; Chen, Jinding; Guo, Xiaofeng; Luo, Yongwen

    2016-01-01

    Rabies remains a major public health concern in many developing countries. The precise neuropathogenesis of rabies is unknown, though it is hypothesized to be due to neuronal death or dysfunction. Mice that received intranasal inoculation of an attenuated rabies virus (RABV) strain HEP-Flury exhibited subtle clinical signs, and eventually recovered, which is different from the fatal encephalitis caused by the virulent RABV strain CVS-11. To understand the neuropathogenesis of rabies and the mechanisms of viral clearance, we applied RNA sequencing (RNA-Seq) to compare the brain transcriptomes of normal mice vs. HEP-Flury or CVS-11 intranasally inoculated mice. Our results revealed that both RABV strains altered positively and negatively the expression levels of many host genes, including genes associated with innate and adaptive immunity, inflammation and cell death. It is found that HEP-Flury infection can activate the innate immunity earlier through the RIG-I/MDA-5 signaling, and the innate immunity pre-activated by HEP-Flury or Newcastle disease virus (NDV) infection can effectively prevent the CVS-11 to invade central nervous system (CNS), but fails to clear the CVS-11 after its entry into the CNS. In addition, following CVS-11 infection, genes implicated in cell adhesion, blood vessel morphogenesis and coagulation were mainly up-regulated, while the genes involved in synaptic transmission and ion transport were significantly down-regulated. On the other hand, several genes involved in the MHC class II-mediated antigen presentation pathway were activated to a greater extent after the HEP-Flury infection as compared with the CVS-11 infection suggesting that the collaboration of CD4+ T cells and MHC class II-mediated antigen presentation is critical for the clearance of attenuated RABV from the CNS. The differentially regulated genes reported here are likely to include potential therapeutic targets for expanding the post-exposure treatment window for RABV

  1. The evolutionary dynamics of canid and mongoose rabies virus in Southern Africa.

    PubMed

    Davis, P L; Rambaut, A; Bourhy, H; Holmes, E C

    2007-01-01

    Two variants of rabies virus (RABV) currently circulate in southern Africa: canid RABV, mainly associated with dogs, jackals, and bat-eared foxes, and mongoose RABV. To investigate the evolutionary dynamics of these variants, we performed coalescent-based analyses of the G-L inter-genic region, allowing for rate variation among viral lineages through the use of a relaxed molecular clock. This revealed that mongoose RABV is evolving more slowly than canid RABV, with mean evolutionary rates of 0.826 and 1.676 x 10(-3) nucleotide substitutions per site, per year, respectively. Additionally, mongoose RABV exhibits older genetic diversity than canid RABV, with common ancestors dating to 73 and 30 years, respectively, and while mongoose RABV has experienced exponential population growth over its evolutionary history in Africa, populations of canid RABV have maintained a constant size. Hence, despite circulating in the same geographic region, these two variants of RABV exhibit striking differences in evolutionary dynamics which are likely to reflect differences in their underlying ecology. PMID:17401615

  2. Evaluation of short-interfering RNAs treatment in experimental rabies due to wild-type virus.

    PubMed

    Appolinario, Camila Michele; Allendorf, Susan Dora; Peres, Marina Gea; Fonseca, Clovis Reynaldo; Vicente, Acacia Ferreira; Antunes, João Marcelo Azevedo de Paula; Pantoja, José Carlos Figueiredo; Megid, Jane

    2015-01-01

    We have evaluated the efficacy of short-interfering RNAs targeting the nucleoprotein gene and also the brain immune response in treated and non-treated infected mice. Mice were inoculated with wild-type virus, classified as dog (hv2) or vampire bat (hv3) variants and both groups were treated or left as controls. No difference was observed in the lethality rate between treated and non-treated groups, although clinical evaluation of hv2 infected mice showed differences in the severity of clinical disease (p=0.0006). Evaluation of brain immune response 5 days post-inoculation in treated hv2 group showed no difference among the analyzed genes, whereas after 10 days post-inoculation there was increased expression of 2',5'-oligoadenylate synthetase 1, tumor necrosis factor alpha, interleukin 12, interferon gamma, and C-X-C motif chemokine 10 associated with higher expression of N gene in the same period (p<0.0001). In hv2 non-treated group only higher interferon beta expression was found at day 5. The observed differences in results of the immune response genes between treated and non-treated groups is not promising as they had neither impact on mortality nor even a reduction in the expression of N gene in siRNA treated animals. This finding suggests that the use of pre-designed siRNA alone may not be useful in rabies treatment. PMID:26254692

  3. Evaluation of short-interfering RNAs treatment in experimental rabies due to wild-type virus.

    PubMed

    Appolinario, Camila Michele; Allendorf, Susan Dora; Peres, Marina Gea; Fonseca, Clovis Reynaldo; Vicente, Acacia Ferreira; Antunes, João Marcelo Azevedo de Paula; Pantoja, José Carlos Figueiredo; Megid, Jane

    2015-01-01

    We have evaluated the efficacy of short-interfering RNAs targeting the nucleoprotein gene and also the brain immune response in treated and non-treated infected mice. Mice were inoculated with wild-type virus, classified as dog (hv2) or vampire bat (hv3) variants and both groups were treated or left as controls. No difference was observed in the lethality rate between treated and non-treated groups, although clinical evaluation of hv2 infected mice showed differences in the severity of clinical disease (p=0.0006). Evaluation of brain immune response 5 days post-inoculation in treated hv2 group showed no difference among the analyzed genes, whereas after 10 days post-inoculation there was increased expression of 2',5'-oligoadenylate synthetase 1, tumor necrosis factor alpha, interleukin 12, interferon gamma, and C-X-C motif chemokine 10 associated with higher expression of N gene in the same period (p<0.0001). In hv2 non-treated group only higher interferon beta expression was found at day 5. The observed differences in results of the immune response genes between treated and non-treated groups is not promising as they had neither impact on mortality nor even a reduction in the expression of N gene in siRNA treated animals. This finding suggests that the use of pre-designed siRNA alone may not be useful in rabies treatment.

  4. The Rabies Virus L Protein Catalyzes mRNA Capping with GDP Polyribonucleotidyltransferase Activity

    PubMed Central

    Ogino, Minako; Ito, Naoto; Sugiyama, Makoto; Ogino, Tomoaki

    2016-01-01

    The large (L) protein of rabies virus (RABV) plays multiple enzymatic roles in viral RNA synthesis and processing. However, none of its putative enzymatic activities have been directly demonstrated in vitro. In this study, we expressed and purified a recombinant form of the RABV L protein and verified its guanosine 5′-triphosphatase and GDP polyribonucleotidyltransferase (PRNTase) activities, which are essential for viral mRNA cap formation by the unconventional mechanism. The RABV L protein capped 5′-triphosphorylated but not 5′-diphosphorylated RABV mRNA-start sequences, 5′-AACA(C/U), with GDP to generate the 5′-terminal cap structure G(5′)ppp(5′)A. The 5′-AAC sequence in the substrate RNAs was found to be strictly essential for RNA capping with the RABV L protein. Furthermore, site-directed mutagenesis showed that some conserved amino acid residues (G1112, T1170, W1201, H1241, R1242, F1285, and Q1286) in the PRNTase motifs A to E of the RABV L protein are required for cap formation. These findings suggest that the putative PRNTase domain in the RABV L protein catalyzes the rhabdovirus-specific capping reaction involving covalent catalysis of the pRNA transfer to GDP, thus offering this domain as a target for developing anti-viral agents. PMID:27213429

  5. The Rabies Virus L Protein Catalyzes mRNA Capping with GDP Polyribonucleotidyltransferase Activity.

    PubMed

    Ogino, Minako; Ito, Naoto; Sugiyama, Makoto; Ogino, Tomoaki

    2016-01-01

    The large (L) protein of rabies virus (RABV) plays multiple enzymatic roles in viral RNA synthesis and processing. However, none of its putative enzymatic activities have been directly demonstrated in vitro. In this study, we expressed and purified a recombinant form of the RABV L protein and verified its guanosine 5'-triphosphatase and GDP polyribonucleotidyltransferase (PRNTase) activities, which are essential for viral mRNA cap formation by the unconventional mechanism. The RABV L protein capped 5'-triphosphorylated but not 5'-diphosphorylated RABV mRNA-start sequences, 5'-AACA(C/U), with GDP to generate the 5'-terminal cap structure G(5')ppp(5')A. The 5'-AAC sequence in the substrate RNAs was found to be strictly essential for RNA capping with the RABV L protein. Furthermore, site-directed mutagenesis showed that some conserved amino acid residues (G1112, T1170, W1201, H1241, R1242, F1285, and Q1286) in the PRNTase motifs A to E of the RABV L protein are required for cap formation. These findings suggest that the putative PRNTase domain in the RABV L protein catalyzes the rhabdovirus-specific capping reaction involving covalent catalysis of the pRNA transfer to GDP, thus offering this domain as a target for developing anti-viral agents.

  6. Developments in rabies vaccines

    PubMed Central

    Hicks, D J; Fooks, A R; Johnson, N

    2012-01-01

    The development of vaccines that prevent rabies has a long and distinguished history, with the earliest preceding modern understanding of viruses and the mechanisms of immune protection against disease. The correct application of inactivated tissue culture-derived vaccines is highly effective at preventing the development of rabies, and very few failures are recorded. Furthermore, oral and parenteral vaccination is possible for wildlife, companion animals and livestock, again using inactivated tissue culture-derived virus. However, rabies remains endemic in many regions of the world and causes thousands of human deaths annually. There also remain no means of prophylaxis for rabies once the virus enters the central nervous system (CNS). One reason for this is the poor immune response within the CNS to infection with rabies virus (RABV). New approaches to vaccination using modified rabies viruses that express components of the innate immune system are being applied to this problem. Preliminary reports suggest that direct inoculation of such viruses could trigger an effective anti-viral response and prevent a fatal outcome from RABV infection. PMID:22861358

  7. Dimeric architecture of the Hendra virus attachment glycoprotein: evidence for a conserved mode of assembly.

    PubMed

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

    2010-06-01

    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 beta-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 alpha-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 degrees ) 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.

  8. Rabies Virus Infection Induces the Formation of Stress Granules Closely Connected to the Viral Factories

    PubMed Central

    Nikolic, Jovan; Civas, Ahmet; Lagaudrière-Gesbert, Cécile; Blondel, Danielle

    2016-01-01

    Stress granules (SGs) are membrane-less dynamic structures consisting of mRNA and protein aggregates that form rapidly in response to a wide range of environmental cellular stresses and viral infections. They act as storage sites for translationally silenced mRNAs under stress conditions. During viral infection, SG formation results in the modulation of innate antiviral immune responses, and several viruses have the ability to either promote or prevent SG assembly. Here, we show that rabies virus (RABV) induces SG formation in infected cells, as revealed by the detection of SG-marker proteins Ras GTPase-activating protein-binding protein 1 (G3BP1), T-cell intracellular antigen 1 (TIA-1) and poly(A)-binding protein (PABP) in the RNA granules formed during viral infection. As shown by live cell imaging, RABV-induced SGs are highly dynamic structures that increase in number, grow in size by fusion events, and undergo assembly/disassembly cycles. Some SGs localize in close proximity to cytoplasmic viral factories, known as Negri bodies (NBs). Three dimensional reconstructions reveal that both structures remain distinct even when they are in close contact. In addition, viral mRNAs synthesized in NBs accumulate in the SGs during viral infection, revealing material exchange between both compartments. Although RABV-induced SG formation is not affected in MEFs lacking TIA-1, TIA-1 depletion promotes viral translation which results in an increase of viral replication indicating that TIA-1 has an antiviral effect. Inhibition of PKR expression significantly prevents RABV-SG formation and favors viral replication by increasing viral translation. This is correlated with a drastic inhibition of IFN-B gene expression indicating that SGs likely mediate an antiviral response which is however not sufficient to fully counteract RABV infection. PMID:27749929

  9. Glycoprotein gp50-negative pseudorabies virus: a novel approach toward a nonspreading live herpesvirus vaccine.

    PubMed Central

    Heffner, S; Kovács, F; Klupp, B G; Mettenleiter, T C

    1993-01-01

    Essential herpesvirus glycoproteins are involved in membrane fusion processes during infection, e.g., viral penetration and direct cell-to-cell transmission. We previously showed that the gD-homologous glycoprotein gp50 of pseudorabies virus (PrV) is essential for virus entry into target cells but proved to be dispensable for direct viral cell-to-cell spread in cell culture (I. Rauh and T. C. Mettenleiter, J. Virol. 65:5348-5456, 1991). For gp50-negative (gp50-) viruses, after phenotypic complementation necessary for primary infection, the only means of viral spread is by way of direct cell-to-cell transmission. In contrast, virus mutants lacking the essential gB-homologous glycoprotein gII after phenotypic complementation are only able to infect primary target cells and are blocked in further viral spread. To analyze how these in vitro phenotypes translate into virus replication in the animal, mice were infected intranasally with gp50- or gII- PrV mutants after prior phenotypic complementation by propagation on cell lines providing the essential glycoprotein in trans. Our results show that whereas the gII- mutants did not cause disease or any symptoms, gp50- mutants derived from two different PrV strains were fully virulent, with animals exhibiting severe symptoms ultimately leading to death. However, free infectious virus could not be recovered from either gp50- or gII- PrV-infected animals. We conclude that direct cell-to-cell transmission as the only means of viral spread of the gp50- mutants is sufficient for a full virulent phenotype in mice. After infection of pigs with phenotypically complemented gp50- PrV, only mild symptoms were observed, whereas the gII- mutant was totally avirulent. In both cases, shedding of infectious virus did not occur, in contrast to results with animals infected by gX- PrV that showed severe signs of disease and extensive virus shedding. After challenge infection with the highly virulent NIA-3 strain, the previously gII- Pr

  10. 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 ...

