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Sample records for infectious junin viruses

  1. Monoclonal antibody therapy for Junin virus infection.

    PubMed

    Zeitlin, Larry; Geisbert, Joan B; Deer, Daniel J; Fenton, Karla A; Bohorov, Ognian; Bohorova, Natasha; Goodman, Charles; Kim, Do; Hiatt, Andrew; Pauly, Michael H; Velasco, Jesus; Whaley, Kevin J; Altmann, Friedrich; Gruber, Clemens; Steinkellner, Herta; Honko, Anna N; Kuehne, Ana I; Aman, M Javad; Sahandi, Sara; Enterlein, Sven; Zhan, Xiaoguo; Enria, Delia; Geisbert, Thomas W

    2016-04-19

    Countermeasures against potential biothreat agents remain important to US Homeland Security, and many of these pharmaceuticals could have dual use in the improvement of global public health. Junin virus, the causative agent of Argentine hemorrhagic fever (AHF), is an arenavirus identified as a category A high-priority agent. There are no Food and Drug Administration (FDA) approved drugs available for preventing or treating AHF, and the current treatment option is limited to administration of immune plasma. Whereas immune plasma demonstrates the feasibility of passive immunotherapy, it is limited in quantity, variable in quality, and poses safety risks such as transmission of transfusion-borne diseases. In an effort to develop a monoclonal antibody (mAb)-based alternative to plasma, three previously described neutralizing murine mAbs were expressed as mouse-human chimeric antibodies and evaluated in the guinea pig model of AHF. These mAbs provided 100% protection against lethal challenge when administered 2 d after infection (dpi), and one of them (J199) was capable of providing 100% protection when treatment was initiated 6 dpi and 92% protection when initiated 7 dpi. The efficacy of J199 is superior to that previously described for all other evaluated drugs, and its high potency suggests that mAbs like J199 offer an economical alternative to immune plasma and an effective dual use (bioterrorism/public health) therapeutic. PMID:27044104

  2. Monoclonal antibody therapy for Junin virus infection

    PubMed Central

    Zeitlin, Larry; Geisbert, Joan B.; Deer, Daniel J.; Fenton, Karla A.; Bohorov, Ognian; Bohorova, Natasha; Goodman, Charles; Kim, Do; Hiatt, Andrew; Pauly, Michael H.; Velasco, Jesus; Whaley, Kevin J.; Altmann, Friedrich; Gruber, Clemens; Steinkellner, Herta; Honko, Anna N.; Kuehne, Ana I.; Aman, M. Javad; Sahandi, Sara; Enterlein, Sven; Zhan, Xiaoguo; Enria, Delia; Geisbert, Thomas W.

    2016-01-01

    Countermeasures against potential biothreat agents remain important to US Homeland Security, and many of these pharmaceuticals could have dual use in the improvement of global public health. Junin virus, the causative agent of Argentine hemorrhagic fever (AHF), is an arenavirus identified as a category A high-priority agent. There are no Food and Drug Administration (FDA) approved drugs available for preventing or treating AHF, and the current treatment option is limited to administration of immune plasma. Whereas immune plasma demonstrates the feasibility of passive immunotherapy, it is limited in quantity, variable in quality, and poses safety risks such as transmission of transfusion-borne diseases. In an effort to develop a monoclonal antibody (mAb)-based alternative to plasma, three previously described neutralizing murine mAbs were expressed as mouse-human chimeric antibodies and evaluated in the guinea pig model of AHF. These mAbs provided 100% protection against lethal challenge when administered 2 d after infection (dpi), and one of them (J199) was capable of providing 100% protection when treatment was initiated 6 dpi and 92% protection when initiated 7 dpi. The efficacy of J199 is superior to that previously described for all other evaluated drugs, and its high potency suggests that mAbs like J199 offer an economical alternative to immune plasma and an effective dual use (bioterrorism/public health) therapeutic. PMID:27044104

  3. Experimental biology and pathogenesis of Junin virus infection in animals and man*

    PubMed Central

    Weissenbacher, M. C.; De Guerrero, L. B.; Boxaca, M. C.

    1975-01-01

    A fatal disease resembling Argentine haemorrhagic fever of man has been produced in guinea-pigs and mice by inoculation with Junin virus. Infected guinea-pigs show macroscopic and microscopic haemorrhagic lesions, marked bone marrow changes, decreased leukocytes and platelets in the peripheral blood, and impairment of immunological response. This response permits differentiation between pathogenic (XJ) and attenuated (XJ Cl3) strains. Guinea-pigs inoculated with the XJ Cl3 strain develop an inapparent infection accompanied by slight haematological changes, the appearance of antibody, and protection against challenge with the pathogenic strain. The attenuated strain has been used successfully as an immunizing antigen in 636 human volunteers. Guinea-pigs infected with Tacaribe virus show cross-protection against Junin virus, with the presence of heterologous neutralizing antibodies. Suckling mice infected with Junin virus develop a typical viral encephalitis; the pathogenicity of the virus decreases with increasing age of the mice. Experiments with thymectomized mice and with mice treated with antithymocyte serum suggest that the pathogenicity of Junin virus in this host is related to the integrity of the thymus-dependent immune system. There is evidence that humoral antibodies do not play any role in the development of the encephalitic lesions but rather protect mice against Junin virus infection. A recent serological survey among laboratory workers and inhabitants of the endemic area has demonstrated the presence of inapparent infection with Junin virus. PMID:182401

  4. Antiviral mode of action of a synthetic brassinosteroid against Junin virus replication.

    PubMed

    Castilla, Viviana; Larzábal, Mariano; Sgalippa, Natalia Aguirre; Wachsman, Mónica B; Coto, Celia E

    2005-11-01

    The antiviral mode of action of the synthetic brassinosteroid (22S,23S)-3beta-bromo-5alpha,22,23-trihydroxystigmastan-6-one (6b) against Junin virus replication in Vero cells was investigated. Time-related experiments showed that 6b mainly affects an early event of virus growth cycle. Neither adsorption nor internalization of viral particles was the target of the inhibitory action. The analysis of the effect of 6b on viral RNA synthesis demonstrated that the presence of the compound adversely affects virus RNA replication by preventing the synthesis of full length antigenomic RNA. Although 6b was most effective the earlier it was added to the cells after infection with JV, a high level of inhibition of JV yield and fusion activity of newly synthesized viral glycoproteins was still detected when the compound was present during the last hours of infection. Therefore, we cannot rule out an inhibitory action of 6b on later events of JV replicative cycle. PMID:16171877

  5. Tests in mice of a dengue vaccine candidate made of chimeric Junin virus-like particles and conserved dengue virus envelope sequences.

    PubMed

    Mareze, Vania Aparecida; Borio, Cristina Silvia; Bilen, Marcos F; Fleith, Renata; Mirazo, Santiago; Mansur, Daniel Santos; Arbiza, Juan; Lozano, Mario Enrique; Bruña-Romero, Oscar

    2016-01-01

    Two new vaccine candidates against dengue virus (DENV) infection were generated by fusing the coding sequences of the self-budding Z protein from Junin virus (Z-JUNV) to those of two cryptic peptides (Z/DENV-P1 and Z/DENV-P2) conserved on the envelope protein of all serotypes of DENV. The capacity of these chimeras to generate virus-like particles (VLPs) and to induce virus-neutralizing antibodies in mice was determined. First, recombinant proteins that displayed reactivity with a Z-JUNV-specific serum by immunofluorescence were detected in HEK-293 cells transfected with each of the two plasmids and VLP formation was also observed by transmission electron microscopy. Next, we determined the presence of antibodies against the envelope peptides of DENV in the sera of immunized C57BL/6 mice. Results showed that those animals that received Z/DENV-P2 DNA coding sequences followed by a boost with DENV-P2 synthetic peptides elicited significant specific antibody titers (≥6.400). Finally, DENV plaque-reduction neutralization tests (PRNT) were performed. Although no significant protective effect was observed when using sera of Z/DENV-P1-immunized animals, antibodies raised against vaccine candidate Z/DENV-P2 (diluted 1:320) were able to reduce in over 50 % the number of viral plaques generated by infectious DENV particles. This reduction was comparable to that of the 4G2 DENV-specific monoclonal cross-reactive (all serotypes) neutralizing antibody. We conclude that Z-JUNV-VLP is a valid carrier to induce antibody-mediated immune responses in mice and that Z/DENV-P2 is not only immunogenic but also protective in vitro against infection of cells with DENV, deserving further studies. On the other side, DENV's fusion peptide-derived chimera Z/DENV-P1 did not display similar protective properties. PMID:26386688