  11. Animal-Associated Exposure to Rabies Virus among Travelers, 1997–2012

    PubMed Central

    Harvey, Kira; Pandey, Prativa; Lim, Poh Lian; Leder, Karin; Piyaphanee, Watcharapong; Shaw, Marc; McDonald, Susan C.; Schwartz, Eli; Esposito, Douglas H.; Parola, Philippe

    2015-01-01

    Among travelers, rabies cases are rare, but animal bites are relatively common. To determine which travelers are at highest risk for rabies, we studied 2,697 travelers receiving care for animal-related exposures and requiring rabies postexposure prophylaxis at GeoSentinel clinics during 1997–2012. No specific demographic characteristics differentiated these travelers from other travelers seeking medical care, making it challenging to identify travelers who might benefit from reinforced pretravel rabies prevention counseling. Median travel duration was short for these travelers: 15 days for those seeking care after completion of travel and 20 days for those seeking care during travel. This finding contradicts the view that preexposure rabies vaccine recommendations should be partly based on longer travel durations. Over half of exposures occurred in Thailand, Indonesia, Nepal, China, and India. International travelers to rabies-endemic regions, particularly Asia, should be informed about potential rabies exposure and benefits of pretravel vaccination, regardless of demographics or length of stay. PMID:25811076

  12. Animal-associated exposure to rabies virus among travelers, 1997-2012.

    PubMed

    Gautret, Philippe; Harvey, Kira; Pandey, Prativa; Lim, Poh Lian; Leder, Karin; Piyaphanee, Watcharapong; Shaw, Marc; McDonald, Susan C; Schwartz, Eli; Esposito, Douglas H; Parola, Philippe

    2015-04-01

    Among travelers, rabies cases are rare, but animal bites are relatively common. To determine which travelers are at highest risk for rabies, we studied 2,697 travelers receiving care for animal-related exposures and requiring rabies postexposure prophylaxis at GeoSentinel clinics during 1997-2012. No specific demographic characteristics differentiated these travelers from other travelers seeking medical care, making it challenging to identify travelers who might benefit from reinforced pretravel rabies prevention counseling. Median travel duration was short for these travelers: 15 days for those seeking care after completion of travel and 20 days for those seeking care during travel. This finding contradicts the view that preexposure rabies vaccine recommendations should be partly based on longer travel durations. Over half of exposures occurred in Thailand, Indonesia, Nepal, China, and India. International travelers to rabies-endemic regions, particularly Asia, should be informed about potential rabies exposure and benefits of pretravel vaccination, regardless of demographics or length of stay. PMID:25811076

  13. Expression of the human immunodeficiency virus envelope glycoprotein is restricted to basolateral surfaces of polarized epithelial cells

    SciTech Connect

    Owens, R.J.; Compans, R.W.

    1989-02-01

    Polarized epithelial cells exhibit apical (lumenal) and basolateral (serosal) membrane domains that are separated by circumferential tight junctions. In such cells, enveloped viruses that mature by budding at cell surfaces are released at particular membrane domains. The authors have used a vaccinia virus recombinant to investigate the site of surface expression of the human immunodeficiency virus type 1 envelope glycoprotein in Madin-Darby canine kidney cells. Cells were infected with the vaccinia virus recombinant, and surface expression of the glycoprotein was analyzed by indirect immunofluorescence, /sup 125/I-protein A binding, and immunoelectron microscopy. The glycoprotein appeared exclusively at the basolateral surface as early as 2 h postinfection and reached a maximum level at 8 h postinfection. The gp120 glycoprotein was found to be secreted efficiently into culture medium, and this secretion occurred exclusively at the basolateral surface.

  14. 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

  15. Amino acid mutations in Ebola virus glycoprotein of the 2014 epidemic.

    PubMed

    Giovanetti, Marta; Grifoni, Alba; Lo Presti, Alessandra; Cella, Eleonora; Montesano, Carla; Zehender, Gianguglielmo; Colizzi, Vittorio; Amicosante, Massimo; Ciccozzi, Massimo

    2015-06-01

    Zaire Ebola virus (EBOV) is an enveloped non-segmented negative strand RNA virus of 19 kb in length belonging to the family Filoviridae. The virus was isolated and identified in 1976 during the epidemic of hemorrhagic fever in Zaire. The most recent outbreak of EBOV among humans, was that occurred in the forested areas of south eastern Guinea, that began in February 2014 and is still ongoing. The recent Ebola outbreak, is affecting other countries in West Africa, in addiction to Guinea: Liberia, Nigeria, and Sierra Leone. In this article, a selective pressure analysis and homology modeling based on the G Glycoprotein (GP) sequences retrieved from public databases were used to investigate the genetic diversity and modification of antibody response in the recent outbreak of Ebola Virus. Structural and the evolutionary analysis underline the 2014 epidemic virus being under negative selective pressure does not change with respect to the old epidemic in terms of genome adaptation.

  16. Rabies virus replication in primary murine bone marrow macrophages and in human and murine macrophage-like cell lines: implications for viral persistence.

    PubMed

    Ray, N B; Ewalt, L C; Lodmell, D L

    1995-02-01

    To determine whether rabies viruses replicate in macrophage or macrophage-like cells, several human and murine macrophage-like cell lines, as well as primary cultures of murine bone marrow macrophages, were incubated with the Evelyn-Rokitnicki-Abelseth (ERA) virus and several different street rabies viruses (SRV). ERA rabies virus replicated well in human monocytic U937 and THP-1 cells and murine macrophage IC-21 cells, as well as primary cultures of murine macrophages. Minimal replication was detected in murine monocytic WEHI-3BD- and PU5-1R cells, and ERA virus did not replicate in murine monocytic P388D1 or J774A.1 cells. A tissue culture-adapted SRV of bat origin also replicated in IC-21 and U937 cells. Non-tissue culture-adapted SRV isolated from different animal species, particularly bats, replicated minimally in U937, THP-1, IC-21 cells and primary murine bone marrow macrophages. To determine whether rabies virus replication is dependent upon the state of differentiation of the macrophage-like cell, human promyelocytic HL-60 cells were differentiated with 12-O-tetradecanoylphorbol-13-acetate (TPA). ERA rabies virus replicated in the differentiated HL-60 cells but not in undifferentiated HL-60 cells. Persistent infections were established in macrophage-like U937 cells with ERA rabies virus and SRV, and infectious SRV was isolated from adherent bone marrow cells of mice that had been infected 96 days previously. Virus harvested from persistently infected U937 cells and the adherent bone marrow cells had specifically adapted to each cell. This specificity was shown by the inability of the viruses to infect macrophages other than U937 cells and primary bone marrow macrophages, respectively. Virus titers of the persistently infected U937 cells fluctuated with extended cell passage. After 30 passages, virus released from the cells had lost virulence as shown by its inability to kill intracranially inoculated mice. However, the avirulent virus released from the

  17. New insights into the Hendra virus attachment and entry process from structures of the virus G glycoprotein and its complex with Ephrin-B2.

    PubMed

    Xu, Kai; Chan, Yee-Peng; Rajashankar, Kanagalaghatta R; Khetawat, Dimple; Yan, Lianying; Kolev, Momchil V; Broder, Christopher C; Nikolov, Dimitar B

    2012-01-01

    Hendra virus and Nipah virus, comprising the genus Henipavirus, are recently emerged, highly pathogenic and often lethal zoonotic agents against which there are no approved therapeutics. Two surface glycoproteins, the attachment (G) and fusion (F), mediate host cell entry. The crystal structures of the Hendra G glycoprotein alone and in complex with the ephrin-B2 receptor reveal that henipavirus uses Tryptophan 122 on ephrin-B2/B3 as a "latch" to facilitate the G-receptor association. Structural-based mutagenesis of residues in the Hendra G glycoprotein at the receptor binding interface document their importance for viral attachments and entry, and suggest that the stability of the Hendra-G-ephrin attachment complex does not strongly correlate with the efficiency of viral entry. In addition, our data indicates that conformational rearrangements of the G glycoprotein head domain upon receptor binding may be the trigger leading to the activation of the viral F fusion glycoprotein during virus infection.

  18. Efficient reverse genetics generation of infectious junin viruses differing in glycoprotein processing.

    PubMed

    Albariño, César G; Bergeron, Eric; Erickson, Bobbie Rae; Khristova, Marina L; Rollin, Pierre E; Nichol, Stuart T

    2009-06-01

    The New World arenaviruses, Junin, Machupo, Guanarito, Sabia, and Chapare, are associated with rapidly progressing severe hemorrhagic fever with a high rate of case fatality in various regions of South America. The threat of natural or deliberate outbreaks associated with these viruses makes the development of preventive or therapeutic measures important. Here we describe a Junin virus functional minigenome system and a reverse genetics system for production of infectious Junin virus. This robust, highly efficient system involves transfection of cells with only two plasmids which transcribe the virus S and L antigenomic RNAs. The utility of the system is demonstrated by generating Junin viruses which encode a glycoprotein precursor (GPC) containing the following: (i) the wild-type (SKI-1/S1P peptidase) cleavage site, (ii) no cleavage site, or (iii) a cleavage site where the SKI-1/S1P motif (RSLK) is replaced by a furin cleavage site (RRKR). In contrast to the wild-type virus, Junin virus lacking a GPC cleavage site replicated within successfully transfected cells but failed to yield infectious virus particles. This confirms observations with other arenaviruses suggesting that GPC cleavage is essential for arenavirus infectivity. In contrast, infectious Junin virus which encoded GPC cleaved by furin-like proteases was easily generated. The two-plasmid, high efficiency aspects of this Junin virus reverse genetics system show great promise for addressing important questions regarding arenavirus hemorrhagic fever disease and for development of precisely attenuated live arenavirus vaccines.

  19. Mutagenesis of the palmitoylation site in vaccinia virus envelope glycoprotein B5.

    PubMed

    Lorenzo, María M; Sánchez-Puig, Juana M; Blasco, Rafael

    2012-04-01

    The outer envelope of vaccinia virus extracellular virions is derived from intracellular membranes that, at late times in infection, are enriched in several virus-encoded proteins. Although palmitoylation is common in vaccinia virus envelope proteins, little is known about the role of palmitoylation in the biogenesis of the enveloped virus. We have studied the palmitoylation of B5, a 42 kDa type I transmembrane glycoprotein comprising a large ectodomain and a short (17 aa) cytoplasmic tail. Mutation of two cysteine residues located in the cytoplasmic tail in close proximity to the transmembrane domain abrogated palmitoylation of the protein. Virus mutants expressing non-palmitoylated versions of B5 and/or lacking most of the cytoplasmic tail were isolated and characterized. Cell-to-cell virus transmission and extracellular virus formation were only slightly affected by those mutations. Notably, B5 versions lacking palmitate showed decreased interactions with proteins A33 and F13, but were still incorporated into the virus envelope. Expression of mutated B5 by transfection into uninfected cells showed that both the cytoplasmic tail and palmitate have a role in the intracellular transport of B5. These results indicate that the C-terminal portion of protein B5, while involved in protein transport and in protein-protein interactions, is broadly dispensable for the formation and egress of infectious extracellular virus and for virus transmission.

  20. 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

  1. Human Rabies - Missouri, 2014.

    PubMed

    Pratt, P Drew; Henschel, Kathleen; Turabelidze, George; Grim, Autumn; Ellison, James A; Orciari, Lillian; Yager, Pamela; Franka, Richard; Wu, Xianfu; Ma, Xiaoyue; Wadhwa, Ashutosh; Smith, Todd G; Petersen, Brett; Shiferaw, Miriam

    2016-03-18

    On September 18, 2014, the Missouri Department of Health and Senior Services (MDHSS) was notified of a suspected rabies case in a Missouri resident. The patient, a man aged 52 years, lived in a rural, deeply wooded area, and bat sightings in and around his home were anecdotally reported. Exposure to bats poses a risk for rabies. After two emergency department visits for severe neck pain, paresthesia in the left arm, upper body tremors, and anxiety, he was hospitalized on September 13 for encephalitis of unknown etiology. On September 24, he received a diagnosis of rabies and on September 26, he died. Genetic sequencing tests confirmed infection with a rabies virus variant associated with tricolored bats. Health care providers need to maintain a high index of clinical suspicion for rabies in patients who have unexplained, rapidly progressive encephalitis, and adhere to recommended infection control practices when examining and treating patients with suspected infectious diseases.

  2. Human Rabies - Missouri, 2014.

    PubMed

    Pratt, P Drew; Henschel, Kathleen; Turabelidze, George; Grim, Autumn; Ellison, James A; Orciari, Lillian; Yager, Pamela; Franka, Richard; Wu, Xianfu; Ma, Xiaoyue; Wadhwa, Ashutosh; Smith, Todd G; Petersen, Brett; Shiferaw, Miriam

    2016-03-18

    On September 18, 2014, the Missouri Department of Health and Senior Services (MDHSS) was notified of a suspected rabies case in a Missouri resident. The patient, a man aged 52 years, lived in a rural, deeply wooded area, and bat sightings in and around his home were anecdotally reported. Exposure to bats poses a risk for rabies. After two emergency department visits for severe neck pain, paresthesia in the left arm, upper body tremors, and anxiety, he was hospitalized on September 13 for encephalitis of unknown etiology. On September 24, he received a diagnosis of rabies and on September 26, he died. Genetic sequencing tests confirmed infection with a rabies virus variant associated with tricolored bats. Health care providers need to maintain a high index of clinical suspicion for rabies in patients who have unexplained, rapidly progressive encephalitis, and adhere to recommended infection control practices when examining and treating patients with suspected infectious diseases. PMID:26985578

  3. Hantavirus Gn and Gc Glycoproteins Self-Assemble into Virus-Like Particles

    PubMed Central

    Acuña, Rodrigo; Cifuentes-Muñoz, Nicolás; Márquez, Chantal L.; Bulling, Manuela; Klingström, Jonas; Mancini, Roberta; Lozach, Pierre-Yves

    2014-01-01

    How hantaviruses assemble and exit infected cells remains largely unknown. Here, we show that the expression of Andes (ANDV) and Puumala (PUUV) hantavirus Gn and Gc envelope glycoproteins lead to their self-assembly into virus-like particles (VLPs) which were released to cell supernatants. The viral nucleoprotein was not required for particle formation. Further, a Gc endodomain deletion mutant did not abrogate VLP formation. The VLPs were pleomorphic, exposed protrusions and reacted with patient sera. PMID:24335294

  4. Rabies and Canine Distemper Virus Epidemics in the Red Fox Population of Northern Italy (2006–2010)

    PubMed Central

    De Benedictis, Paola; Citterio, Carlo; Obber, Federica; Lorenzetto, Monica; Pozza, Manuela Dalla; Cauchemez, Simon; Cattoli, Giovanni

    2013-01-01

    Since 2006 the red fox (Vulpes vulpes) population in north-eastern Italy has experienced an epidemic of canine distemper virus (CDV). Additionally, in 2008, after a thirteen-year absence from Italy, fox rabies was re-introduced in the Udine province at the national border with Slovenia. Disease intervention strategies are being developed and implemented to control rabies in this area and minimise risk to human health. Here we present empirical data and the epidemiological picture relating to these epidemics in the period 2006–2010. Of important significance for epidemiological studies of wild animals, basic mathematical models are developed to exploit information collected from the surveillance program on dead and/or living animals in order to assess the incidence of infection. These models are also used to estimate the rate of transmission of both diseases and the rate of vaccination, while correcting for a bias in early collection of CDV samples. We found that the rate of rabies transmission was roughly twice that of CDV, with an estimated effective contact between infected and susceptible fox leading to a new infection occurring once every 3 days for rabies, and once a week for CDV. We also inferred that during the early stage of the CDV epidemic, a bias in the monitoring protocol resulted in a positive sample being almost 10 times more likely to be collected than a negative sample. We estimated the rate of intake of oral vaccine at 0.006 per day, allowing us to estimate that roughly 68% of the foxes would be immunised. This was confirmed by field observations. Finally we discuss the implications for the eco-epidemiological dynamics of both epidemics in relation to control measures. PMID:23630599

  5. [Rabies in bats].