  6. In vitro inactivation of complement by a serum factor present in Junin-virus infected guinea-pigs.

    PubMed Central

    Rimoldi, M T; de Bracco, M M

    1980-01-01

    A serum factor(s) of guinea-pigs infected with Junin virus, the etiological agent of Argentine haemorrhagic fever, is endowed with a potent anticomplementary activity. It is resistant to heat (56 degrees, 30 min) and elutes from a Sephadex G-200 column between albumin and haemoglobin. It is ineffective in the presence of EDTA or EGTA and does not sediment at 82,000 g. It has no direct effect on C4 unless functional Cl is present. However, it induces Cl activation that consumes C4 haemolytic activity in normal human and guinea-pig sera. The evidence presented in this report demonstrates that the complement activation observed in experimental Argentine haemorrhagic fever is at least in part due to a direct effect of this serum factor on the classical complement pathway. PMID:6247264

  7. [New transmission scenarios of the Argentine hemorrhagic fever since the introduction of the live attenuated junin virus vaccine (Candid #1): an experience in migrant workers].

    PubMed

    Briggiler, Ana; Sinchi, Anabel; Coronel, Florencia; Sánchez, Zaida; Levis, Silvana; Taylor, Jorge; Enria, Delia

    2015-01-01

    The Argentine hemorrhagic fever (AHF) is a severe acute viral disease caused by the Junin virus of the Arenaviridae family. The AHF endemic area coincides geographically with the largest grain export agro-industrial complex of the country [Argentina]. Since the implementation of vaccination with the Candid #1 vaccine, a significant reduction in incidence was achieved and risk patterns were modified. A previous study allowed characterizing these changes and identifying three transmission scenarios: classic, emergent-reemergent, and traveler. The latter scenario includes seasonal migrant workers who move each year, mainly from the province of Santiago del Estero, the endemic area to work in the detasseling of maize. With the objective of protecting this group of workers, a prevention campaign was initiated which included: capacity building of health personnel in the province, health education, and immunization with the vaccine Candid #1. 3,021 workers were vaccinated. Prior to vaccination, serum samples were taken from a group of 104 volunteers. Tests for neutralizing antibodies specific to the Junin virus were performed and 6 (5.76%) tested positive. The unexpected finding of a high percentage of workers with antibodies suggests the need to evaluate several hypotheses: a) that the result is the product of non-probabilistic sampling; b) that it could be people who fell ill in previous travels, c) or who were vaccinated in previous travels; or d) consider this region as an emerging scenario. PMID:26102120

  8. The Ectodomain of Glycoprotein from the Candid#1 Vaccine Strain of Junin Virus Rendered Machupo Virus Partially Attenuated in Mice Lacking IFN-αβ/γ Receptor.

    PubMed

    Koma, Takaaki; Huang, Cheng; Aronson, Judith F; Walker, Aida G; Miller, Milagros; Smith, Jeanon N; Patterson, Michael; Paessler, Slobodan

    2016-08-01

    Machupo virus (MACV), a New World arenavirus, is the etiological agent of Bolivian hemorrhagic fever (BHF). Junin virus (JUNV), a close relative, causes Argentine hemorrhagic fever (AHF). Previously, we reported that a recombinant, chimeric MACV (rMACV/Cd#1-GPC) expressing glycoprotein from the Candid#1 (Cd#1) vaccine strain of JUNV is completely attenuated in a murine model and protects animals from lethal challenge with MACV. A rMACV with a single F438I substitution in the transmembrane domain (TMD) of GPC, which is equivalent to the F427I attenuating mutation in Cd#1 GPC, was attenuated in a murine model but genetically unstable. In addition, the TMD mutation alone was not sufficient to fully attenuate JUNV, indicating that other domains of the GPC may also contribute to the attenuation. To investigate the requirement of different domains of Cd#1 GPC for successful attenuation of MACV, we rescued several rMACVs expressing the ectodomain of GPC from Cd#1 either alone (MCg1), along with the TMD F438I substitution (MCg2), or with the TMD of Cd#1 (MCg3). All rMACVs exhibited similar growth curves in cultured cells. In mice, the MCg1 displayed significant reduction in lethality as compared with rMACV. The MCg1 was detected in brains and spleens of MCg1-infected mice and the infection was associated with tissue inflammation. On the other hand, all animals survived MCg2 and MCg3 infection without detectable levels of virus in various organs while producing neutralizing antibody against Cd#1. Overall our data suggest the indispensable role of each GPC domain in the full attenuation and immunogenicity of rMACV/Cd#1 GPC. PMID:27580122

  9. Infectious laryngotracheitis virus in chickens.

    PubMed

    Ou, Shan-Chia; Giambrone, Joseph J

    2012-10-12

    Infectious laryngotracheitis (ILT) is an important respiratory disease of chickens and annually causes significant economic losses in the poultry industry world-wide. ILT virus (ILTV) belongs to alphaherpesvirinae and the Gallid herpesvirus 1 species. The transmission of ILTV is via respiratory and ocular routes. Clinical and post-mortem signs of ILT can be separated into two forms according to its virulence. The characteristic of the severe form is bloody mucus in the trachea with high mortality. The mild form causes nasal discharge, conjunctivitis, and reduced weight gain and egg production. Conventional polymerase chain reaction (PCR), nested PCR, real-time PCR, and loop-mediated isothermal amplification were developed to detect ILTV samples from natural or experimentally infected birds. The PCR combined with restriction fragment length polymorphism (RFLP) can separate ILTVs into several genetic groups. These groups can separate vaccine from wild type field viruses. Vaccination is a common method to prevent ILT. However, field isolates and vaccine viruses can establish latent infected carriers. According to PCR-RFLP results, virulent field ILTVs can be derived from modified-live vaccines. Therefore, modified-live vaccine reversion provides a source for ILT outbreaks on chicken farms. Two recently licensed commercial recombinant ILT vaccines are also in use. Other recombinant and gene-deficient vaccine candidates are in the developmental stages. They offer additional hope for the control of this disease. However, in ILT endemic regions, improved biosecurity and management practices are critical for improved ILT control. PMID:24175219

  10. Infectious laryngotracheitis virus in chickens

    PubMed Central

    Ou, Shan-Chia; Giambrone, Joseph J

    2012-01-01

    Infectious laryngotracheitis (ILT) is an important respiratory disease of chickens and annually causes significant economic losses in the poultry industry world-wide. ILT virus (ILTV) belongs to alphaherpesvirinae and the Gallid herpesvirus 1 species. The transmission of ILTV is via respiratory and ocular routes. Clinical and post-mortem signs of ILT can be separated into two forms according to its virulence. The characteristic of the severe form is bloody mucus in the trachea with high mortality. The mild form causes nasal discharge, conjunctivitis, and reduced weight gain and egg production. Conventional polymerase chain reaction (PCR), nested PCR, real-time PCR, and loop-mediated isothermal amplification were developed to detect ILTV samples from natural or experimentally infected birds. The PCR combined with restriction fragment length polymorphism (RFLP) can separate ILTVs into several genetic groups. These groups can separate vaccine from wild type field viruses. Vaccination is a common method to prevent ILT. However, field isolates and vaccine viruses can establish latent infected carriers. According to PCR-RFLP results, virulent field ILTVs can be derived from modified-live vaccines. Therefore, modified-live vaccine reversion provides a source for ILT outbreaks on chicken farms. Two recently licensed commercial recombinant ILT vaccines are also in use. Other recombinant and gene-deficient vaccine candidates are in the developmental stages. They offer additional hope for the control of this disease. However, in ILT endemic regions, improved biosecurity and management practices are critical for improved ILT control. PMID:24175219

  11. Genome of infectious bronchitis virus.

    PubMed Central

    Lomniczi, B; Kennedy, I

    1977-01-01

    Techniques are described for the growth and rapid purification of the avian coronavirus infectious bronchitis virus (IBV). Purified IBV has a sedimentation coefficient of 320S and a buoyant density of 1.22 g/ml in sucrose-deuterium oxide equilibrium gradients. IBV RNA extracted by proteinase K in the presence of sodium dodecyl sulfate and further purified by phenol extraction and gradient centrifugation is single stranded and has a sedimentation coefficient of 64S, as determined by isokinetic gradient centrifugation. Analysis on sucrose gradients under both aqueous and denaturing conditions together with agarose gel electrophoresis in the presence of the chaotropic agent methylmercuric hydroxide gave a value of 8 X 10(6) for the moleclar weight of IBV RNA. This value was confirmed by RNase T1 fingerprinting, which also indicated that IBV RNA is haploid. No evidence was found of subunit structure in IBV RNA. From these results together with the recently reported observation that IBV RNA is infectious and contains a tract of polyadenylic acid (Lomniczi, J. Gen. Virol., in press), we conclude that the genome of the coronaviruses is a single continuous chain of about 23,000 mononucleotides that is of messenger polarity. Images PMID:198590

  12. Infectious Viral Quantification of Chikungunya Virus-Virus Plaque Assay.

    PubMed

    Kaur, Parveen; Lee, Regina Ching Hua; Chu, Justin Jang Hann

    2016-01-01

    The plaque assay is an essential method for quantification of infectious virus titer. Cells infected with virus particles are overlaid with a viscous substrate. A suitable incubation period results in the formation of plaques, which can be fixed and stained for visualization. Here, we describe a method for measuring Chikungunya virus (CHIKV) titers via virus plaque assays. PMID:27233264

  13. Heterotypic protection to infectious bronchitis virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We developed Newcastle disease virus (NDV) LaSota (rLS) expressing a distinct spike (S) protein gene of infectious bronchitis virus (IBV). This recombinant vaccine technology confers cross-protection among different IBV strains. We also experimentally demonstrated that the recombinant construct main...

  14. Thermal inactivation of infectious hematopoietic necrosis and infectious pancreatic necrosis virus

    USGS Publications Warehouse

    Gosting, L.; Gould, R.W.

    1981-01-01

    A plaque assay was used to follow the inactivation kinetics of infectious hematopoietic necrosis virus and infectious pancreatic necrosis virus in cell culture media at various temperatures. Inactivation of infectious hematopoietic necrosis virus in a visceral organ slurry was compared with that in culture media.

  15. Rescue from Cloned cDNAs and In Vivo Characterization of Recombinant Pathogenic Romero and Live-Attenuated Candid #1 Strains of Junin Virus, the Causative Agent of Argentine Hemorrhagic Fever Disease ▿

    PubMed Central

    Emonet, Sebastien F.; Seregin, Alexey V.; Yun, Nadezhda E.; Poussard, Allison L.; Walker, Aida G.; de la Torre, Juan C.; Paessler, Slobodan

    2011-01-01

    The New World arenavirus Junin virus (JUNV) is the causative agent of Argentine hemorrhagic fever (AHF), which is associated with high morbidity and significant mortality. Several pathogenic strains of JUNV have been documented, and a highly attenuated vaccine strain (Candid #1) was generated and used to vaccinate the human population at risk. The identification and functional characterization of viral genetic determinants associated with AHF and Candid #1 attenuation would contribute to the elucidation of the mechanisms contributing to AHF and the development of better vaccines and therapeutics. To this end, we used reverse genetics to rescue the pathogenic Romero and the attenuated Candid #1 strains of JUNV from cloned cDNAs. Both recombinant Candid #1 (rCandid #1) and Romero (rRomero) had the same growth properties and phenotypic features in cultured cells and in vivo as their corresponding parental viruses. Infection with rRomero caused 100% lethality in guinea pigs, whereas rCandid #1 infection was asymptomatic and provided protection against a lethal challenge with Romero. Notably, Romero and Candid #1 trans-acting proteins, L and NP, required for virus RNA replication and gene expression were exchangeable in a minigenome rescue assay. These findings support the feasibility of studies aimed at determining the contribution of each viral gene to JUNV pathogenesis and attenuation. In addition, we rescued Candid #1 viruses with three segments that efficiently expressed foreign genes introduced into their genomes. This finding opens the way for the development of a safe multivalent arenavirus vaccine. PMID:21123388

  16. Immune responses to infectious laryngotracheitis virus.

    PubMed

    Coppo, Mauricio J C; Hartley, Carol A; Devlin, Joanne M

    2013-11-01

    Infectious laryngotracheitis (ILT) is an upper respiratory tract disease in chickens caused by infectious laryngotracheitis virus (ILTV), an alphaherpesvirus. Despite the extensive use of attenuated, and more recently recombinant, vaccines for the control of this disease, ILT continues to affect the intensive poultry industries worldwide. Innate and cell-mediated, rather than humoral immune responses, have been identified as responsible for protection against disease. This review examines the current understandings in innate and adaptive immune responses towards ILTV, as well as the role of ILTV glycoprotein G in modulating the host immune response towards infection. Protective immunity induced by ILT vaccines is also examined. The increasing availability of tools and reagents for the characterisation of avian innate and cell-mediated immune responses are expected to further our understanding of immunity against ILTV and drive the development of new generation vaccines towards enhanced control of this disease. PMID:23567343

  17. Newcastle disease virus as a vaccine vector for infectious laryngotracheitis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Effective, safe, and incapable of reverting to virulence are characteristics desirable for infectious laryngotracheitis virus (ILTV) vaccines. Recombinant Newcastle disease virus (NDV) expressing foreign antigens of avian and mammalian pathogens have been demonstrated to elicit protective immunity....

  18. Reaction of goats to infection with infectious bovine rhinotracheitis virus.

    PubMed

    Wafula, J S; Mushi, E Z; Wamwayi, H

    1985-07-01

    Intranasal exposure of goats to infectious bovine rhinotracheitis virus resulted in mild respiratory disease and virus reisolation from nasal secretions. No disease was produced in goats exposed to the same virus by the genital or ocular routes. There was serological evidence of contact transmission of infection from infected goats to cattle. Virus recrudescence was not detected in goats treated with dexamethasone two months after virus inoculation. PMID:2994191

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

    PubMed

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

    2011-02-01

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

  20. Morphological and quantitative comparison between infectious and non-infectious forms of influenza virus.

    PubMed

    WERNER, G H; SCHLESINGER, R W

    1954-08-01

    Electron microscopic study has revealed the morphological entity responsible for the rise in viral hemagglutinin observed in brains of mice after intracerebral inoculation of non-neurotropic strains of influenza virus. This rise in hemagglutinin, although dependent on inoculation of fully infectious virus, is not associated with an increase in infectious titer. The hemagglutinating principle is functionally similar to the "incomplete" influenza virus which can be obtained from chick embryos by serial egg-to-egg transfer of undiluted, infected allantoic fluid according to the method of von Magnus. A method has been described which facilitates selective adsorption of viral particles recovered from organ extracts on saponine-lysed ghosts of fowl erythrocytes. This procedure has been utilized in studying the morphology of non-infectious, hemagglutinating virus from chorio-allantoic membranes or mouse brains and in comparing these two forms with each other and with ordinary, infectious (standard) influenza virus. Standard virus isolated from allantoic fluids or membranes of infected eggs was found to contain uniform particles of predominantly spherical shape with smooth surface and even density, resembling those described by others. The appearance of such particles was not affected by the procedure of extraction and concentration used. In contrast, non-infectious, hemagglutinating virus obtained either from allantoic sacs ("undiluted passages") or from mouse brain was pleomorphic and seemed to consist of disintegrating particles. The majority appeared flattened and bag-like and had a rough, granular surface and reduced, uneven density. 37 per cent of the non-infectious particles isolated from mouse brain infected with the non-neurotropic strain WS had diameters in excess of 170 mmicro, as compared with only 2 per cent of the particles of the parent strain itself. Regardless of whether or not the contrast in appearance of standard and of non-infectious particles was due

  1. The Glycoprotein Precursor Gene of Junin Virus Determines the Virulence of the Romero Strain and the Attenuation of the Candid #1 Strain in a Representative Animal Model of Argentine Hemorrhagic Fever

    PubMed Central

    Seregin, Alexey V.; Yun, Nadezhda E.; Miller, Milagros; Aronson, Judith; Smith, Jennifer K.; Walker, Aida G.; Smith, Jeanon N.; Huang, Cheng; Manning, John T.; de la Torre, Juan C.