    PubMed

    Beranová, Kateřina; Zendulková, Dagmar

    2016-06-01

    Rabies is a zoonosis ending fatally in all mammals, including humans. Unlike the other mammals, this disease is usually not fatal in bats. Rabies is caused by lyssaviruses which are divided into several distinct phylogroups comprising 15 known viruses. It is believed that the original hosts of all lyssaviruses are bats. Classical rabies virus (RABV) occurs in bats across Americas and represents the major cause of rabies in humans and domestic animals there. European bat lyssavirus type 1 (EBLV-1) and European bat lyssavirus type 2 (EBLV-2) are the most frequently diagnosed lyssaviruses in Eurasia. The transmission of EBLV-1 and EBLV-2 from bats to other mammals is very rare. As of now, more detailed information is missing about the other Eurasian lyssaviruses - West Caucasian bat virus (WCBV), Bokeloh bat lyssavirus (BBLV), Aravan virus (ARAV), Irkut virus (IRKV), Khujand virus (KHUV) and Lleida virus. The lyssavirus most frequently found in Africa is Lagos bat virus (LBV). In Australia, only Australian bat lyssavirus (ABLV) has been demonstrated as yet. In the Czech Republic, a total of five cases of rabies in bats were confirmed between 1994 and 2015. Rabies can be transmitted from bats mainly by biting or scratching. Clinically ill bats suffer from nervous disorders or produce abnormal sounds. If rabies is suspected, laboratory tests are essential. Protection of human health is based on pre-exposure and/or post-exposure vaccination. However, the available vaccines do not protect against some newly identified lyssaviruses such as WCBV. Nevertheless, most bat species pose a minimal risk to humans.

  6. [Rabies in bats].

    PubMed

    Beranová, Kateřina; Zendulková, Dagmar

    2016-06-01

    Rabies is a zoonosis ending fatally in all mammals, including humans. Unlike the other mammals, this disease is usually not fatal in bats. Rabies is caused by lyssaviruses which are divided into several distinct phylogroups comprising 15 known viruses. It is believed that the original hosts of all lyssaviruses are bats. Classical rabies virus (RABV) occurs in bats across Americas and represents the major cause of rabies in humans and domestic animals there. European bat lyssavirus type 1 (EBLV-1) and European bat lyssavirus type 2 (EBLV-2) are the most frequently diagnosed lyssaviruses in Eurasia. The transmission of EBLV-1 and EBLV-2 from bats to other mammals is very rare. As of now, more detailed information is missing about the other Eurasian lyssaviruses - West Caucasian bat virus (WCBV), Bokeloh bat lyssavirus (BBLV), Aravan virus (ARAV), Irkut virus (IRKV), Khujand virus (KHUV) and Lleida virus. The lyssavirus most frequently found in Africa is Lagos bat virus (LBV). In Australia, only Australian bat lyssavirus (ABLV) has been demonstrated as yet. In the Czech Republic, a total of five cases of rabies in bats were confirmed between 1994 and 2015. Rabies can be transmitted from bats mainly by biting or scratching. Clinically ill bats suffer from nervous disorders or produce abnormal sounds. If rabies is suspected, laboratory tests are essential. Protection of human health is based on pre-exposure and/or post-exposure vaccination. However, the available vaccines do not protect against some newly identified lyssaviruses such as WCBV. Nevertheless, most bat species pose a minimal risk to humans. PMID:27450525

  7. Bioecological Drivers of Rabies Virus Circulation in a Neotropical Bat Community

    PubMed Central

    de Thoisy, Benoit; Bourhy, Hervé; Delaval, Marguerite; Pontier, Dominique; Dacheux, Laurent; Darcissac, Edith; Donato, Damien; Guidez, Amandine; Larrous, Florence; Lavenir, Rachel; Salmier, Arielle; Lacoste, Vincent; Lavergne, Anne

    2016-01-01

    Introduction In addition to the commonly accepted importance of the vampire bat in the maintenance and transmission of the rabies virus (RABV) in South America, RABV infection of other species is widely evidenced, challenging their role in the viral cycle. Methodology / Principles findings To identify the bioecological drivers of RABV circulation in neotropical bat communities, we conducted a molecular and serological survey on almost 1,000 bats from 30 species, and a 4-year longitudinal survey in two colonies of vampire bats in French Guiana. RABV was molecularly detected in a common vampire and in a frugivorous bat. The sequences corresponded to haematophagous bat-related strains and were close to viruses circulating in the Brazilian Amazon region. Species’ seroprevalence ranged from 0 to 20%, and the risk of seropositivity was higher in bats with a haematophagous diet, living in monospecific colonies and in dense forests. The longitudinal survey showed substantial temporal fluctuations, with individual waves of seroconversions and waning immunity. The high prevalences observed in bat communities, in most habitats and in species that do not share the same microhabitats and bioecological patterns, the temporal variations, and a rather short period of detectable antibodies as observed in recaptured vampires suggest (i) frequent exposure of animals, (ii) an ability of the infected host to control and eliminate the virus, (iii) more relaxed modes of exposure between bats than the commonly assumed infection via direct contact with saliva of infected animals, all of which should be further investigated. Conclusions / significance We hypothesize that RABV circulation in French Guiana is mainly maintained in the pristine forest habitats that may provide sufficient food resources to allow vampire bats, the main prevalent species, to survive and RABV to be propagated. However, on the forest edge and in disturbed areas, human activities may induce more insidious effects

  8. Rabies virus and canine distemper virus in wild and domestic carnivores in Northern Kenya: are domestic dogs the reservoir?

    PubMed

    Prager, K C; Mazet, Jonna A K; Dubovi, Edward J; Frank, Laurence G; Munson, Linda; Wagner, Aaron P; Woodroffe, Rosie

    2012-12-01

    Rabies virus (RV) and canine distemper virus (CDV) can cause significant mortality in wild carnivore populations, and RV threatens human lives. We investigated serological patterns of exposure to CDV and RV in domestic dogs (Canis familiaris), African wild dogs (Lycaon pictus), black-backed jackals (Canis mesomelas), spotted hyenas (Crocuta crocuta), striped hyenas (Hyaena hyaena) and African lions (Panthera leo), over a 10-year period, in a Kenyan rangeland to assess the role domestic dogs may play in the transmission dynamics of these two important canid pathogens. Observed patterns of RV exposure suggested that repeated introduction, rather than maintenance, occurred in the wild carnivore species studied. However, RV appeared to have been maintained in domestic dogs: exposure was more likely in domestic dogs than in the wild carnivores; was detected consistently over time without variation among years; and was detected in juveniles (≤1-year-old) as well as adults (>1-year-old). We conclude that this domestic dog population could be a RV reservoir. By contrast, the absence of evidence of CDV exposure for each carnivore species examined in the study area, for specific years, suggested repeated introduction, rather than maintenance, and that CDV may require a larger reservoir population than RV. This reservoir could be a larger domestic dog population; another wildlife species; or a "metareservoir" consisting of multiple interconnected carnivore populations. Our findings suggest that RV risks to people and wild carnivores might be controlled by domestic dog vaccination, but that CDV control, if required, would need to target the species of concern. PMID:23459924

  9. Rabies virus and canine distemper virus in wild and domestic carnivores in Northern Kenya: are domestic dogs the reservoir?

    PubMed

    Prager, K C; Mazet, Jonna A K; Dubovi, Edward J; Frank, Laurence G; Munson, Linda; Wagner, Aaron P; Woodroffe, Rosie

    2012-12-01

    Rabies virus (RV) and canine distemper virus (CDV) can cause significant mortality in wild carnivore populations, and RV threatens human lives. We investigated serological patterns of exposure to CDV and RV in domestic dogs (Canis familiaris), African wild dogs (Lycaon pictus), black-backed jackals (Canis mesomelas), spotted hyenas (Crocuta crocuta), striped hyenas (Hyaena hyaena) and African lions (Panthera leo), over a 10-year period, in a Kenyan rangeland to assess the role domestic dogs may play in the transmission dynamics of these two important canid pathogens. Observed patterns of RV exposure suggested that repeated introduction, rather than maintenance, occurred in the wild carnivore species studied. However, RV appeared to have been maintained in domestic dogs: exposure was more likely in domestic dogs than in the wild carnivores; was detected consistently over time without variation among years; and was detected in juveniles (≤1-year-old) as well as adults (>1-year-old). We conclude that this domestic dog population could be a RV reservoir. By contrast, the absence of evidence of CDV exposure for each carnivore species examined in the study area, for specific years, suggested repeated introduction, rather than maintenance, and that CDV may require a larger reservoir population than RV. This reservoir could be a larger domestic dog population; another wildlife species; or a "metareservoir" consisting of multiple interconnected carnivore populations. Our findings suggest that RV risks to people and wild carnivores might be controlled by domestic dog vaccination, but that CDV control, if required, would need to target the species of concern.

  10. 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

  11. Instructive even after a decade: Complete results of initial virological diagnostics and re-evaluation of molecular data in the German rabies virus "outbreak" caused by transplantations.

    PubMed

    Ross, R Stefan; Wolters, Bernd; Hoffmann, Bernd; Geue, Lutz; Viazov, Sergei; Grüner, Nico; Roggendorf, Michael; Müller, Thomas

    2015-10-01

    In 2005, six patients in Germany received solid organs and both corneas from a donor with an unrecognized rabies infection. Initial virological diagnostics with the machinery available at the two national reference laboratories could quickly clarify the situation. Rabies virus antigen was detected in the organ donor's brain. In two of the three recipients with neurological alterations, intra vitam diagnosis was achieved by conventional RT-PCRs. Comparison of the phylogenetic relatedness of the different viral isolates proved transmission from the donor and, consequently, also established the diagnosis for the third patient. As indicated by the titre of neutralizing antibodies, the liver transplant recipient was protected from the lethal infection due to a vaccination against rabies virus, which he had received more than 15 years ago. All samples from the recipients of the corneas were invariably negative. Re-evaluation of the molecular data by real-time PCR did not lead to an improvement of intra vitam diagnosis but provided intriguing insights regarding the relative amounts of rabies virus RNA in different body fluids and peripheral organs. In saliva and skin, they were 250-200,000 times lower than in the infected patient's brains. Furthermore, in saliva samples taken serially from the same patient fluctuations by a factor of 160-500 were recorded. These findings highlight the problems of intra vitam diagnosis of rabies virus infections and make understandable why the virus can escape from all diagnostic attempts. Finally, in this context one should recall an almost trivial fact: Simple and appropriate postexposure prophylaxis could not only have saved the young organ donor's life but would also have prevented the whole transplantation-associated rabies "outbreak" in Germany.

  12. Mutational analysis of the vesicular stomatitis virus glycoprotein G for membrane fusion domains.

    PubMed

    Li, Y; Drone, C; Sat, E; Ghosh, H P

    1993-07-01

    The spike glycoprotein G of vesicular stomatitis virus (VSV) induces membrane fusion at low pH. We used linker insertion mutagenesis to characterize the domain(s) of G glycoprotein involved in low-pH-induced membrane fusion. Two or three amino acids were inserted in frame into various positions in the extracellular domain of G, and 14 mutants were isolated. All of the mutants expressed fully glycosylated proteins in COS cells. However, only seven mutant G glycoproteins were transported to the cell surface. Two of these mutants, D1 and A6, showed wild-type fusogenic properties. The mutant A2 had a temperature-sensitive defect in the transport of the mutant G glycoprotein to the cell surface. The other four mutants, H2, H5, H10, and A4, although present in cell surface, failed to induce cell fusion when cells expressing these mutant glycoproteins were exposed to acidic pH. These four mutant G proteins could form trimers, indicating that the defect in fusion was not due to defective oligomerization. One of these mutations, H2, is within a region of conserved, uncharged amino acids that has been proposed as a possible fusogenic sequence. The mutation in H5 was about 70 amino acids downstream of the mutation in H2, while mutations in H10 and A4 were about 300 amino acids downstream of the mutation in H2. Conserved sequences were also noted in the H10 and A4 segment. The results suggest that in the case of VSV G glycoprotein, the fusogenic activity may involve several spatially separated regions in the extracellular domain of the protein.

  13. Characterization of virulence-associated determinants in the envelope glycoprotein of Pichinde virus.

    PubMed

    Kumar, Naveen; Wang, Jialong; Lan, Shuiyun; Danzy, Shamika; McLay Schelde, Lisa; Seladi-Schulman, Jill; Ly, Hinh; Liang, Yuying

    2012-11-10

    We use a small animal model, based on guinea pigs infected with a non-pathogenic Pichinde virus (PICV), to understand the virulence mechanisms of arenavirus infections in the hosts. PICV P2 strain causes a mild febrile reaction in guinea pigs, while P18 causes severe disease with clinical and pathological features reminiscent of Lassa hemorrhagic fever in humans. The envelope glycoproteins (GPC) of P2 and P18 viruses differ at positions 119, 140, and 164, all localized to the receptor-binding G1 subunit. We found that lentiviral pseudotyped virions (VLPs) bearing P18 GPC show more efficient cell entry than those with P2 GPC, and that the E140 residue plays a critical role in this process. Infection of guinea pigs with the recombinant viruses containing the E140K change demonstrated that E140 of GPC is a necessary virulence determinant of P18 infections, possibly by enhancing the ability of virus to enter target cells.

  14. 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.

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

    PubMed

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

    2015-12-01

    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.

  16. 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

  17. 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

  18. Immunogenic glycoproteins of laboratory and vaccine strains of Varicella-Zoster virus.

    PubMed Central

    Grose, C; Edmond, B J; Friedrichs, W E

    1981-01-01

    High-titered antisera were prepared in guinea pigs and rabbits against two strains of varicella-zoster virus (VZV): VZV-32, a low-passage laboratory strain, and VZV-Oka, a vaccine strain attenuated by passage in both human and guinea pig embryo cells. When the animal VZV-immune sera, as well as a human zoster serum, were used to precipitate radiolabeled glycoproteins from VZV-infected cells and the immune precipitates were analyzed by polyacrylamide gel electrophoresis and fluorography, it was observed that cell cultures infected with either strain had similar electrophoretic profiles containing major glycoproteins of approximate molecular weights 62,000, 98,000, and 118,000. A prominent high-molecular-weight (approximately 150,000) nonglycosylated polypeptide was identified in both strains also. These determinants were demonstrable by both indirect (staphylococcal protein A-antibody adsorbent) and direct immunoprecipitation, as long as VZV-immune sera with an antibody titer greater than or equal to 1:128 were used. Further analysis of individual caviid VZV antisera demonstrated some heterogeneity which appeared to be related to the method of immunization rather than the level of virus-specific antibody. VZV extracts emulsified with complete Freund adjuvant elicited an antibody response to all major immunogenic viral glycoproteins, whereas guinea pigs inoculated with virus alone during the primary immunization initially produced VZV antibody which failed to precipitate the highest-molecular-weight glycoprotein (gp118). Thus, Freund-type adjuvants promoted the maturation of the humoral immune response after VZV immunization in outbred guinea pigs. Images PMID:6262245

  19. Influenza Virus Assembly and Lipid Raft Microdomains: a Role for the Cytoplasmic Tails of the Spike Glycoproteins

    PubMed Central

    Zhang, Jie; Pekosz, Andrew; Lamb, Robert A.

    2000-01-01

    Influenza viruses encoding hemagglutinin (HA) and neuraminidase (NA) glycoproteins with deletions in one or both cytoplasmic tails (HAt− or NAt−) have a reduced association with detergent-insoluble glycolipids (DIGs). Mutations which eliminated various combinations of the three palmitoylation sites in HA exhibited reduced amounts of DIG-associated HA in virus-infected cells. The influenza virus matrix (M1) protein was also found to be associated with DIGs, but this association was decreased in cells infected with HAt− or NAt− virus. Regardless of the amount of DIG-associated protein, the HA and NA glycoproteins were targeted primarily to the apical surface of virus-infected, polarized cells. The uncoupling of DIG association and apical transport was augmented by the observation that the influenza A virus M2 protein as well as the influenza C virus HA-esterase-fusion glycoprotein were not associated with DIGs but were apically targeted. The reduced DIG association of HAt− and NAt− is an intrinsic property of the glycoproteins, as similar reductions in DIG association were observed when the proteins were expressed from cDNA. Examination of purified virions indicated reduced amounts of DIG-associated lipids in the envelope of HAt− and NAt− viruses. The data indicate that deletion of both the HA and NA cytoplasmic tails results in reduced DIG association and changes in both virus polypeptide and lipid composition. PMID:10775599

  20. Infection with street strain rabies virus induces modulation of the microRNA profile of the mouse brain

    PubMed Central

    2012-01-01

    Background Rabies virus (RABV) causes a fatal infection of the central nervous systems (CNS) of warm-blooded animals. Once the clinical symptoms develop, rabies is almost invariably fatal. The mechanism of RABV pathogenesis remains poorly understood. Recent studies have shown that microRNA (miRNA) plays an important role in the pathogenesis of viral infections. Our recent findings have revealed that infection with laboratory-fixed rabies virus strain can induce modulation of the microRNA profile of mouse brains. However, no previous report has evaluated the miRNA expression profile of mouse brains infected with RABV street strain. Results The results of microarray analysis show that miRNA expression becomes modulated in the brains of mice infected with street RABV. Quantitative real-time PCR assay of the differentially expressed miRNAs confirmed the results of microarray assay. Functional analysis showed the differentially expressed miRNAs to be involved in many immune-related signaling pathways, such as the Jak-STAT signaling pathway, the MAPK signaling pathway, cytokine-cytokine receptor interactions, and Fc gamma R-mediated phagocytosis. The predicted expression levels of the target genes of these modulated miRNAs were found to be correlated with gene expression as measured by DNA microarray and qRT-PCR. Conclusion RABV causes significant changes in the miRNA expression profiles of infected mouse brains. Predicted target genes of the differentially expression miRNAs are associated with host immune response, which may provide important information for investigation of RABV pathogenesis and therapeutic method. PMID:22882874

  1. Heminested reverse-transcriptase polymerase chain reaction (hnRT-PCR) as a tool for rabies virus detection in stored and decomposed samples

    PubMed Central

    Araújo, Danielle B; Langoni, Helio; Almeida, Marilene F; Megid, Jane

    2008-01-01

    Background The use of methods, both sensitive and specific, for rabies diagnosis are important tools for the control and prophylaxis of the disease. Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR) has been used in rabies diagnosis with good results, even in decomposed materials. Additionally, molecular techniques have been used for epidemiological studies and to gain a better knowledge of viral epidemiology. Findings The aim of this work was to evaluate the RT-PCR and hnRT-PCR for rabies virus detection in original tissues stored at -20°C for different periods considering their use for rabies virus detection in stored and decomposed samples. RT-PCR and hnRT-PCR were evaluated in 151 brain samples from different animal species, thawed and left at room temperature for 72 hours for decomposition. The RT-PCR and hnRT-PCR results were compared with previous results from Direct Fluorescent Antibody Test and Mouse Inoculation Test. From the 50 positive fresh samples, 26 (52%) were positive for RT-PCR and 45 (90%) for hnRT-PCR. From the 48 positive decomposed samples, 17 (34, 3%) were positive for RT-PCR and 36 (75%) for hnRT-PCR. No false-positives results were found in the negatives samples evaluated to the molecular techniques. Conclusion These results show that the hnRT-PCR was more sensitive than RT-PCR, and both techniques presented lower sensibility in decomposed samples. The hnRT-PCR demonstrated efficacy in rabies virus detection in stored and decomposed materials suggesting it's application for rabies virus retrospective epidemiological studies. PMID:18710536

  2. High-Throughput Differentiation and Screening of a Library of Mutant Stem Cell Clones Defines New Host-Based Genes Involved in Rabies Virus Infection.

    PubMed

    Wallis, Deeann; Loesch, Kimberly; Galaviz, Stacy; Sun, Qingan; DeJesus, Michael; Ioerger, Thomas; Sacchettini, James C

    2015-08-01

    We used a genomic library of mutant murine embryonic stem cells (ESCs) and report the methodology required to simultaneously culture, differentiate, and screen more than 3,200 heterozygous mutant clones to identify host-based genes involved in both sensitivity and resistance to rabies virus infection. Established neuronal differentiation protocols were miniaturized such that many clones could be handled simultaneously, and molecular markers were used to show that the resultant cultures were pan-neuronal. Next, we used a green fluorescent protein (GFP) labeled rabies virus to develop, validate, and implement one of the first host-based, high-content, high-throughput screens for rabies virus. Undifferentiated cell and neuron cultures were infected with GFP-rabies and live imaging was used to evaluate GFP intensity at time points corresponding to initial infection/uptake and early and late replication. Furthermore, supernatants were used to evaluate viral shedding potential. After repeated testing, 63 genes involved in either sensitivity or resistance to rabies infection were identified. To further explore hits, we used a completely independent system (siRNA) to show that reduction in target gene expression leads to the observed phenotype. We validated the immune modulatory gene Unc13d and the dynein adapter gene Bbs4 by treating wild-type ESCs and primary neurons with siRNA; treated cultures were resistant to rabies infection/replication. Overall, the potential of such in vitro functional genomics screens in stem cells adds additional value to other libraries of stem cells. This technique is applicable to any bacterial or virus interactome and any cell or tissue types that can be differentiated from ESCs.

  3. 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

  4. 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

  5. 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

  6. The Lyssavirus glycoprotein: A key to cross-immunity.

    PubMed

    Buthelezi, Sindisiwe G; Dirr, Heini W; Chakauya, Ereck; Chikwamba, Rachel; Martens, Lennart; Tsekoa, Tsepo L; Stoychev, Stoyan H; Vandermarliere, Elien

    2016-11-01

    Rabies is an acute viral encephalomyelitis in warm-blooded vertebrates, caused by viruses belonging to Rhabdovirus family and genus Lyssavirus. Although rabies is categorised as a neglected disease, the rabies virus (RABV) is the most studied amongst Lyssaviruses which show nearly identical infection patterns. In efforts to improving post-exposure prophylaxis, several anti-rabies monoclonal antibodies (mAbs) targeting the glycoprotein (G protein) sites I, II, III and G5 have been characterized. To explore cross-neutralization capacity of available mAbs and discover new possible B-cell epitopes, we have analyzed all available glycoprotein sequences from Lyssaviruses with a focus on sequence variation and conservation. This information was mapped on the structure of a representative G protein. We proposed several possible cross-neutralizing B-cell epitopes (GUVTTTF, WLRTV, REECLD and EHLVVEEL) in complement to the already well-characterized antigenic sites. The research could facilitate development of novel cross-reactive mAbs against RABV and even more broad, against possibly all Lyssavirus members. PMID:27614701

  7. Aberrant virion assembly and limited glycoprotein C production in varicella-zoster virus-infected neurons.

    PubMed

    Grose, Charles; Yu, Xiaoli; Cohrs, Randall J; Carpenter, John E; Bowlin, Jacqueline L; Gilden, Don

    2013-09-01

    Highly pure (>95%) terminally differentiated neurons derived from pluripotent stem cells appear healthy at 2 weeks after infection with varicella-zoster virus (VZV), and the cell culture medium contains no infectious virus. Analysis of the healthy-appearing neurons revealed VZV DNA, transcripts, and proteins corresponding to the VZV immediate early, early, and late kinetic phases of replication. Herein, we further characterized virus in these neuronal cells, focusing on (i) transcription and expression of late VZV glycoprotein C (gC) open reading frame 14 (ORF14) and (ii) ultrastructural features of virus particles in neurons. The analysis showed that gC was not expressed in most infected neurons and gC expression was markedly reduced in a minority of VZV-infected neurons. In contrast, expression of the early-late VZV gE glycoprotein (ORF68) was abundant. Transcript analysis also showed decreased gC transcription compared with gE. Examination of viral structure by high-resolution transmission electron microscopy revealed fewer viral particles than typically observed in cells productively infected with VZV. Furthermore, viral particles were more aberrant, in that most capsids in the nuclei lacked a dense core and most enveloped particles in the cytoplasm were light particles (envelopes without capsids). Together, these results suggest a considerable deficiency in late-phase replication and viral assembly during VZV infection of neurons in culture.

  8. 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

  9. Steric Shielding of Surface Epitopes and Impaired Immune Recognition Induced by the Ebola Virus Glycoprotein

    PubMed Central

    Francica, Joseph R.; Varela-Rohena, Angel; Medvec, Andrew; Plesa, Gabriela; Riley, James L.; Bates, Paul

    2010-01-01

    Many viruses alter expression of proteins on the surface of infected cells including molecules important for immune recognition, such as the major histocompatibility complex (MHC) class I and II molecules. Virus-induced downregulation of surface proteins has been observed to occur by a variety of mechanisms including impaired transcription, blocks to synthesis, and increased turnover. Viral infection or transient expression of the Ebola virus (EBOV) glycoprotein (GP) was previously shown to result in loss of staining of various host cell surface proteins including MHC1 and β1 integrin; however, the mechanism responsible for this effect has not been delineated. In the present study we demonstrate that EBOV GP does not decrease surface levels of β1 integrin or MHC1, but rather impedes recognition by steric occlusion of these proteins on the cell surface. Furthermore, steric occlusion also occurs for epitopes on the EBOV glycoprotein itself. The occluded epitopes in host proteins and EBOV GP can be revealed by removal of the surface subunit of GP or by removal of surface N- and O- linked glycans, resulting in increased surface staining by flow cytometry. Importantly, expression of EBOV GP impairs CD8 T-cell recognition of MHC1 on antigen presenting cells. Glycan-mediated steric shielding of host cell surface proteins by EBOV GP represents a novel mechanism for a virus to affect host cell function, thereby escaping immune detection. PMID:20844579

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

    PubMed

    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

    2016-02-01

    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.

  11. A Particle-Associated Glycoprotein Signal Peptide Essential for Virus Maturation and Infectivity

    PubMed Central

    Lindemann, Dirk; Pietschmann, Thomas; Picard-Maureau, Marcus; Berg, Angelika; Heinkelein, Martin; Thurow, Jana; Knaus, Petra; Zentgraf, Hanswalter; Rethwilm, Axel

    2001-01-01

    Signal peptides (SP) are key determinants for targeting glycoproteins to the secretory pathway. Here we describe the involvement in particle maturation as an additional function of a viral glycoprotein SP. The SP of foamy virus (FV) envelope glycoprotein is predicted to be unusually long. Using an SP-specific antiserum, we demonstrate that its proteolytic removal occurs posttranslationally by a cellular protease and that the major N-terminal cleavage product, gp18, is found in purified viral particles. Analysis of mutants in proposed signal peptidase cleavage positions and N-glycosylation sites revealed an SP about 148 amino acids (aa) in length. FV particle release from infected cells requires the presence of cognate envelope protein and cleavage of its SP sequence. An N-terminal 15-aa SP domain with two conserved tryptophan residues was found to be essential for the egress of FV particles. While the SP N terminus was found to mediate the specificity of FV Env to interact with FV capsids, it was dispensable for Env targeting to the secretory pathway and FV envelope-mediated infectivity of murine leukemia virus pseudotypes. PMID:11390578

  12. Partial protection by vaccination with recombinant feline immunodeficiency virus surface glycoproteins.

    PubMed

    Leutenegger, C M; Hofmann-Lehmann, R; Holznagel, E; Cuisinier, A M; Wolfensberger, C; Duquesne, V; Cronier, J; Allenspach, K; Aubert, A; Ossent, P; Lutz, H

    1998-02-10

    In an effort to induce a strong immune response that might protect against feline immunodeficiency virus (FIV) challenge infection, three groups of five specified pathogen-free (spf) cats each were immunized subcutaneously with different FIV antigen preparations. Immunizations were done at weeks 0, 2, and 4 with 100 microg of recombinant SU from an FIV Zurich 2 (FIV Z2) strain expressed by E. coli (group 1) or the baculovirus expression system (groups 2 and 3) adsorbed on aluminum hydroxyde and administered with QS-21 (groups 1 and 2) or Freund's adjuvant together with the recombinant nucleocapsid protein (protein NC) of rabies virus (group 3). Protein NC was described to act as an exogenous superantigen. Group 3 cats demonstrated the highest detectable antibody response to the vaccine antigen as determined by ELISA and Western blot analysis. All immunized cats together with seven control animals were challenged with 20 CID50 of cat lymphocyte-grown FIV Z2 3 weeks following the last immunization. Whereas virus was readily recovered from peripheral blood lymphocytes of seven of seven nonvaccinated control cats following this challenge dose, virus was not recovered from two cats of groups 1 and 2. All cats in groups 2 and 3 showed a provirus load significantly decreased to 3% of that of controls up to week 8 after challenge infection. Eleven of 15 vaccinated cats and 5 of 7 control cats developed virus-neutralizing antibodies by week 8 after challenge infection. The two cats negative on virus isolation remained seronegative, developed no detectable virus-neutralizing activities, but were repeatedly positive in provirus PCR. Moreover, starting at week 1 after challenge, both cats showed the lowest provirus load in their respective groups. These results indicate that immunization with recombinant FIV SU in conjunction with appropriate adjuvants may lead to partial protection against FIV challenge infection.