    2015-01-01

    ABSTRACT The New World arenavirus Junin virus (JUNV) is the causative agent of Argentine hemorrhagic fever (AHF), a potentially deadly disease endemic to central regions of Argentina. The live-attenuated Candid #1 (Can) strain of JUNV is currently used to vaccinate the human population at risk. However, the mechanism of attenuation of this strain is still largely unknown. Therefore, the identification and functional characterization of viral genetic determinants dictating JUNV virulence or attenuation would significantly improve the understanding of the mechanisms underlying AHF and facilitate the development of novel, more effective, and safer vaccines. Here, we utilized a reverse genetics approach to generate recombinant JUNV (rJUNV) strains encoding different gene combinations of the pathogenic Romero (Rom) and attenuated Can strains of JUNV. All strains of rJUNV exhibited in vitro growth kinetics similar to those of their parental counterparts. Analysis of virulence of the rJUNV in a guinea pig model of lethal infection that closely reproduces the features of AHF identified the envelope glycoproteins (GPs) as the major determinants of pathogenesis and attenuation of JUNV. Accordingly, rJUNV strains expressing the full-length GPs of Rom and Can exhibited virulent and attenuated phenotypes, respectively, in guinea pigs. Mutation F427I in the transmembrane region of JUNV envelope glycoprotein GP2 has been shown to attenuate the neurovirulence of JUNV in suckling mice. We document that in the guinea pig model of AHF, mutation F427I in GP2 is also highly attenuating but insufficient to prevent virus dissemination and development of mild clinical and pathological symptoms, indicating that complete attenuation of JUNV requires additional mutations present in Can glycoprotein precursor (GPC). IMPORTANCE Development of antiviral strategies against viral hemorrhagic fevers, including AHF, is one of the top priorities within the Implementation Plan of the U.S. Department

  2. Minimum intravenous infectious dose of ovine progressive pneumonia virus (OPPV)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The minimum intravenous infectious dose for ovine progressive pneumonia virus (OPPV) WLC1 was determined using twenty-four 6 month-old lambs. Twelve groups of two 6 month-old lambs were inoculated intravenously with tissue culture fluid containing ovine progressive pneumonia virus (OPPV) WLC1 titer...

  3. Influenza Virus Aerosols in the Air and Their Infectiousness

    PubMed Central

    2014-01-01

    Influenza is one of the most contagious and rapidly spreading infectious diseases and an important global cause of hospital admissions and mortality. There are some amounts of the virus in the air constantly. These amounts is generally not enough to cause disease in people, due to infection prevention by healthy immune systems. However, at a higher concentration of the airborne virus, the risk of human infection increases dramatically. Early detection of the threshold virus concentration is essential for prevention of the spread of influenza infection. This review discusses different approaches for measuring the amount of influenza A virus particles in the air and assessing their infectiousness. Here we also discuss the data describing the relationship between the influenza virus subtypes and virus air transmission, and distribution of viral particles in aerosol drops of different sizes. PMID:25197278

  4. Morphological method for estimation of simian virus 40 infectious titer.

    PubMed

    Landau, S M; Nosach, L N; Pavlova, G V

    1982-01-01

    The cytomorphologic method previously reported for titration of adenoviruses has been employed for estimating the infectious titer of simian virus 40 (SV 40). Infected cells forming intranuclear inclusions were determined. The method examined possesses a number of advantages over virus titration by plaque assay and cytopathic effect. The virus titer estimated by the method of inclusion counting and expressed as IFU/ml (Inclusion Forming Units/ml) corresponds to that estimated by plaque count and expressed as PFU/ml. PMID:6289780

  5. Generation of an infectious Negev virus cDNA clone

    PubMed Central

    Gorchakov, Rodion V.; Tesh, Robert B.; Weaver, Scott C.

    2014-01-01

    The genus Negevirus consists of insect-only viruses isolated from mosquitoes and sandflies. Here, we report the successful construction of a full-length infectious cDNA clone of Negev virus (NEGV) strain M30957. Viral RNA was transcribed in vitro and virus was readily rescued with or without the use of a cap analogue. These results strongly suggest that NEGV, and likely other members within the genus, is a non-segmented, single-stranded, positive-sense RNA virus. PMID:24878640

  6. Comparative nucleotide sequence analysis of three virulent strains of infectious laryngotracheitis virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Infectious laryngotracheitis is a very serious and widespread respiratory disease of chickens caused by gallid herpesvirus type 1, commonly named infectious laryngotracheitis virus. For protection from infectious laryngotracheitis, chickens have traditionally been vaccinated with live-attenuated str...

  7. Zika virus: A rapidly emerging infectious disease.

    PubMed

    Borchardt, Roy A

    2016-04-01

    Zika virus is a flavivirus transmitted to humans via the bite of infected mosquitoes. A recent outbreak in Brazil has spread to several surrounding countries, and the virus also has been reported in the United States. The virus is associated with microcephaly among newborns whose mothers were infected. Because no vaccine or treatment is available, efforts have focused on preventing mosquito bites and advising pregnant women and women trying to get pregnant to avoid active areas of Zika virus transmission. Clinicians should understand the infection, its diagnosis and testing, and monitor pregnant women for travel history to outbreak regions and for the presence of clinical symptoms. Patient education on preventive measures offers the best option to avoid Zika virus infection. PMID:26953673

  8. S2 expressed from recombinant virus confers broad protection against infectious bronchitis virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We previously demonstrated that overexposing the IBV (infectious bronchitis virus) S2 to the chicken immune system by means of a vectored vaccine, followed by boost with whole virus, protects chickens against IBV showing dissimilar S1. We developed recombinant Newcastle disease virus (NDV) LaSota (...

  9. Avian infectious bronchitis virus in Africa: a review.

    PubMed

    Khataby, Khadija; Fellahi, Siham; Loutfi, Chafiqa; Mustapha, Ennaji Moulay

    2016-06-01

    Infectious bronchitis virus (IBV) is worldwide in distribution, highly infectious, and extremely difficult to control because it has extensive genetic diversity, a short generation time, and a high mutation rate. IBV is a Gammacoronavirus, single-stranded, and positive-sense RNA virus. Avian infectious bronchitis is well studied in European countries with identification of a large number of IBV variants, whereas in African countries epidemiological and scientific data are poor and not updated. However, previous studies reported that an IBV variant continues to appear regularly in Africa, as currently described in Morocco. No cross-protection between IBV strains was reported, some being unique to a particular country, others having a more general distribution. This review aims to provide a general overview on IB disease distribution in African countries and an update on the available studies of IBV variants in each country. PMID:27150555

  10. Infectious Maize rayado fino virus from cloned cDNA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Maize rayado fino virus (MRFV) is the type member of the marafiviruses within the family Tymoviridae. A cDNA clone from which infectious RNA can be transcribed was produced from a US isolate of MRFV (MRFV-US). Infectivity of transcripts derived from cDNA clones was demonstrated by infection of mai...

  11. Genome Sequence of a Distinct Infectious Bursal Disease Virus

    PubMed Central

    Tomás, Gonzalo; Hernández, Martín; Marandino, Ana; Hernández, Diego; Techera, Claudia; Grecco, Sofía; Panzera, Yanina

    2015-01-01

    Infectious bursal disease virus is a relevant avian pathogen that affects poultry production. Here, we report the full-length coding sequence of the Uruguayan strain dIBDV/UY/2014/2202, isolated from a commercial broiler flock. The strain belongs to the distinct IBDV lineage that is widely distributed in South America. PMID:26430025

  12. Natural infection of turkeys by infectious laryngotracheitis virus.

    PubMed

    Portz, Cristiana; Beltrão, Nilzane; Furian, Thales Quedi; Júnior, Alfredo Bianco; Macagnan, Marisa; Griebeler, Josiane; Lima Rosa, Carlos André Veiga; Colodel, Edson Moleta; Driemeier, David; Back, Alberto; Barth Schatzmayr, Ortrud Monika; Canal, Cláudio Wageck

    2008-09-18

    The infectious laryngotracheitis virus (ILTV) is an important respiratory pathogen of chickens that also infects pheasants and peafowl. Epidemiologically non-related commercial turkey flocks with clinical signs such as tracheitis, swollen sinuses, conjunctivitis and expectoration of bloody mucus were examined for the presence of the virus. Laboratory ILTV detection was performed by virus isolation in embryonated eggs and cell cultures, PCR and sequencing of amplification products, histopathology, indirect immunofluorescence and electron microscopy. One ILTV turkey isolate was also experimentally inoculated into susceptible chickens and turkeys, reproducing a mild respiratory disease. This is the first description of natural infections with ILTV in turkeys. PMID:18436397

  13. Characterization of infectious dose and lethal dose of two strains of infectious hematopoietic necrosis virus (IHNV).