  13. Spatio-temporal Analysis of the Genetic Diversity of Arctic Rabies Viruses and Their Reservoir Hosts in Greenland.

    PubMed

    Hanke, Dennis; Freuling, Conrad M; Fischer, Susanne; Hueffer, Karsten; Hundertmark, Kris; Nadin-Davis, Susan; Marston, Denise; Fooks, Anthony R; Bøtner, Anette; Mettenleiter, Thomas C; Beer, Martin; Rasmussen, Thomas B; Müller, Thomas F; Höper, Dirk

    2016-07-01

    There has been limited knowledge on spatio-temporal epidemiology of zoonotic arctic fox rabies among countries bordering the Arctic, in particular Greenland. Previous molecular epidemiological studies have suggested the occurrence of one particular arctic rabies virus (RABV) lineage (arctic-3), but have been limited by a low number of available samples preventing in-depth high resolution phylogenetic analysis of RABVs at that time. However, an improved knowledge of the evolution, at a molecular level, of the circulating RABVs and a better understanding of the historical perspective of the disease in Greenland is necessary for better direct control measures on the island. These issues have been addressed by investigating the spatio-temporal genetic diversity of arctic RABVs and their reservoir host, the arctic fox, in Greenland using both full and partial genome sequences. Using a unique set of 79 arctic RABV full genome sequences from Greenland, Canada, USA (Alaska) and Russia obtained between 1977 and 2014, a description of the historic context in relation to the genetic diversity of currently circulating RABV in Greenland and neighboring Canadian Northern territories has been provided. The phylogenetic analysis confirmed delineation into four major arctic RABV lineages (arctic 1-4) with viruses from Greenland exclusively grouping into the circumpolar arctic-3 lineage. High resolution analysis enabled distinction of seven geographically distinct subclades (3.I - 3.VII) with two subclades containing viruses from both Greenland and Canada. By combining analysis of full length RABV genome sequences and host derived sequences encoding mitochondrial proteins obtained simultaneously from brain tissues of 49 arctic foxes, the interaction of viruses and their hosts was explored in detail. Such an approach can serve as a blueprint for analysis of infectious disease dynamics and virus-host interdependencies. The results showed a fine-scale spatial population structure in

  14. Spatio-temporal Analysis of the Genetic Diversity of Arctic Rabies Viruses and Their Reservoir Hosts in Greenland.

    PubMed

    Hanke, Dennis; Freuling, Conrad M; Fischer, Susanne; Hueffer, Karsten; Hundertmark, Kris; Nadin-Davis, Susan; Marston, Denise; Fooks, Anthony R; Bøtner, Anette; Mettenleiter, Thomas C; Beer, Martin; Rasmussen, Thomas B; Müller, Thomas F; Höper, Dirk

    2016-07-01

    There has been limited knowledge on spatio-temporal epidemiology of zoonotic arctic fox rabies among countries bordering the Arctic, in particular Greenland. Previous molecular epidemiological studies have suggested the occurrence of one particular arctic rabies virus (RABV) lineage (arctic-3), but have been limited by a low number of available samples preventing in-depth high resolution phylogenetic analysis of RABVs at that time. However, an improved knowledge of the evolution, at a molecular level, of the circulating RABVs and a better understanding of the historical perspective of the disease in Greenland is necessary for better direct control measures on the island. These issues have been addressed by investigating the spatio-temporal genetic diversity of arctic RABVs and their reservoir host, the arctic fox, in Greenland using both full and partial genome sequences. Using a unique set of 79 arctic RABV full genome sequences from Greenland, Canada, USA (Alaska) and Russia obtained between 1977 and 2014, a description of the historic context in relation to the genetic diversity of currently circulating RABV in Greenland and neighboring Canadian Northern territories has been provided. The phylogenetic analysis confirmed delineation into four major arctic RABV lineages (arctic 1-4) with viruses from Greenland exclusively grouping into the circumpolar arctic-3 lineage. High resolution analysis enabled distinction of seven geographically distinct subclades (3.I - 3.VII) with two subclades containing viruses from both Greenland and Canada. By combining analysis of full length RABV genome sequences and host derived sequences encoding mitochondrial proteins obtained simultaneously from brain tissues of 49 arctic foxes, the interaction of viruses and their hosts was explored in detail. Such an approach can serve as a blueprint for analysis of infectious disease dynamics and virus-host interdependencies. The results showed a fine-scale spatial population structure in

  15. Spatio-temporal Analysis of the Genetic Diversity of Arctic Rabies Viruses and Their Reservoir Hosts in Greenland

    PubMed Central

    Hanke, Dennis; Freuling, Conrad M.; Fischer, Susanne; Hueffer, Karsten; Hundertmark, Kris; Nadin-Davis, Susan; Marston, Denise; Fooks, Anthony R.; Bøtner, Anette; Mettenleiter, Thomas C.; Beer, Martin; Rasmussen, Thomas B.; Müller, Thomas F.; Höper, Dirk

    2016-01-01

    There has been limited knowledge on spatio-temporal epidemiology of zoonotic arctic fox rabies among countries bordering the Arctic, in particular Greenland. Previous molecular epidemiological studies have suggested the occurrence of one particular arctic rabies virus (RABV) lineage (arctic-3), but have been limited by a low number of available samples preventing in-depth high resolution phylogenetic analysis of RABVs at that time. However, an improved knowledge of the evolution, at a molecular level, of the circulating RABVs and a better understanding of the historical perspective of the disease in Greenland is necessary for better direct control measures on the island. These issues have been addressed by investigating the spatio-temporal genetic diversity of arctic RABVs and their reservoir host, the arctic fox, in Greenland using both full and partial genome sequences. Using a unique set of 79 arctic RABV full genome sequences from Greenland, Canada, USA (Alaska) and Russia obtained between 1977 and 2014, a description of the historic context in relation to the genetic diversity of currently circulating RABV in Greenland and neighboring Canadian Northern territories has been provided. The phylogenetic analysis confirmed delineation into four major arctic RABV lineages (arctic 1–4) with viruses from Greenland exclusively grouping into the circumpolar arctic-3 lineage. High resolution analysis enabled distinction of seven geographically distinct subclades (3.I – 3.VII) with two subclades containing viruses from both Greenland and Canada. By combining analysis of full length RABV genome sequences and host derived sequences encoding mitochondrial proteins obtained simultaneously from brain tissues of 49 arctic foxes, the interaction of viruses and their hosts was explored in detail. Such an approach can serve as a blueprint for analysis of infectious disease dynamics and virus-host interdependencies. The results showed a fine-scale spatial population structure

  16. Prevalence of neutralizing antibodies to rabies virus in serum of seven species of insectivorous bats from Colorado and New Mexico, United States

    USGS Publications Warehouse

    Bowen, Richard A.; O'Shea, Thomas J.; Shankar, Vidya; Neubaum, Melissa A.; Neubaum, Daniel J.; Rupprecht, Charles E.

    2013-01-01

    We determined the presence of rabies-virus-neutralizing antibodies (RVNA) in serum of 721 insectivorous bats of seven species captured, sampled, and released in Colorado and New Mexico, United States in 2003-2005. A subsample of 160 bats was tested for rabies-virus RNA in saliva. We sampled little brown bats (Myotis lucifugus) at two maternity roosts in Larimer County, Colorado; big brown bats (Eptesicus fuscus) at three maternity roosts in Morgan County, Colorado; and big brown bats at five maternity roosts in Larimer County. We also sampled hoary bats (Lasiurus cinereus) and silver-haired bats (Lasionycteris noctivagans) captured while drinking or foraging over water in Bernalillo County, New Mexico and at various locations in Larimer County. Big brown bats, little brown bats, long-legged myotis (Myotis volans), long-eared myotis (Myotis evotis), and fringed myotis (Myotis thysanodes) were also sampled over water in Larimer County. All species except long-eared myotis included individuals with RVNA, with prevalences ranging from 7% in adult female silver-haired bats to 32% in adult female hoary bats. None of the bats had detectable rabies-virus RNA in oropharyngeal swabs, including 51 bats of 5 species that had RVNA in serum. Antibody-positive bats were present in nine of the 10 maternity colonies sampled. These data suggest that wild bats are commonly exposed to rabies virus and develop a humoral immune response suggesting some degree of viral replication, but many infections fail to progress to clinical disease.

  17. Complete Genome Sequence of a Rabies Virus Strain Isolated from a Brown Bear (Ursus arctos) in Primorsky Krai, Russia (November 2014)

    PubMed Central

    Deviatkin, Andrei A.; Ananiev, Vasily Y.; Dedkov, Vladimir G.; Shipulin, German A.; Sokol, Nataliya N.; Dombrovskaya, Irina E.; Galkina, Irina V.; Shmelev, Mikhail E.; Gorelikov, Vladimir N.; Kozhan, Valentina N.; Prosyannikova, Marina N.; Aramilev, Sergei V.; Fomenko, Pavel V.

    2016-01-01

    We report here the complete genome sequence (GenBank KP997032) of rabies virus strain RABV/Ursus arctos/Russia/Primorye/PO-01/2014, isolated in November 2014 from a brown bear (Ursus arctos) that attacked a person in Primorsky Krai (Russian Federation). This strain was clustered into the Eurasian genetic subgroup of genotype 1 (street rage). PMID:27389270

  18. Complete Genome Sequence of a Rabies Virus Strain Isolated from a Brown Bear (Ursus arctos) in Primorsky Krai, Russia (November 2014).

    PubMed

    Shchelkanov, Michael Y; Deviatkin, Andrei A; Ananiev, Vasily Y; Dedkov, Vladimir G; Shipulin, German A; Sokol, Nataliya N; Dombrovskaya, Irina E; Galkina, Irina V; Shmelev, Mikhail E; Gorelikov, Vladimir N; Kozhan, Valentina N; Prosyannikova, Marina N; Aramilev, Sergei V; Fomenko, Pavel V

    2016-01-01

    We report here the complete genome sequence (GenBank KP997032) of rabies virus strain RABV/Ursus arctos/Russia/Primorye/PO-01/2014, isolated in November 2014 from a brown bear (Ursus arctos) that attacked a person in Primorsky Krai (Russian Federation). This strain was clustered into the Eurasian genetic subgroup of genotype 1 (street rage). PMID:27389270

  19. Herpes simplex virus 1 glycoprotein M and the membrane-associated protein UL11 are required for virus-induced cell fusion and efficient virus entry.

    PubMed

    Kim, In-Joong; Chouljenko, Vladimir N; Walker, Jason D; Kousoulas, Konstantin G

    2013-07-01

    Herpes simplex virus 1 (HSV-1) facilitates virus entry into cells and cell-to-cell spread by mediating fusion of the viral envelope with cellular membranes and fusion of adjacent cellular membranes. Although virus strains isolated from herpetic lesions cause limited cell fusion in cell culture, clinical herpetic lesions typically contain large syncytia, underscoring the importance of cell-to-cell fusion in virus spread in infected tissues. Certain mutations in glycoprotein B (gB), gK, UL20, and other viral genes drastically enhance virus-induced cell fusion in vitro and in vivo. Recent work has suggested that gB is the sole fusogenic glycoprotein, regulated by interactions with the viral glycoproteins gD, gH/gL, and gK, membrane protein UL20, and cellular receptors. Recombinant viruses were constructed to abolish either gM or UL11 expression in the presence of strong syncytial mutations in either gB or gK. Virus-induced cell fusion caused by deletion of the carboxyl-terminal 28 amino acids of gB or the dominant syncytial mutation in gK (Ala to Val at amino acid 40) was drastically reduced in the absence of gM. Similarly, syncytial mutations in either gB or gK did not cause cell fusion in the absence of UL11. Neither the gM nor UL11 gene deletion substantially affected gB, gC, gD, gE, and gH glycoprotein synthesis and expression on infected cell surfaces. Two-way immunoprecipitation experiments revealed that the membrane protein UL20, which is found as a protein complex with gK, interacted with gM while gM did not interact with other viral glycoproteins. Viruses produced in the absence of gM or UL11 entered into cells more slowly than their parental wild-type virus strain. Collectively, these results indicate that gM and UL11 are required for efficient membrane fusion events during virus entry and virus spread.

  20. 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.

  1. Crystal structure of west nile virus envelope glycoprotein reveals viral surface epitopes.

    PubMed

    Kanai, Ryuta; Kar, Kalipada; Anthony, Karen; Gould, L Hannah; Ledizet, Michel; Fikrig, Erol; Marasco, Wayne A; Koski, Raymond A; Modis, Yorgo

    2006-11-01

    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.

  2. 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.

  3. Glycoprotein D protects mice against lethal challenge with herpes simplex virus types 1 and 2.

    PubMed Central

    Long, D; Madara, T J; Ponce de Leon, M; Cohen, G H; Montgomery, P C; Eisenberg, R J

    1984-01-01

    Glycoprotein D is a virion envelope component of herpes simplex virus types 1 and 2. Sets of mice were immunized with purified gD-1 or gD-2 and were challenged with a lethal dose of herpes simple virus, either type 1 or type 2. All or virtually all of the immunized mice survived challenge with either agent, whereas challenge of sham-immunized mice was almost always fatal. Serum samples taken before challenge contained gD-specific antibodies which had 50% neutralization titers ranging from 1:16 to 1:512 against homologous and heterologous virus types. We conclude that either gD-1 or gD-2 is a potential candidate for a subunit vaccine against herpetic infections. Images PMID:6319291

  4. Immunization with DNA vaccines encoding glycoprotein D or glycoprotein B, alone or in combination, induces protective immunity in animal models of herpes simplex virus-2 disease.

    PubMed

    McClements, W L; Armstrong, M E; Keys, R D; Liu, M A

    1996-10-15

    DNA vaccines expressing herpes simplex virus type 2 (HSV-2) full-length glycoprotein D (gD), or a truncated form of HSV-2 glycoprotein B (gB) were evaluated for protective efficacy in two experimental models of HSV-2 infection. Intramuscular (i.m.) injection of mice showed that each construction induced neutralizing serum antibodies and protected the mice from lethal HSV-2 infection. Dose-titration studies showed that low doses (< or = 1 microgram) of either DNA construction induced protective immunity, and that a single immunization with the gD construction was effective. The two DNAs were then tested in a low-dosage combination in guinea pigs. Immune sera from DNA-injected animals had antibodies to both gD and gB, and virus neutralizing activity. When challenged by vaginal infection with HSV-2, the DNA-immunized animals were significantly protected from primary genital disease.

  5. 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

  6. 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.

  7. Cross-protection conferred by filovirus virus-like particles containing trimeric hybrid glycoprotein.

    PubMed

    Martins, Karen; Carra, John H; Cooper, Christopher L; Kwilas, Steven A; Robinson, Camenzind G; Shurtleff, Amy C; Schokman, Rowena D; Kuehl, Kathleen A; Wells, Jay B; Steffens, Jesse T; van Tongeren, Sean A; Hooper, Jay W; Bavari, Sina

    2015-02-01

    Filoviruses are causative agents of hemorrhagic fever, and to date no effective vaccine or therapeutic has been approved to combat infection. Filovirus glycoprotein (GP) is the critical immunogenic component of filovirus vaccines, eliciting high levels of antibody after successful vaccination. Previous work has shown that protection against both Ebola virus (EBOV) and Marburg virus (MARV) can be achieved by vaccinating with a mixture of virus-like particles (VLPs) expressing either EBOV GP or MARV GP. In this study, the potential for eliciting effective immune responses against EBOV, Sudan virus, and MARV with a single GP construct was tested. Trimeric hybrid GPs were produced that expressed the sequence of Marburg GP2 in conjunction with a hybrid GP1 composed EBOV and Sudan virus GP sequences. VLPs expressing these constructs, along with EBOV VP40, provided comparable protection against MARV challenge, resulting in 75 or 100% protection. Protection from EBOV challenge differed depending upon the hybrid used, however, with one conferring 75% protection and one conferring no protection. By comparing the overall antibody titers and the neutralizing antibody titers specific for each virus, it is shown that higher antibody responses were elicited by the C terminal region of GP1 than by the N terminal region, and this correlated with protection. These data collectively suggest that GP2 and the C terminal region of GP1 are highly immunogenic, and they advance progress toward the development of a pan-filovirus vaccine.

  8. Demyelination determinants map to the spike glycoprotein gene of coronavirus mouse hepatitis virus.

    PubMed

    Das Sarma, J; Fu, L; Tsai, J C; Weiss, S R; Lavi, E

    2000-10-01

    Demyelination is the pathologic hallmark of the human immune-mediated neurologic disease multiple sclerosis, which may be triggered or exacerbated by viral infections. Several experimental animal models have been developed to study the mechanism of virus-induced demyelination, including coronavirus mouse hepatitis virus (MHV) infection in mice. The envelope spike (S) glycoprotein of MHV contains determinants of properties essential for virus-host interactions. However, the molecular determinants of MHV-induced demyelination are still unknown. To investigate the mechanism of MHV-induced demyelination, we examined whether the S gene of MHV contains determinants of demyelination and whether demyelination is linked to viral persistence. Using targeted RNA recombination, we replaced the S gene of a demyelinating virus (MHV-A59) with the S gene of a closely related, nondemyelinating virus (MHV-2). Recombinant viruses containing an S gene derived from MHV-2 in an MHV-A59 background (Penn98-1 and Penn98-2) exhibited a persistence-positive, demyelination-negative phenotype. Thus, determinants of demyelination map to the S gene of MHV. Furthermore, viral persistence is insufficient to induce demyelination, although it may be a prerequisite for the development of demyelination.

  9. Site-directed ELISA identifies a highly antigenic region of the simian immunodeficiency virus transmembrane glycoprotein.

    PubMed

    Johnson, P R; Parks, D E; Norrby, E; Lerner, R A; Purcell, R H; Chanock, R M

    1988-06-01

    The transmembrane glycoprotein (gp32) of the simian immunodeficiency virus (SIV) contains a highly antigenic region that includes amino acid residues 606-628. A synthetic peptide representing this region was highly immunoreactive with sera from SIV-infected primates in a site-directed enzyme-linked immunosorbent assay (ELISA). This reactivity extended across four primate species from three genera and identified infection with at least two distinct isolates of SIV. This site-directed ELISA represents a simple, accessible method with broad specificity for screening large numbers of primates for antibodies against SIV.

  10. 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.

  11. Comparison of primary skunk brain and kidney and raccoon kidney cells with established cell lines for isolation and propagation of street rabies virus.

    PubMed

    Umoh, J U; Blenden, D C

    1983-09-01

    Cell cultures prepared from skunk kidney, raccoon kidney, and skunk brain were compared with CER, murine neuroblastoma (C1300, clone NA), baby hamster kidney (BHK-21, S-13), and dog kidney (MDCK) cell lines for virus isolation and propagation of street and fixed rabies virus. The skunk brain cells were suitable for efficient replication of all the virus isolates. They were comparable to CER and murine neuroblastoma cells for virus isolation and propagation. None of the other cell cultures was satisfactory. Further work is under way to refine the skunk brain cell cultures.

  12. Skunk rabies surveillance in Illinois.

    PubMed

    Barllett, P C; Martin, R J

    1982-06-15

    Surveillance data indicated that an increased incidence of skunk rabies in Illinois during 1979-1980 was not attributable to increased reporting or submission of skunks to the state laboratories for rabies examination. Available road-kill data suggested that the skunk population increased prior to the increase in skunk rabies incidence. An increased skunk population was hypothesized to have caused the increased incidence by facilitating transmission of the virus through increased skunk density. Analysis of the temporal distribution of skunk rabies revealed a bimodal seasonal cycle, with peak occurrence in the spring and fall, and the possible beginning of a secular cycle, with peak incidence every 6 to 8 years.

  13. 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...

  14. The co-chaperone Cdc37 regulates the rabies virus phosphoprotein stability by targeting to Hsp90AA1 machinery

    PubMed Central

    Xu, Yunbin; Liu, Fei; Liu, Juan; Wang, Dandan; Yan, Yan; Ji, Senlin; Zan, Jie; Zhou, Jiyong

    2016-01-01

    Cdc37, as a kinase-specific co-chaperone of the chaperone Hsp90AA1 (Hsp90), actively aids with the maturation, stabilization and activation of the cellular or viral kinase/kinase-like targets. Phosphoprotein (P) of rabies virus (RABV) is a multifunctional, non-kinase protein involved in interferon antagonism, viral transcription and replication. Here, we demonstrated that the RABV non-kinase P is chaperoned by Cdc37 and Hsp90 during infection. We found that Cdc37 and Hsp90 affect the RABV life cycle directly. Activity inhibition and knockdown of Cdc37 and Hsp90 increased the instability of the viral P protein. Overexpression of Cdc37 and Hsp90 maintained P’s stability but did not increase the yield of infectious RABV virions. We further demonstrated that the non-enzymatic polymerase cofactor P protein of all the genotypes of lyssaviruses is a target of the Cdc37/Hsp90 complex. Cdc37, phosphorylated or unphosphorylated on Ser13, aids the P protein to load onto the Hsp90 machinery, with or without Cdc37 binding to Hsp90. However, the interaction between Cdc37 and Hsp90 appears to have additional allosteric regulation of the conformational switch of Hsp90. Our study highlighted a novel mechanism in which Cdc37/Hsp90 chaperones a non-kinase target, which has significant implications for designing therapeutic targets against Rabies. PMID:27251758

  15. Critical Role of K1685 and K1829 in the Large Protein of Rabies Virus in Viral Pathogenicity and Immune Evasion

    PubMed Central

    Tian, Dayong; Luo, Zhaochen; Zhou, Ming; Li, Mingming; Yu, Lan; Wang, Chong; Yuan, Jiaolong; Li, Fang; Tian, Bin; Sui, Baokun; Chen, Huanchun

    2015-01-01

    ABSTRACT Rabies, one of the oldest infectious diseases, still presents a public health threat in most parts of the world today. Its pathogen, rabies virus (RABV), can utilize its viral proteins, such as the nucleoprotein and phosphorylation protein, to subvert the host innate immune system. For a long time, the large (L) protein was believed to be essential for RABV transcription and replication, but its role in viral pathogenicity and immune evasion was not known. Recent studies have found that the conserved K-D-K-E tetrad motif in the L protein is related to the methyltransferase (MTase) activity in the viral mRNA process. In the present study, a series of RABV mutations in this motif was constructed with the recombinant CVS-B2c (rB2c) virus. Two of these mutants, rB2c-K1685A and rB2c-K1829A, were found to be stable and displayed an attenuated phenotype in both in vitro growth and in vivo pathogenicity in adult and suckling mice. Further studies demonstrated that these two mutants were more sensitive to the expression of the interferon-stimulated gene product IFIT2 than the parent virus. Taken together, our results suggest that K1685 and K1829 in the L protein play important roles in pathogenicity and immune evasion during RABV infection. IMPORTANCE Rabies continues to present a public health threat in most areas of the world, especially in the developing countries of Asia and Africa. The pathogenic mechanisms for rabies are not well understood. In the present study, it was found that the recombinant rabies viruses rB2c-K1685A and rB2c-K1829A, carrying mutations at the predicted MTase catalytic sites in the L protein, were highly attenuated both in vitro and in vivo. Further studies showed that these mutants were more sensitive to the expression of the interferon-stimulated gene product IFIT2 than the parent virus. These findings improve our understanding of rabies pathogenesis, which may help in developing potential therapeutics and an avirulent rabies vaccine

  16. Roles of bovine viral diarrhea virus envelope glycoproteins in inducing autophagy in MDBK cells.

    PubMed

    Fu, Qiang; Shi, Huijun; Shi, Mengting; Meng, Luping; Bao, Haiyang; Zhang, Guoqi; Ren, Yan; Zhang, Hui; Guo, Fei; Qiao, Jun; Jia, Bin; Wang, Pengyan; Ni, Wei; Sheng, Jinliang; Chen, Chuangfu

    2014-11-01

    Macroautophagy (autophagy) is an evolutionarily conserved control process that maintains cellular homeostasis in eukaryotic cells. Autophagy principally serves an adaptive role to degrade dysfunctional proteins and to clean damaged organelles in response to pathogenic, viral, or microbial infection, nutrient deprivation and endoplasmic reticulum (ER) stress. In previous study, we showed bovine viral diarrhea virus (BVDV) NADL infection induced autophagy and significantly elevated the expression levels of autophagy-related genes, Beclin1 and ATG14, at 12 h post-infection in MDBK cells. However, the specific mechanisms involved in controlling autophagic activity remain unclear. Here, we investigate the effects of BVDV NADL envelope glycoproteins overexpression on inducing autophagy. The results show that viral envelope glycoproteins E(rns) and E2 overexpression mediated by lentivirus increase the formation of autophagosome, the percentage of GFP-LC3 puncta-positive cells and the expression levels of Beclin1 and ATG14. Whereas E1 overexpression doesn't affect autophagic activity. Collectively, these findings suggest that the viral envelope glycoproteins E(rns) and E2 are involved in inducing autophagy, and provide a mechanistic insight into the regulation of autophagy in viral infected cells.

  17. Opposite polarity of virus budding and of viral envelope glycoprotein distribution in epithelial cells derived from different tissues

    PubMed Central

    1992-01-01

    We compared the surface envelope glycoprotein distribution and the budding polarity of four RNA viruses in Fischer rat thyroid (FRT) cells and in CaCo-2 cells derived from a human colon carcinoma. Whereas both FRT and CaCo-2 cells sort similarly influenza hemagglutinin and vesicular stomatitis virus (VSV) G protein, respectively, to apical and basolateral membrane domains, they differ in their handling of two togaviruses, Sindbis and Semliki Forest virus (SFV). By conventional EM Sindbis virus and SFV were shown to bud apically in FRT cells and basolaterally in CaCo-2 cells. Consistent with this finding, the distribution of the p62/E2 envelope glycoprotein of SFV, assayed by immunoelectronmicroscopy and by domain-selective surface biotinylation was predominantly apical on FRT cells and basolateral on CaCo-2 cells. We conclude that a given virus and its envelope glycoprotein can be delivered to opposite membrane domains in epithelial cells derived from different tissues. The tissue specificity in the polarity of virus budding and viral envelope glycoprotein distribution indicate that the sorting machinery varies considerably between different epithelial cell types. PMID:1572895

  18. 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.

  19. Development of infectious clones of a wild-type Korean rabies virus and evaluation of their pathogenic potential.

    PubMed

    Park, Jun-Sun; Kim, Chi-Kyeong; Um, Ji-Hye; Ju, Young Ran; Lee, Yeong Seon; Choi, Young-Ki; Kim, Su Yeon

    2016-09-01

    Most reverse genetic (RG) systems for rabies viruses (RVs) have been constructed on the genome background of laboratory-adapted strains. In this study, we developed an RG system using a Korean wild type (KGH) strain to investigate the pathogenic potential of different strains. We developed a RG system with the KGH strain for the first time. Following the complete genome sequencing of the KGH strain, pKGH infectious clones were constructed using the CMV/T7 promoter, and HamRz and HdvRz were introduced to allow self-cleavage of the synthesized RNA. We successfully recovered the rescued virus by constructing chimeric RVs in which we replaced a part of the construct with the partial gene from the fixed RC-HL strain. The rescued viruses formed clearer and countable plaques in an immunostaining plaque assay, with a distinct plaque morphology. Furthermore, compared with the chimeric RVs, the pKGH/RCinsΔ4 strain containing the KGH strain G protein exhibited a decreased efficiency of cell-to-cell spreading in BHK-21 cells and significantly reduced (100-1000 fold) replication kinetics. However, pKGH/RCinsΔ4 strain-infected mice revealed 100% morbidity at 11days post-infection, whereas other chimeric RV strains showed no mortality. Our RG system is a useful tool for studying differences in the cell-to-cell spreading efficiency and replication with respect to the different internalization patterns of street and fixed laboratory-adapted viruses. PMID:27397101

  20. Effect of nicotinic acetylcholine receptor alpha 1 (nAChRα1) peptides on rabies virus infection in neuronal cells.

    PubMed

    Sajjanar, Basavaraj; Saxena, Shikha; Bisht, Deepika; Singh, Arvind Kumar; Manjunatha Reddy, G B; Singh, Rajendra; Singh, R P; Kumar, Satish

    2016-06-01

    Rabies virus (RABV) is neurotropic and causes acute progressive encephalitis. Herein, we report the interaction of nAChRα1-subunit peptides with RABV and the effect of these peptides on RABV infection in cultured neuronal cells. Peptide sequences derived from torpedo, bovine, human and rats were synthesized and studied for their interactions with RABV using virus capture ELISA and peptide immunofluorescence. The results showed specific binding of the nAChRα1-subunit peptides to the RABV. In the virus adsorption assay, these peptides were found to inhibit the attachment of the RABV to the neuronal cells. The nAChRα1-subunit peptides inhibited the RABV infection and reduced viral gene expression in the cultured neuroblastoma (N2A) cells. Torpedo peptide sequence (T-32) had highest antiviral effect (IC50=14±3.01μM) compared to the other peptides studied. The results of the study indicated that nAChRα1-subunit peptides may act as receptor decoy molecules and inhibit the binding of virus to the native host cell receptors and hence may reduce viral infection. PMID:26656837

  1. Rabies Vaccine

    MedlinePlus

    ... common source of human rabies infection in the United States. Skunks, raccoons, dogs, and cats can also transmit the disease.Human rabies is rare in the United States. There have been only 55 cases diagnosed since ...

  2. 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.