    PubMed

    McKenney, Douglas G; Kurath, Gael; Wargo, Andrew R

    2016-03-01

    The ability to infect a host is a key trait of a virus, and differences in infectivity could put one virus at an evolutionary advantage over another. In this study we have quantified the infectivity of two strains of infectious hematopoietic necrosis virus (IHNV) that are known to differ in fitness and virulence. By exposing juvenile rainbow trout (Oncorhynchus mykiss) hosts to a wide range of virus doses, we were able to calculate the infectious dose in terms of ID50 values for the two genotypes. Lethal dose experiments were also conducted to confirm the virulence difference between the two virus genotypes, using a range of virus doses and holding fish either in isolation or in batch so as to calculate LD50 values. We found that infectivity is positively correlated with virulence, with the more virulent genotype having higher infectivity. Additionally, infectivity increases more steeply over a short range of doses compared to virulence, which has a shallower increase. We also examined the data using models of virion interaction and found no evidence to suggest that virions have either an antagonistic or a synergistic effect on each other, supporting the independent action hypothesis in the process of IHNV infection of rainbow trout. PMID:26752429

  14. Characterization of infectious dose and lethal dose of two strains of infectious hematopoietic necrosis virus (IHNV)

    USGS Publications Warehouse

    McKenney, Douglas; Kurath, Gael; Wargo, Andrew

    2016-01-01

    The ability to infect a host is a key trait of a virus, and differences in infectivity could put one virus at an evolutionary advantage over another. In this study we have quantified the infectivity of two strains of infectious hematopoietic necrosis virus (IHNV) that are known to differ in fitness and virulence. By exposing juvenile rainbow trout (Oncorhynchus mykiss) hosts to a wide range of virus doses, we were able to calculate the infectious dose in terms of ID50 values for the two genotypes. Lethal dose experiments were also conducted to confirm the virulence difference between the two virus genotypes, using a range of virus doses and holding fish either in isolation or in batch so as to calculate LD50values. We found that infectivity is positively correlated with virulence, with the more virulent genotype having higher infectivity. Additionally, infectivity increases more steeply over a short range of doses compared to virulence, which has a shallower increase. We also examined the data using models of virion interaction and found no evidence to suggest that virions have either an antagonistic or a synergistic effect on each other, supporting the independent action hypothesis in the process of IHNV infection of rainbow trout.

  15. Evaluation of endocrine and immune disruption of infectious bovine rhinotracheitis virus vaccinated steers challenged with infectious bovine rhinotracheitis virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this study was to evaluate the endocrine and immune responses of steers challenged with infectious bovine rhinotracheitis virus (IBRV). For the study, twelve crossbred beef steers weighing approximately 228.82 kg were fitted with indwelling rectal temperature monitoring devices and ...

  16. Norwalk virus: How infectious is it?

    EPA Science Inventory

    Noroviruses are major agents of viral gastroenteritis worldwide. The infectivity of Norwalk virus, the prototype norovirus, has been studied in susceptible human volunteers. A new variant of the hit theory model of microbial infection was developed to estimate the variation in No...

  17. Propagation of infectious salmon anaemia (ISA) virus in cell culture.

    PubMed

    Dannevig, B H; Falk, K; Press, C M

    1995-01-01

    A long-term cell line supporting growth of the infectious salmon anaemia (ISA) virus has been established. The cell line (SHK-1) was developed from a culture of head kidney leucocytes from Atlantic salmon, and exhibited macrophage-like enzyme reactivities. By means of transmission experiments, ISA infectivity of cell culture medium could be demonstrated from day 5 after infection of SHK-1 cells with ISA-infective tissue homogenate. ISA infectivity of cell culture medium increased following repeated passages of virus. ISA-infected cell cultures develop cytopathic effects (CPE), making quantitation of virus possible. The development of CPE in ISA virus infected cells was inhibited by ammonium chloride, chloroquine and bafilomycin A, suggesting that infection of SHK-1 cells with ISA virus requires a low-pH step. PMID:8581019

  18. Subclinical Shed of Infectious Varicella zoster Virus in Astronauts

    NASA Technical Reports Server (NTRS)

    Cohrs, Randall J.; Mehta, Satish K.; Schmid, D. Scott; Gilden, Donald H.; Pierson, Duane L.

    2007-01-01

    Aerosol borne varicella zoster virus (VZV) enters the nasopharynx and replicates in tonsillar T-cells, resulting in viremia and varicella (chickenpox). Virus then becomes latent in cranial nerve, dorsal root and autonomic nervous system ganglia along the entire neuraxis (1). Decades later, as cell-mediated immunity to VZV declines (4), latent VZV can reactivate to produce zoster (shingles). Infectious VZV is present in patients with varicella or zoster, but shed of infectious virus in the absence of disease has not been shown. We previously detected VZV DNA in saliva of astronauts during and shortly after spaceflight, suggesting stress induced subclinical virus reactivation (3). We show here that VZV DNA as well as infectious virus in present in astronaut saliva. VZV DNA was detected in saliva during and after a 13-day spaceflight in 2 of 3 astronauts (Fig. panel A). Ten days before liftoff, there was a rise in serum anti-VZV antibody in subjects 1 and 2, consistent with virus reactivation. In subject 3, VZV DNA was not detected in saliva, and there was no rise in anti-VZV antibody titer. Subject 3 may have been protected from virus reactivation by having zoster <10 years ago, which provides a boost in cell-medicated immunity to VZV (2). No VZV DNA was detected in astronaut saliva months before spaceflight, or in saliva of 10 age/sex-matched healthy control subjects sampled on alternate days for 3 weeks (88 saliva samples). Saliva taken 2-6 days after landing from all 3 subjects was cultured on human fetal lung cells (Fig. panel B). Infectious VZV was recovered from saliva of subjects 1 and 2 on the second day after landing. Virus specificity was confirmed by antibody staining and DNA analysis which showed it to be VZV of European descent, common in the US (5). Further, both antibody staining and DNA PCR demonstrated that no HSV-1 was detected in any infected culture. This is the first report of infectious VZV shedding in the absence of clinical disease

  19. Infectious salmon anaemia virus (ISAV) mucosal infection in Atlantic salmon.

    PubMed

    Aamelfot, Maria; McBeath, Alastair; Christiansen, Debes H; Matejusova, Iveta; Falk, Knut

    2015-01-01

    All viruses infecting fish must cross the surface mucosal barrier to successfully enter a host. Infectious salmon anaemia virus (ISAV), the causative agent of the economically important infectious salmon anaemia (ISA) in Atlantic salmon, Salmo salar L., has been shown to use the gills as its entry point. However, other entry ports have not been investigated despite the expression of virus receptors on the surface of epithelial cells in the skin, the gastrointestinal (GI) tract and the conjunctiva. Here we investigate the ISAV mucosal infection in Atlantic salmon after experimental immersion (bath) challenge and in farmed fish collected from a confirmed outbreak of ISA in Norway. We show for the first time evidence of early replication in several mucosal surfaces in addition to the gills, including the pectoral fin, skin and GI tract suggesting several potential entry points for the virus. Initially, the infection is localized and primarily infecting epithelial cells, however at later stages it becomes systemic, infecting the endothelial cells lining the circulatory system. Viruses of low and high virulence used in the challenge revealed possible variation in virus progression during infection at the mucosal surfaces. PMID:26490835

  20. Structural and growth characteristics of infectious bursal disease virus.

    PubMed Central

    Nick, H; Cursiefen, D; Becht, H

    1976-01-01

    The infectious bursal disease virus is not enveloped and has a diameter of 60 nm and a density of about 1.32 g/ml. It contains two pieces of single-stranded RNA with molecular weights close to 2 X 10(6). The capsid is made up of four major polypeptides with molecular weights of 110,000, 50,000, 35,000, and 25,000. The virus replicates in chicken embryo fibroblasts rather than in epitheloid cells. After an eclipse period of 4 h, virus production reaches a maximum about 12 h later. The virus has no structural or biological similarities with defined avian reoviruses, and it cannot be classified in one of the established taxonomic groups. Images PMID:176463

  1. The affect of infectious bursal disease virus on avian influenza virus vaccine efficacy

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Immunosuppressive viruses are known to affect vaccinal immunity, however the impact of virally induced immunosuppression on avian influenza vaccine efficacy has not been quantified. In order to determine the effect of exposure to infectious bursal disease virus (IBDV) on vaccinal immunity to highly ...

  2. Virus like particle-based vaccines against emerging infectious disease viruses.

    PubMed

    Liu, Jinliang; Dai, Shiyu; Wang, Manli; Hu, Zhihong; Wang, Hualin; Deng, Fei

    2016-08-01

    Emerging infectious diseases are major threats to human health. Most severe viral disease outbreaks occur in developing regions where health conditions are poor. With increased international travel and business, the possibility of eventually transmitting infectious viruses between different countries is increasing. The most effective approach in preventing viral diseases is vaccination. However, vaccines are not currently available for numerous viral diseases. Virus-like particles (VLPs) are engineered vaccine candidates that have been studied for decades. VLPs are constructed by viral protein expression in various expression systems that promote the selfassembly of proteins into structures resembling virus particles. VLPs have antigenicity similar to that of the native virus, but are non-infectious as they lack key viral genetic material. VLP vaccines have attracted considerable research interest because they offer several advantages over traditional vaccines. Studies have shown that VLP vaccines can stimulate both humoral and cellular immune responses, which may offer effective antiviral protection. Here we review recent developments with VLP-based vaccines for several highly virulent emerging or re-emerging infectious diseases. The infectious agents discussed include RNA viruses from different virus families, such as the Arenaviridae, Bunyaviridae, Caliciviridae, Coronaviridae, Filoviridae, Flaviviridae, Orthomyxoviridae, Paramyxoviridae, and Togaviridae families. PMID:27405928

  3. Spread of the newly emerging infectious laryngotracheitis viruses in Australia.

    PubMed

    Agnew-Crumpton, Rebecca; Vaz, Paola K; Devlin, Joanne M; O'Rourke, Denise; Blacker-Smith, Hayley Patricia; Konsak-Ilievski, Barbara; Hartley, Carol A; Noormohammadi, Amir H

    2016-09-01

    Infectious laryngotracheitis (ILT) is a significant viral disease of chickens in many countries around the globe. In this report the status of ILT in Australia has been used as a model to evaluate the evolution of the ILT viruses (ILTVs). Due to its geographical isolation, Australia harbored a distinct lineage of ILT viruses (ILTV) up to 2007. However examination of the ILT viruses (ILTV) involved in outbreaks between 2007 and 2009 has revealed that many of the outbreaks were caused by two new viral genotypes, class 8 and class 9. These two recombinant viruses were found to emerge as a result of recombination between previously existing live vaccine strains (SA2 and A20), and another live vaccine strain (Serva) introduced into the country in 2007. The new recombinant ILTVs were also shown to possess significantly higher virulence and replication capacity compared with a previously predominant ILTV, class 2. In the current study, examination of a large number of ILTVs isolated from outbreaks between 2009 and 2015 revealed the emergence of yet another recombinant virus (class 10) that appears to have become a predominant genotype in New South Wales. In Victoria however, the recombinant class 9 gradually became the predominant virus, replacing class 2. Therefore, there was an unusual pattern in geographical spread of the newly emerged viruses in different states of the country. These results suggest that ILTV is fast evolving towards a greater transmissibility and therefore greater capacity to spread into ILTV-free areas. PMID:27223632

  4. Infectious and Non-infectious Etiologies of Cardiovascular Disease in Human Immunodeficiency Virus Infection

    PubMed Central

    Chastain, Daniel B.; King, Travis S.; Stover, Kayla R.

    2016-01-01

    Background: Increasing rates of HIV have been observed in women, African Americans, and Hispanics, particularly those residing in rural areas of the United States. Although cardiovascular (CV) complications in patients infected with human immunodeficiency virus (HIV) have significantly decreased following the introduction of antiretroviral therapy on a global scale, in many rural areas, residents face geographic, social, and cultural barriers that result in decreased access to care. Despite the advancements to combat the disease, many patients in these medically underserved areas are not linked to care, and fewer than half achieve viral suppression. Methods: Databases were systematically searched for peer-reviewed publications reporting infectious and non-infectious etiologies of cardiovascular disease in HIV-infected patients. Relevant articles cited in the retrieved publications were also reviewed for inclusion. Results: A variety of outcomes studies and literature reviews were included in the analysis. Relevant literature discussed the manifestations, diagnosis, treatment, and outcomes of infectious and non-infectious etiologies of cardiovascular disease in HIV-infected patients. Conclusion: In these medically underserved areas, it is vital that clinicians are knowledgeable in the manifestations, diagnosis, and treatment of CV complications in patients with untreated HIV. This review summarizes the epidemiology and causes of CV complications associated with untreated HIV and provide recommendations for management of these complications. PMID:27583063

  5. Structure of equine infectious anemia virus matrix protein.

    PubMed

    Hatanaka, Hideki; Iourin, Oleg; Rao, Zihe; Fry, Elizabeth; Kingsman, Alan; Stuart, David I

    2002-02-01

    The Gag polyprotein is key to the budding of retroviruses from host cells and is cleaved upon virion maturation, the N-terminal membrane-binding domain forming the matrix protein (MA). The 2.8-A resolution crystal structure of MA of equine infectious anemia virus (EIAV), a lentivirus, reveals that, despite showing no sequence similarity, more than half of the molecule can be superimposed on the MAs of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV). However, unlike the structures formed by HIV-1 and SIV MAs, the oligomerization state observed is not trimeric. We discuss the potential of this molecule for membrane binding in the light of conformational differences between EIAV MA and HIV or SIV MA. PMID:11799182

  6. Structure of Equine Infectious Anemia Virus Matrix Protein

    PubMed Central

    Hatanaka, Hideki; Iourin, Oleg; Rao, Zihe; Fry, Elizabeth; Kingsman, Alan; Stuart, David I.

    2002-01-01

    The Gag polyprotein is key to the budding of retroviruses from host cells and is cleaved upon virion maturation, the N-terminal membrane-binding domain forming the matrix protein (MA). The 2.8-Å resolution crystal structure of MA of equine infectious anemia virus (EIAV), a lentivirus, reveals that, despite showing no sequence similarity, more than half of the molecule can be superimposed on the MAs of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV). However, unlike the structures formed by HIV-1 and SIV MAs, the oligomerization state observed is not trimeric. We discuss the potential of this molecule for membrane binding in the light of conformational differences between EIAV MA and HIV or SIV MA. PMID:11799182

  7. Phylogeography of infectious haematopoietic necrosis virus in North America

    USGS Publications Warehouse

    Kurath, Gael; Garver, Kyle A.; Troyer, Ryan M.; Emmenegger, Eveline J.; Einer-Jensen, Katja; Anderson, Eric D.

    2003-01-01

    Infectious hematopoietic necrosis virus (IHNV) is a rhabdoviral pathogen that infects wild and cultured salmonid fish throughout the Pacific Northwest of North America. IHNV causes severe epidemics in young fish and can cause disease or occur asymptomatically in adults. In a broad survey of 323 IHNV field isolates, sequence analysis of a 303 nucleotide variable region within the glycoprotein gene revealed a maximum nucleotide diversity of 8.6 %, indicating low genetic diversity overall for this virus. Phylogenetic analysis revealed three major virus genogroups, designated U, M and L, which varied in topography and geographical range. Intragenogroup genetic diversity measures indicated that the M genogroup had three- to fourfold more diversity than the other genogroups and suggested relatively rapid evolution of the M genogroup and stasis within the U genogroup. We speculate that factors influencing IHNV evolution may have included ocean migration ranges of their salmonid host populations and anthropogenic effects associated with fish culture.