  3. Human carcinoembryonic antigen and biliary glycoprotein can serve as mouse hepatitis virus receptors.

    PubMed Central

    Chen, D S; Asanaka, M; Chen, F S; Shively, J E; Lai, M M

    1997-01-01

    Receptors for murine coronavirus mouse hepatitis virus (MHV) are members of the murine carcinoembryonic antigen (CEA) gene family. Since MHV can also infect primates and cause central nervous system lesions (G. F. Cabirac et al., Microb. Pathog. 16:349-357, 1994; R. S. Murray et al., Virology 188:274-284, 1992), we examined whether human CEA-related molecules can be used by MHV as potential receptors. Transfection of plasmids expressing human carcinoembryonic antigen (hCEA) and human biliary glycoprotein into COS-7 cells, which lack a functional MHV receptor, conferred susceptibility to two MHV strains, A59 and MHV-2. Domain exchange experiments between human and murine CEA-related molecules identified the immunoglobulin-like loop I of hCEA as the region conferring the virus-binding specificity. This finding expands the potential MHV receptors to primate species. PMID:8995701

  4. Molecular association of herpes simplex virus type 1 glycoprotein E with membrane protein Us9.

    PubMed

    Awasthi, Sita; Friedman, Harvey M

    2016-11-01

    Herpes simplex virus type 1 (HSV-1) glycoprotein E (gE), glycoprotein I (gI), and Us9 promote efficient anterograde axonal transport of virus from the neuron cytoplasm to the axon terminus. HSV-1 and PRV gE and gI form a heterodimer that is required for anterograde transport, but an association that includes Us9 has not been demonstrated. NS-gE380 is an HSV-1 mutant that has five amino acids inserted after gE residue 380, rendering it defective in anterograde axonal transport. We demonstrated that gE, gI and Us9 form a trimolecular complex in Vero cells infected with NS-gE380 virus in which gE binds to both Us9 and gI. We detected the complex using immunoprecipitation with anti-gE or anti-gI monoclonal antibodies in the presence of ionic detergents. Under these conditions, Us9 did not associate with gE in cells infected with wild-type HSV-1; however, using a nonionic detergent, TritonX-100, an association between Us9 and gE was detected in immunoprecipitates of both wild-type and NS-gE380-infected cells. The results suggest that the interaction between Us9 and gE is weak and disrupted by ionic detergents in wild-type infected cells. We postulate that the tight interaction between Us9 and gE leads to the anterograde spread defect in the NS-gE380 virus. PMID:27568015

  5. An analysis of correspondence between unique rabies virus variants and divergent big brown bat (Eptesicus fuscus) mitochondrial DNA lineages

    USGS Publications Warehouse

    Neubaum, M.A.; Shankar, V.; Douglas, M.R.; Douglas, M.E.; O'Shea, T.J.; Rupprecht, C.E.

    2008-01-01

    The literature supports that unique rabies virus (RABV) variants are often compartmentalized in different species of bats. In Colorado, two divergent mtDNA lineages of big brown bats (Eptesicus fuscus) co-occur. RABV associated with this species also segregates into two clades. We hypothesized that unique RABV variants might be associated with mtDNA lineages of Colorado big brown bats. DNA was extracted from brain tissue of rabid big brown bats, the ND2 gene was amplified to determine mtDNA lineage, and the lineage was compared to a previously derived phylogenetic analysis of the RABV N gene. No correspondence was found between host bat lineage and RABV variant. ?? 2008 Springer-Verlag.

  6. 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.

  7. Improving immunogenicity and efficacy of vaccines for genital herpes containing herpes simplex virus glycoprotein D.

    PubMed

    Awasthi, Sita; Shaw, Carolyn; Friedman, Harvey

    2014-12-01

    No vaccines are approved for prevention or treatment of genital herpes. The focus of genital herpes vaccine trials has been on prevention using herpes simplex virus type 2 (HSV-2) glycoprotein D (gD2) alone or combined with glycoprotein B. These prevention trials did not achieve their primary end points. However, subset analyses reported some positive outcomes in each study. The most recent trial was the Herpevac Trial for Women that used gD2 with monophosphoryl lipid A and alum as adjuvants in herpes simplex virus type 1 (HSV-1) and HSV-2 seronegative women. Unexpectedly, the vaccine prevented genital disease by HSV-1 but not HSV-2. Currently, HSV-1 causes more first episodes of genital herpes than HSV-2, highlighting the importance of protecting against HSV-1. The scientific community is conflicted between abandoning vaccine efforts that include gD2 and building upon the partial successes of previous trials. We favor building upon success and present approaches to improve outcomes of gD2-based subunit antigen vaccines.

  8. Evaluation of Measles Vaccine Virus as a Vector to Deliver Respiratory Syncytial Virus Fusion Protein or Epstein-Barr Virus Glycoprotein gp350

    PubMed Central

    Mok, Hoyin; Cheng, Xing; Xu, Qi; Zengel, James R; Parhy, Bandita; Zhao, Jackie; Wang, C. Kathy; Jin, Hong

    2012-01-01

    Live attenuated recombinant measles vaccine virus (MV) Edmonston-Zagreb (EZ) strain was evaluated as a viral vector to express the ectodomains of fusion protein of respiratory syncytial virus (RSV F) or glycoprotein 350 of Epstein-Barr virus (EBV gp350) as candidate vaccines for prophylaxis of RSV and EBV. The glycoprotein gene was inserted at the 1st or the 3rd position of the measles virus genome and the recombinant viruses were generated. Insertion of the foreign gene at the 3rd position had a minimal impact on viral replication in vitro. RSV F or EBV gp350 protein was secreted from infected cells. In cotton rats, EZ-RSV F and EZ-EBV gp350 induced MV- and insert-specific antibody responses. In addition, both vaccines also induced insert specific interferon gamma (IFN-γ) secreting T cell response. EZ-RSV F protected cotton rats from pulmonary replication of RSV A2 challenge infection. In rhesus macaques, although both EZ-RSV F and EZ-EBV gp350 induced MV specific neutralizing antibody responses, only RSV F specific antibody response was detected. Thus, the immunogenicity of the foreign antigens delivered by measles vaccine virus is dependent on the nature of the insert and the animal models used for vaccine evaluation. PMID:22383906

  9. A Systematic Review of Human Bat Rabies Virus Variant Cases: Evaluating Unprotected Physical Contact with Claws and Teeth in Support of Accurate Risk Assessments

    PubMed Central

    Campagnolo, Enzo R.; Long, Jonah; Rupprecht, Charles E.

    2016-01-01

    In the United States and Canada, the most recent documented cases of rabies have been attributed to bat rabies viruses (RABV). We undertook this systematic review in an effort to summarize and enhance understanding of the risk of infection for individuals who have been potentially exposed to a suspect or confirmed rabid bat. United States rabies surveillance summaries documented a total of 41 human bat-rabies virus variant verified non-transplant cases between 1990 and 2015. All cases were fatal. Seven (17.1%) of 41 cases reported a bite from a bat. Ten (24.3%) cases had unprotected physical contact (UPC); these included seven cases that had a bat land or crawl on them (contact with claws) and one case that touched a bat’s teeth. Seven (17.1%) cases had probable UPC. Insectivorous bat teeth are extremely sharp and highly efficient for predation upon arthropod prey. Bats also have sharp claws on the end of their thumbs and feet. One of the most common bat RABV variants has an ability to replicate in non-neural cells. Questioning individuals about unprotected contact with bat teeth and claws (including a bat landing or crawling on a person) may help identify additional exposures. PMID:27459720

  10. A Systematic Review of Human Bat Rabies Virus Variant Cases: Evaluating Unprotected Physical Contact with Claws and Teeth in Support of Accurate Risk Assessments.

    PubMed

    Dato, Virginia M; Campagnolo, Enzo R; Long, Jonah; Rupprecht, Charles E

    2016-01-01

    In the United States and Canada, the most recent documented cases of rabies have been attributed to bat rabies viruses (RABV). We undertook this systematic review in an effort to summarize and enhance understanding of the risk of infection for individuals who have been potentially exposed to a suspect or confirmed rabid bat. United States rabies surveillance summaries documented a total of 41 human bat-rabies virus variant verified non-transplant cases between 1990 and 2015. All cases were fatal. Seven (17.1%) of 41 cases reported a bite from a bat. Ten (24.3%) cases had unprotected physical contact (UPC); these included seven cases that had a bat land or crawl on them (contact with claws) and one case that touched a bat's teeth. Seven (17.1%) cases had probable UPC. Insectivorous bat teeth are extremely sharp and highly efficient for predation upon arthropod prey. Bats also have sharp claws on the end of their thumbs and feet. One of the most common bat RABV variants has an ability to replicate in non-neural cells. Questioning individuals about unprotected contact with bat teeth and claws (including a bat landing or crawling on a person) may help identify additional exposures. PMID:27459720

  11. 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.

  12. Development of an edible rabies vaccine in maize using the Vnukovo strain.

    PubMed

    Loza-Rubio, E; Rojas, E; Gómez, L; Olivera, M T J; Gómez-Lim, M A

    2008-01-01

    The objective of this study was to obtain transgenic maize expressing the rabies virus glycoprotein (G) of the Vnukovo strain and to evaluate its immunogenicity in mice, by the oral route. The ubiquitin maize promoter fused to the whole coding region of the rabies virus G gene, and a constitutive promoter from cauliflowermosaic virus (CaMV)were used. Maize embryogenic callus were transformed with the above construct by biolistics. Regenerated maize plants were recovered and grown in a greenhouse. The presence of the G gene and its product was detected by PCR and western blot, respectively. The amount of G protein detected in the grains was approximately 1% of the total soluble plant protein. Transformed kernels containing 50 microg of G protein were given once by the oral route in adult mice (BALB-C strain). Challenge was undertaken at 90-days post-vaccination using a lethal dose of a vampire bat rabies virus (100 LD 50% in mice); vampire bats are one of the main reservoirs in Latin America. The edible vaccine induced viral neutralizing antibodies (VNA) which, protected mice 100% against challenge. The control group did not survive. The G protein of the Vnukovo strain expressed in transgenic maize may be considered as an oral immunogen against rabies, conferring cross-protection. PMID:18634510

  13. Microarray for Identification of the Chiropteran Host Species of Rabies Virus in Canada

    PubMed Central

    Lung, Oliver; Nadin-Davis, Susan; Fisher, Mathew; Erickson, Anthony; Knowles, M. Kimberly; Furukawa-Stoffer, Tara; Ambagala, Aruna

    2013-01-01

    Species identification through genetic barcoding can augment traditional taxonomic methods, which rely on morphological features of the specimen. Such approaches are especially valuable when specimens are in poor condition or comprise very limited material, a situation that often applies to chiropteran (bat) specimens submitted to the Canadian Food Inspection Agency for rabies diagnosis. Coupled with phenotypic plasticity of many species and inconclusive taxonomic keys, species identification using only morphological traits can be challenging. In this study, a microarray assay with associated PCR of the mitochondrial cytochrome c oxidase subunit I (COI) gene was developed for differentiation of 14 bat species submitted to the Canadian Food Inspection Agency from 1985–2012 for rabies diagnosis. The assay was validated with a reference collection of DNA from 153 field samples, all of which had been barcoded previously. The COI gene from 152 samples which included multiple specimens of each target species were successfully amplified by PCR and accurately identified by the microarray. One sample that was severely decomposed failed to amplify with PCR primers developed in this study, but amplified weakly after switching to alternate primers and was accurately typed by the microarray. Thus, the chiropteran microarray was able to accurately differentiate between the 14 species of Canadian bats targeted. This PCR and microarray assay would allow unequivocal identification to species of most, if not all, bat specimens submitted for rabies diagnosis in Canada. PMID:27605186

  14. Binding of rabies virus polymerase cofactor to recombinant circular nucleoprotein-RNA complexes.

    PubMed

    Ribeiro, Euripedes de Almeida; Leyrat, Cédric; Gérard, Francine C A; Albertini, Aurélie A V; Falk, Caroline; Ruigrok, Rob W H; Jamin, Marc

    2009-12-01

    In rabies virus, the attachment of the L polymerase (L) to the viral nucleocapsids (NCs)-a nucleoprotein (N)-RNA complex that serves as template for RNA transcription and replication-is mediated by the polymerase cofactor, the phosphoprotein (P). P forms dimers (P(2)) that bind through their C-terminal domains (P(CTD)) to the C-terminal region of the N. Recombinant circular N(m)-RNA complexes containing 9 to 12 protomers of N (hereafter, the subscript m denotes the number of N protomers) served here as model systems for studying the binding of P to NC-like N(m)-RNA complexes. Titration experiments show that there are only two equivalent and independent binding sites for P dimers on the N(m)-RNA rings and that each P dimer binds through a single P(CTD). A dissociation constant in the nanomolar range (160+/-20 nM) was measured by surface plasmon resonance, indicating a strong interaction between the two partners. Small-angle X-ray scattering (SAXS) data and small-angle neutron scattering data showed that binding of two P(CTD) had almost no effect on the size and shape of the N(m)-RNA rings, whereas binding of two P(2) significantly increased the size of the complexes. SAXS data and molecular modeling were used to add flexible loops (N(NTD) loop, amino acids 105-118; N(CTD) loop, amino acids 376-397) missing in the recently solved crystal structure of the circular N(11)-RNA complex and to build a model for the N(10)-RNA complex. Structural models for the N(m)-RNA-(P(CTD))(2) complexes were then built by docking the known P(CTD) structure onto the completed structures of the circular N(10)-RNA and N(11)-RNA complexes. A multiple-stage flexible docking procedure was used to generate decoys, and SAXS and biochemical data were used for filtering the models. In the refined model, the P(CTD) is bound to the C-terminal top of one N protomer (N(i)), with the C-terminal helix (alpha(6)) of P(CTD) lying on helix alpha(14) of N(i). By an induced-fit mechanism, the N(CTD) loop of

  15. Comparison of affinity chromatography and adsorption to vaccinia virus recombinant infected cells for depletion of antibodies directed against respiratory syncytial virus glycoproteins present in a human immunoglobulin preparation.

    PubMed

    Sastre, Patricia; Melero, José A; García-Barreno, Blanca; Palomo, Concepción

    2005-06-01

    Antibodies directed against human respiratory syncytial virus (HRSV) glycoproteins were depleted from a commercial immunoglobulin preparation (RespiGam) by two different methods. The first method consisted of repeated adsorption of RespiGam to Sepharose beads with covalently bound soluble forms of the two major viral glycoproteins (F or G). The second method consisted of adsorption of immunoglobulins to live cells expressing F or G glycoproteins on their surfaces after infection with vaccinia virus recombinants. While the first method removed efficiently antibodies that reacted with F and/or G glycoproteins by ELISA, it was inefficient in the elimination of anti-HRSV neutralizing antibodies. In contrast, the second method removed efficiently anti-HRSV antibodies that both reacted by ELISA and neutralized virus infectivity. These results confirm that human neutralizing antibodies are directed exclusively against HRSV F and G glycoproteins, and, they raise the possibility that F and G glycoproteins inserted into cell membranes differ antigenically from their soluble forms linked covalently to Sepharose beads.

  16. Safety and immunogenicity of Ontario Rabies Vaccine Bait (ONRAB) in the first us field trial in raccoons (Procyon lotor).

    PubMed

    Slate, Dennis; Chipman, Richard B; Algeo, Timothy P; Mills, Samuel A; Nelson, Kathleen M; Croson, Christopher K; Dubovi, Edward J; Vercauteren, Kurt; Renshaw, Randall W; Atwood, Todd; Johnson, Shylo; Rupprecht, Charles E

    2014-07-01

    In 2011, we conducted a field trial in rural West Virginia, USA to evaluate the safety and immunogenicity of a live, recombinant human adenovirus (AdRG1.3) rabies virus glycoprotein vaccine (Ontario Rabies Vaccine Bait; ONRAB) in wild raccoons (Procyon lotor) and striped skunks (Mephitis mephitis). We selected ONRAB for evaluation because of its effectiveness in raccoon rabies management in Ontario and Quebec, Canada, and significantly higher antibody prevalence rates in raccoons compared with a recombinant vaccinia-rabies glycoprotein (V-RG) vaccine, Raboral V-RG®, in US-Canada border studies. Raccoon rabies was enzootic and oral rabies vaccination (ORV) had never been used in the study area. We distributed 79,027 ONRAB baits at 75 baits/km(2) mostly by fixed-wing aircraft along parallel flight lines at 750-m intervals. Antibody prevalence was significantly higher at 49.2% (n=262) in raccoons after ONRAB was distributed than the 9.6% (n=395) before ORV. This was the highest antibody prevalence observed in raccoons by US Department of Agriculture Wildlife Services for areas with similar management histories evaluated before and after an initial ORV campaign at 75 baits/km(2) with Raboral V-RG. Tetracycline biomarker (TTCC) was significantly higher among antibody-positive raccoons after ONRAB baiting and was similar among raccoons before ORV had been conducted, an indication of vaccine-induced rabies virus-neutralizing antibody production following consumption of bait containing TTCC. Skunk sample size was inadequate to assess ONRAB effects. Safety and immunogenicity results supported replication of this field trial and led to a recommendation for expanded field trials in 2012 to evaluate safety and immunogenicity of ground-distributed ONRAB at 150 baits/km(2) in residential and commercial habitats in Ohio, USA and aerially distributed ONRAB at 75 baits/km(2) in rural habitats along US-Quebec border. PMID:24807178

  17. Safety and immunogenicity of Ontario Rabies Vaccine Bait (ONRAB) in the first us field trial in raccoons (Procyon lotor).

    PubMed

    Slate, Dennis; Chipman, Richard B; Algeo, Timothy P; Mills, Samuel A; Nelson, Kathleen M; Croson, Christopher K; Dubovi, Edward J; Vercauteren, Kurt; Renshaw, Randall W; Atwood, Todd; Johnson, Shylo; Rupprecht, Charles E

    2014-07-01

    In 2011, we conducted a field trial in rural West Virginia, USA to evaluate the safety and immunogenicity of a live, recombinant human adenovirus (AdRG1.3) rabies virus glycoprotein vaccine (Ontario Rabies Vaccine Bait; ONRAB) in wild raccoons (Procyon lotor) and striped skunks (Mephitis mephitis). We selected ONRAB for evaluation because of its effectiveness in raccoon rabies management in Ontario and Quebec, Canada, and significantly higher antibody prevalence rates in raccoons compared with a recombinant vaccinia-rabies glycoprotein (V-RG) vaccine, Raboral V-RG®, in US-Canada border studies. Raccoon rabies was enzootic and oral rabies vaccination (ORV) had never been used in the study area. We distributed 79,027 ONRAB baits at 75 baits/km(2) mostly by fixed-wing aircraft along parallel flight lines at 750-m intervals. Antibody prevalence was significantly higher at 49.2% (n=262) in raccoons after ONRAB was distributed than the 9.6% (n=395) before ORV. This was the highest antibody prevalence observed in raccoons by US Department of Agriculture Wildlife Services for areas with similar management histories evaluated before and after an initial ORV campaign at 75 baits/km(2) with Raboral V-RG. Tetracycline biomarker (TTCC) was significantly higher among antibody-positive raccoons after ONRAB baiting and was similar among raccoons before ORV had been conducted, an indication of vaccine-induced rabies virus-neutralizing antibody production following consumption of bait containing TTCC. Skunk sample size was inadequate to assess ONRAB effects. Safety and immunogenicity results supported replication of this field trial and led to a recommendation for expanded field trials in 2012 to evaluate safety and immunogenicity of ground-distributed ONRAB at 150 baits/km(2) in residential and commercial habitats in Ohio, USA and aerially distributed ONRAB at 75 baits/km(2) in rural habitats along US-Quebec border.

  18. 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

  19. Dual Transneuronal Tracing in the Rat Entorhinal-Hippocampal Circuit by Intracerebral Injection of Recombinant Rabies Virus Vectors

    PubMed Central

    Ohara, Shinya; Inoue, Ken-ichi; Yamada, Masahiro; Yamawaki, Takuma; Koganezawa, Noriko; Tsutsui, Ken-Ichiro; Witter, Menno P.; Iijima, Toshio

    2008-01-01

    Dual transneuronal tracing is a novel viral tracing methodology which employs two recombinant viruses, each expressing a different reporter protein. Peripheral injection of recombinant pseudorabies viruses has been used as a powerful method to define neurons that coordinate outputs to various peripheral targets of motor and autonomic systems. Here, we assessed the feasibility of recombinants of rabies virus (RV) vector for dual transneuronal tracing in the central nervous system. First, we examined whether two different RV-vectors can double label cells in vitro, and showed that efficient double labeling can be realized by infecting targeted cells with the two RV-vectors within a short time interval. The potential of dual transneuronal tracing was then examined in vivo in the entorhinal-hippocampal circuit, using the chain of projections from CA3 pyramidal cells to CA1 pyramidal cells and subsequently to entorhinal cortex. Six days after the injection of two RV-vectors into the left and right entorhinal cortex respectively, double-labeled neurons were observed in CA3 bilaterally. Some double-labeled neurons showed a Golgi-like labeling. Dual transneuronal tracing potentially provides a powerful and sensitive method to study issues such as the amount of convergence and divergence within and between circuits in the central nervous system. Using this sensitive technique, we established that single neurons in CA3 are connected to the entorhinal cortex bilaterally with only one synaptic relay. PMID:19169410

  20. Viperin inhibits rabies virus replication via reduced cholesterol and sphingomyelin and is regulated upstream by TLR4.

    PubMed

    Tang, Hai-Bo; Lu, Zhuan-Ling; Wei, Xian-Kai; Zhong, Tao-Zhen; Zhong, Yi-Zhi; Ouyang, Ling-Xuan; Luo, Yang; Xing, Xing-Wei; Liao, Fang; Peng, Ke-Ke; Deng, Chao-Qian; Minamoto, Nobuyuki; Luo, Ting Rong

    2016-01-01

    Viperin (virus inhibitory protein, endoplasmic reticulum-associated, IFN-inducible) is an interferon-inducible protein that mediates antiviral activity. Generally, rabies virus (RABV) multiplies extremely well in susceptible cells, leading to high virus titres. In this study, we found that viperin was significantly up-regulated in macrophage RAW264.7 cells but not in NA, BHK-21 or BSR cells. Transient viperin overexpression in BSR cells and stable expression in BHK-21 cells could inhibit RABV replication, including both attenuated and street RABV. Furthermore, the inhibitory function of viperin was related to reduce cholesterol/sphingomyelin on the membranes of RAW264.7 cells. We explored the up-stream regulation pathway of viperin in macrophage RAW264.7 cells in the context of RABV infection. An experiment confirmed that a specific Toll-like receptor 4 (TLR4) inhibitor, TAK-242, could inhibit viperin expression in RABV-infected RAW264.7 cells. These results support a regulatory role for TLR4. Geldanamycin, a specific inhibitor of interferon regulatory factor 3 (IRF3) (by inhibiting heat-shock protein 90 (Hsp90) of the IRF3 phosphorylation chaperone), significantly delayed and reduced viperin expression, indicating that IRF3 is involved in viperin induction in RAW264.7 cells. Taken together, our data support the therapeutic potential for viperin to inhibit RABV replication, which appears to involve upstream regulation by TLR4. PMID:27456665

  1. Viperin inhibits rabies virus replication via reduced cholesterol and sphingomyelin and is regulated upstream by TLR4

    PubMed Central

    Tang, Hai-Bo; Lu, Zhuan-Ling; Wei, Xian-Kai; Zhong, Tao-Zhen; Zhong, Yi-Zhi; Ouyang, Ling-Xuan; Luo, Yang; Xing, Xing-Wei; Liao, Fang; Peng, Ke-Ke; Deng, Chao-Qian; Minamoto, Nobuyuki; Luo, Ting Rong

    2016-01-01

    Viperin (virus inhibitory protein, endoplasmic reticulum-associated, IFN-inducible) is an interferon-inducible protein that mediates antiviral activity. Generally, rabies virus (RABV) multiplies extremely well in susceptible cells, leading to high virus titres. In this study, we found that viperin was significantly up-regulated in macrophage RAW264.7 cells but not in NA, BHK-21 or BSR cells. Transient viperin overexpression in BSR cells and stable expression in BHK-21 cells could inhibit RABV replication, including both attenuated and street RABV. Furthermore, the inhibitory function of viperin was related to reduce cholesterol/sphingomyelin on the membranes of RAW264.7 cells. We explored the up-stream regulation pathway of viperin in macrophage RAW264.7 cells in the context of RABV infection. An experiment confirmed that a specific Toll-like receptor 4 (TLR4) inhibitor, TAK-242, could inhibit viperin expression in RABV-infected RAW264.7 cells. These results support a regulatory role for TLR4. Geldanamycin, a specific inhibitor of interferon regulatory factor 3 (IRF3) (by inhibiting heat-shock protein 90 (Hsp90) of the IRF3 phosphorylation chaperone), significantly delayed and reduced viperin expression, indicating that IRF3 is involved in viperin induction in RAW264.7 cells. Taken together, our data support the therapeutic potential for viperin to inhibit RABV replication, which appears to involve upstream regulation by TLR4. PMID:27456665

  2. Evaluation of pseudorabies virus glycoprotein gp50 as a vaccine for Aujeszky's disease in mice and swine: expression by vaccinia virus and Chinese hamster ovary cells.

    PubMed Central

    Marchioli, C C; Yancey, R J; Petrovskis, E A; Timmins, J G; Post, L E

    1987-01-01

    Pseudorabies virus (PRV) is an alphaherpesvirus which causes an economically important disease of swine. One of the PRV glycoproteins, gp50, was previously identified as the sequence homolog of herpes simplex virus glycoprotein gD (E.A. Petrovskis, J.G. Timmins, M.A. Armentrout, C.C. Marchioli, R.J. Yancey, Jr., and L.E. Post, J. Virol. 59:216-223, 1986). gp50 was evaluated as a PRV subunit vaccine candidate. gp50 protected mice from PRV-induced mortality either when delivered via infection with a recombinant vaccinia virus or when administered as a subunit vaccine produced in a eucaryotic cell line, Chinese hamster ovary (CHO) cells. In addition, gp50 synthesized in CHO cells protected pigs from lethal infection with PRV. This result demonstrates that a single viral glycoprotein could induce a protective immune response in the natural host of a herpesvirus infection. Images PMID:2824827

  3. 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

  4. The ERA Strain of Rabies Vaccine

    PubMed Central

    Lawson, K. F.; Crawley, J. F.

    1972-01-01

    An antigenic extinction trial in cats showed that the ERA rabies vaccine had superior antigenic properties over Flury H.E.P. C.E.O. and killed tissue culture rabies vaccine. Dogs and cats on a duration of immunity study of ERA rabies vaccine were challenged with fox salivary gland “street” rabies virus. The results of this challenge show a duration of immunity of five years in dogs and four years in cats. Vaccination of dams in late pregnancy with ERA rabies vaccine resulted in transference of maternal antibody to the newborn, in both cattle and dogs. This maternally derived antibody interfered with the successful active immunization of the young calf. Calves free of antibodies for rabies could be successfully vaccinated as early as 17 days of age and were able to withstand a challenge with virulent “street” rabies virus two years later. PMID:4263912

  5. Ngaingan virus, a macropod-associated rhabdovirus, contains a second glycoprotein gene and seven novel open reading frames.

    PubMed

    Gubala, Aneta; Davis, Steven; Weir, Richard; Melville, Lorna; Cowled, Chris; Walker, Peter; Boyle, David

    2010-03-30

    Ngaingan virus (NGAV) was isolated from a pool of biting midges that were collected in the tropics of northern Australia. Reported here is the full-length sequence of the NGAV genome, which, at over 15.7 kb, is the largest in any rhabdovirus described to date and contains 13 genes, the highest number of genes observed in any (-) ssRNA virus. Seven of these putative genes show no significant homology to known proteins. Like viruses in the genus Ephemerovirus, NGAV possesses a second glycoprotein gene (G(NS)). Phylogenetic analyses, however, place NGAV within the yet to be classified "Hart Park" group containing Wongabel and Flanders viruses, which do not contain a second glycoprotein gene. Screening of various animal sera from northern Australia has indicated that NGAV is currently circulating in macropods (wallabies, wallaroos and kangaroos), highlighting the need for further studies to determine its potential to cause disease in these species.

  6. Molecular epidemiology of rabies: focus on domestic dogs (Canis familiaris) and black-backed jackals (Canis mesomelas) from northern South Africa.

    PubMed

    Zulu, G C; Sabeta, C T; Nel, L H

    2009-03-01

    Phylogenetic relationships of rabies viruses recovered from black-backed jackals (Canis mesomelas) and domestic dogs (Canis familiaris) in northern South Africa were investigated to determine whether the black-backed jackal is an emerging maintenance host species for rabies in this region. A panel of 123 rabies viruses obtained from the two host species between 1980 and 2006 were characterised by nucleotide sequencing of the cytoplasmic domain of the glycoprotein gene and the non-coding G-L intergenic region. Through phylogenetic analysis a viral cluster specific to black-backed jackals and spanning a 5-year period was delineated in western Limpopo. Virus strains associated with domestic dogs prevail in densely populated communal areas in north-eastern Limpopo and in south and eastern Mpumalanga. The data presented in this study indicated the likelihood that black-backed jackals are capable of sustaining rabies cycles independent of domestic dogs. It is proposed that wildlife rabies control strategies, in synergy with domestic animal vaccination should be considered for effective control of rabies in South Africa. PMID:19061924

  7. Molecular epidemiology of rabies: focus on domestic dogs (Canis familiaris) and black-backed jackals (Canis mesomelas) from northern South Africa.

    PubMed

    Zulu, G C; Sabeta, C T; Nel, L H

    2009-03-01

    Phylogenetic relationships of rabies viruses recovered from black-backed jackals (Canis mesomelas) and domestic dogs (Canis familiaris) in northern South Africa were investigated to determine whether the black-backed jackal is an emerging maintenance host species for rabies in this region. A panel of 123 rabies viruses obtained from the two host species between 1980 and 2006 were characterised by nucleotide sequencing of the cytoplasmic domain of the glycoprotein gene and the non-coding G-L intergenic region. Through phylogenetic analysis a viral cluster specific to black-backed jackals and spanning a 5-year period was delineated in western Limpopo. Virus strains associated with domestic dogs prevail in densely populated communal areas in north-eastern Limpopo and in south and eastern Mpumalanga. The data presented in this study indicated the likelihood that black-backed jackals are capable of sustaining rabies cycles independent of domestic dogs. It is proposed that wildlife rabies control strategies, in synergy with domestic animal vaccination should be considered for effective control of rabies in South Africa.

  8. 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 wh