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

    PubMed

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

    2011-11-01

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

  9. Construction of infectious cDNA clones for RNA viruses: Turnip crinkle virus.

    PubMed

    Ryabov, Eugene V

    2008-01-01

    Reverse genetic approach is widely used in virology as it makes possible direct identification of viral gene function and uses RNA genomes as vectors. Production of infectious cDNA clones is an essential step in developing a reverse genetic system for an RNA virus. Here, we present rapid method for generation of infectious cDNA clone for Turnip crinkle virus (TCV). The infectious cDNA clone could be used for production of in vitro transcripts with the T7 RNA polymerase which could be used for infection of plants or plant cell protoplasts. The procedure described here includes purification of TCV, viral RNA extraction, reverse transcription, PCR amplification of the full-length cDNA copy of TCV linked to a T7 RNA polymerase promoter, cloning into a plasmid vector, in vitro transcription, and selection of infectious clones. PMID:18370276

  10. Epidemiological characteristics of infectious hematopoietic necrosis virus (IHNV): a review.

    PubMed

    Dixon, Peter; Paley, Richard; Alegria-Moran, Raul; Oidtmann, Birgit

    2016-01-01

    Infectious hematopoietic necrosis virus (IHNV, Rhabdoviridae), is the causative agent of infectious hematopoietic necrosis (IHN), a disease notifiable to the World Organisation for Animal Health, and various countries and trading areas (including the European Union). IHNV is an economically important pathogen causing clinical disease and mortalities in a wide variety of salmonid species, including the main salmonid species produced in aquaculture, Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss). We reviewed the scientific literature on IHNV on a range of topics, including geographic distribution; host range; conditions required for infection and clinical disease; minimum infectious dose; subclinical infection; shedding of virus by infected fish; transmission via eggs; diagnostic tests; pathogen load and survival of IHNV in host tissues. This information is required for a range of purposes including import risk assessments; parameterisation of disease models; for surveillance planning; and evaluation of the chances of eradication of the pathogen to name just a few. The review focuses on issues that are of relevance for the European context, but many of the data summarised have relevance to IHN globally. Examples for application of the information is presented and data gaps highlighted. PMID:27287024

  11. Cloning and identification of the infectious salmon anaemia virus haemagglutinin.

    PubMed

    Krossøy, B; Devold, M; Sanders, L; Knappskog, P M; Aspehaug, V; Falk, K; Nylund, A; Koumans, S; Endresen, C; Biering, E

    2001-07-01

    Infectious salmon anaemia virus (ISAV) is an orthomyxo-like virus that causes serious disease in Atlantic salmon (Salmo salar). Like the orthomyxoviruses, ISAV has been shown to possess haemagglutinin (HA) activity. This study presents the cloning, expression and identification of the ISAV HA gene, which was isolated from a cDNA library by immunoscreening. The HA gene contained an ISAV-specific conserved nucleotide motif in the 5' region and a 1167 bp open reading frame encoding a protein with a predicted molecular mass of 42.4 kDa. The HA gene was expressed in a baculovirus system. A monoclonal antibody (MAb) shown previously to be directed against the ISAV HA reacted with insect cells infected with recombinant baculovirus. Salmon erythrocytes also adsorbed to these cells and adsorption was inhibited by the addition of either the ISAV-specific MAb or a polyclonal rabbit serum prepared against purified virus, confirming the virus specificity of the reaction. Immunoblot analyses indicated that ISAV HA, in contrast to influenza virus HA, is not posttranslationally cleaved. Sequence comparisons of the HA gene from five Norwegian, one Scottish and one Canadian isolate revealed a highly polymorphic region that may be useful in epidemiological studies. PMID:11413388

  12. Characterization of the equine infectious anaemia virus S2 protein.

    PubMed

    Yoon, S; Kingsman, S M; Kingsman, A J; Wilson, S A; Mitrophanous, K A

    2000-09-01

    S2 is an accessory protein of equine infectious anaemia virus (EIAV), the function of which is unknown. In order to gain insight into the function of S2, the intracellular localization of the protein, its interaction with viral proteins and its incorporation into viral particles have been investigated. Immunolocalization of S2 revealed punctate staining in the cytoplasm and the S2 protein co-precipitated with the EIAV Gag precursor. Despite overexpression of S2 through the use of a codon-optimized sequence, there was no preferential association of S2 with EIAV particles. These data suggest that S2 may function to organize the Gag protein during particle assembly in the cytoplasm but that it is unlikely to be involved in the early stages of the virus life-cycle. PMID:10950976

  13. Transcriptome analysis of feline infectious peritonitis virus infection.

    PubMed

    Mehrbod, Parvaneh; Harun, Mohammad Syamsul Reza; Shuid, Ahmad Naqib; Omar, Abdul Rahman

    2015-01-01

    Feline infectious peritonitis (FIP) is a lethal systemic disease caused by FIP virus (FIPV). There are no effective vaccines or treatment available, and the virus virulence determinants and pathogenesis are not fully understood. Here, we describe the sequencing of RNA extracted from Crandell Rees Feline Kidney (CRFK) cells infected with FIPV using the Illumina next-generation sequencing approach. Bioinformatics analysis, based on Felis catus 2X annotated shotgun reference genome, using CLC bio Genome Workbench is used to map both control and infected cells. Kal's Z test statistical analysis is used to analyze the differentially expressed genes from the infected CRFK cells. In addition, RT-qPCR analysis is used for further transcriptional profiling of selected genes in infected CRFK cells and Peripheral Blood Mononuclear Cells (PBMCs) from healthy and FIP-diagnosed cats. PMID:25720485

  14. Preliminary crystallographic analysis of avian infectious bronchitis virus main protease

    SciTech Connect

    Li, Jun; Shen, Wei; Liao, Ming; Bartlam, Mark

    2007-01-01

    The avian infectious bronchitis virus main protease has been crystallized; crystals diffract to 2.7 Å resolution. Infectious bronchitis virus (IBV) is the prototype of the genus Coronavirus. It causes a highly contagious disease which affects the respiratory, reproductive, neurological and renal systems of chickens, resulting great economic losses in the poultry industry worldwide. The coronavirus (CoV) main protease (M{sup pro}), which plays a pivotal role in viral gene expression and replication through a highly complex cascade involving the proteolytic processing of replicase polyproteins, is an attractive target for antiviral drug design. In this study, IBV M{sup pro} was overexpressed in Escherichia coli. Crystals suitable for X-ray crystallography have been obtained using microseeding techniques and belong to space group P6{sub 1}22. X-ray diffraction data were collected in-house to 2.7 Å resolution from a single crystal. The unit-cell parameters were a = b = 119.1, c = 270.7 Å, α = β = 90, γ = 120°. Three molecules were predicted to be present in the asymmetric unit from a calculated self-rotation function.

  15. Marek's Disease Virus As a Vectored Vaccine for Infectious Laryngotracheitis and Marek's Disease

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We replaced the MEQ gene from a bacterial artificial chromosome clone of Marek’s disease virus with gJ and gB genes from infectious laryngotracheitis virus. We will compare the efficacy of these vectored vaccines with commercial vaccines for Marek’s disease and infectious laryngotracheitis....

  16. Challenges and recent advancements in infectious laryngotracheitis virus vaccines.

    PubMed

    Coppo, Mauricio J C; Noormohammadi, Amir H; Browning, Glenn F; Devlin, Joanne M

    2013-01-01

    Over the past 80 years, biosecurity measures and vaccines have been used to prevent the occurrence of outbreaks of infectious laryngotracheitis (ILT). Despite these control strategies, ILT continues to have an impact on intensive poultry industries. Attenuated vaccines, particularly those derived by passage in chicken embryos, have been associated with a number of side effects, including residual virulence, transmission to naïve birds, establishment of latent infections with subsequent reactivation and shedding of virus, and reversion to virulence after in vivo passage. Most recently, recombination between attenuated ILT vaccines in the field has been shown to be responsible for the emergence of new virulent viruses that have caused widespread disease. To address some of these issues, new-generation virally vectored recombinant vaccines have been developed and recently released in some countries. In addition, recombinant deletion mutants of ILT virus have been proposed as vaccine candidates. In this review, recent advances in the understanding of the epidemiology of traditionally attenuated ILT vaccines as well as in the development and use of new generation vaccines are examined. Next-generation vaccines, along with more appropriate immunological screening strategies, are identified as particularly promising options to enhance ILT control in the future. PMID:23718807

  17. Recombination in Avian Gamma-Coronavirus Infectious Bronchitis Virus

    PubMed Central

    Thor, Sharmi W.; Hilt, Deborah A.; Kissinger, Jessica C.; Paterson, Andrew H.; Jackwood, Mark W.

    2011-01-01

    Recombination in the family Coronaviridae has been well documented and is thought to be a contributing factor in the emergence and evolution of different coronaviral genotypes as well as different species of coronavirus. However, there are limited data available on the frequency and extent of recombination in coronaviruses in nature and particularly for the avian gamma-coronaviruses where only recently the emergence of a turkey coronavirus has been attributed solely to recombination. In this study, the full-length genomes of eight avian gamma-coronavirus infectious bronchitis virus (IBV) isolates were sequenced and along with other full-length IBV genomes available from GenBank were analyzed for recombination. Evidence of recombination was found in every sequence analyzed and was distributed throughout the entire genome. Areas that have the highest occurrence of recombination are located in regions of the genome that code for nonstructural proteins 2, 3 and 16, and the structural spike glycoprotein. The extent of the recombination observed, suggests that this may be one of the principal mechanisms for generating genetic and antigenic diversity within IBV. These data indicate that reticulate evolutionary change due to recombination in IBV, likely plays a major role in the origin and adaptation of the virus leading to new genetic types and strains of the virus. PMID:21994806

  18. Morphology of certain viruses of Salmonid fishes. II. In vivo studies of infectious Hematopoietic Necrosis Virus

    USGS Publications Warehouse

    Amend, Donald F.; Chambers, Velma C.

    1970-01-01

    Juvenile sockeye salmon (Oncorhynchus nerka) were injected with the infectious hematopoietic necrosis (IHN) virus, and tissue samples from the anterior kidney, spleen, liver, intestine, and pyloric caeca of moribund fish were prepared for electron microscopy. Bullet-shaped virus particles measuring 158 × 90 mμ were observed in the hematopoietic tissues of the anterior kidney and spleen. Virus particles were also observed in the outer connective tissues of the pancreas or pyloric caeca, or both. No virus was found in the intestine or liver. The healthy appearance of erythrocytes, reticular cells, and endothelial cells in necrotic areas of the spleen and anterior kidney, and the absence of lymphocytes in these areas, suggested that lymphocytes might be one source of the virus.

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

    PubMed Central

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

    1995-01-01

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

  20. Immune complex vaccines for chicken infectious anemia virus.

    PubMed

    Schat, Karel A; Martins, Nelson Rodrigo da Silva; O'Connell, Priscilla H; Piepenbrink, Michael S

    2011-03-01

    Infection of maternal, antibody-negative chickens with chicken infectious anemia virus (CIAV) can cause clinical disease, while infection after maternal antibodies wane often results in subclinical infection and immunosuppression. Currently, vaccines are not available for vaccination in ovo or in newly hatched chickens. Development of CIAV vaccines for in ovo use depends on the ability to generate vaccines that do not cause lesions in newly hatched chicks and that can induce an immune response regardless of maternal immunity. Immune complex (IC) vaccines have been successfully used for control of infectious bursal disease, and we used a similar approach to determine if an IC vaccine is feasible for CIAV. Immune complexes were prepared that consisted of 0.1 ml containing 10(5.4) tissue culture infective dose 50% of CIA-1 and 0.1 ml containing 10 to 160 neutralizing units (IC Positive [ICP]10 to ICP160), in which one neutralizing unit is the reciprocal of the serum dilution required to protect 50% of CU147 cells from the cytopathic effects caused by CIA-1. Virus replication was delayed comparing ICP80 and ICP160 with combinations using negative serum (IC Negative [ICN]80 or ICN160). In addition, the number of birds with hematocrit values <28% were decreased with ICP80 or ICP160 compared to ICN80 or ICN160. Seroconversion was delayed in ICP80 and ICP160 groups. To determine if ICP80 or ICN 160 protected against challenge, we vaccinated maternal, antibody-free birds at 1 day of age and challenged at 2 wk or 3 wk of age with the 01-4201 strain. Both ICP80 and ICP160 protected against replication of the challenge virus, which was measured using differential quantitative PCR with primers distinguishing between the two isolates. Thus, in principle, immune complex vaccines may offer a method to protect newly hatched chicks against challenge with field virus. However, additional studies using maternal, antibody-positive chicks in combination with in ovo vaccination will be

  1. Molecular characterization of infectious bursal disease viruses from Pakistan.

    PubMed

    Shabbir, Muhammad Zubair; Ali, Muhammad; Abbas, Muhammad; Chaudhry, Umer Naveed; Zia-Ur-Rehman; Munir, Muhammad

    2016-07-01

    Since the first report of infectious bursal disease in Pakistan in 1987, outbreaks have been common even in vaccinated flocks. Despite appropriate administration of vaccines, concerns arise if the circulating strains are different from the ones used in the vaccine. Here, we sequenced the hypervariable region (HVR) of the VP2 gene of circulating strains of infectious bursal disease virus (IBDV) originating from outbreaks (n = 4) in broiler flocks in Pakistan. Nucleotide sequencing followed by phylogeny and deduced amino acid sequence analysis showed the circulating strains to be very virulent (vv) and identified characteristic residues at position 222 (A), 242 (I), 256 (I), 294 (I) and 299 (S). In addition, a substitution at positions 221 (Q→H) was found to be exclusive to Pakistani strains in our analysis, although a larger dataset is required to confirm this finding. Compared to vaccine strains that are commonly used in Pakistan, substitution mutations were found at key amino acid positions in VP2 that may be responsible for potential changes in neutralization epitopes and vaccine failure. PMID:27107876

  2. Hepatitis in skunks caused by the virus of infectious canine hepatitis.

    PubMed

    Karstad, L; Ramsden, R; Berry, T J; Binn, L N

    1975-10-01

    Two cases of acute, fatal, hepatitis occurred in young, striped skunks (Mephitis mephitis) trapped in southern Ontario. Histologically, lesions in the liver were similar to infectious canine hepatitis. A virus was isolated which produced large intranuclear inclusions in dog kidney cell cultures. These inclusions were Feulgen-positive and fluoresced green with acridine orange stain. The skunk hepatitis isolate was identified as the virus of infectious canine hepatitis by virus neutralization tests. PMID:172663

  3. Naturally occurring reassortant infectious bursal disease virus in northern China.

    PubMed

    Lu, Zhen; Zhang, Lizhou; Wang, Nian; Chen, Yuming; Gao, Li; Wang, Yongqiang; Gao, Honglei; Gao, Yulong; Li, Kai; Qi, Xiaole; Wang, Xiaomei

    2015-05-01

    Infectious bursal disease virus (IBDV) is a bi-segmented, double-stranded RNA virus that belongs to the genus Avibirnavirus of the family of Birnavirideae. The co-evolution of genome segments is a major evolutionary feature for IBDV. However, in recent years, some strains exhibited markedly different genetic relationships for segments A and B. In this study, we firstly isolated a new type of reassortment IBDV strain named IBD13HeB01 from northern China. The full-length genomes of segments A and B were cloned and identified. Sequence analysis revealed that IBD13HeB01 was a segment-reassortment strain, whose segment A was derived from very virulent strain and segment B from attenuated IBDV. In addition, the virulence of IBD13HeB01 strain was evaluated using SPF chickens. This study is not only beneficial for further understanding of the viral evolution but also suggests the potential risk of application of the live vaccines of IBDV. PMID:25869881

  4. Molecular and phenotypic characterization of infectious bursal disease virus isolates.

    PubMed

    Dormitorio, T V; Giambrone, J J; Guo, K; Jackwood, D J

    2007-06-01

    Two infectious bursal disease viruses (IBDVs 1174 and V1) were isolated from IBDV-vaccinated broiler flocks in California and Georgia. These flocks had a history of subclinical immunosuppression. These isolates are commonly used in IBDV progeny challenge studies at Auburn, AL, as well as vaccine manufacturer's vaccine efficacy studies, because they come from populated poultry-producing states, and are requested by poultry veterinarians from those states. Nested polymerase chain reaction (PCR) generated viral genome products for sequencing. A 491-bp segment from the VP2 gene, covering the hypervariable region, from each isolate was analyzed and compared with previously sequenced isolates. Sequence analysis showed that they were more closely related to the Delaware (Del) E antigenic variant than they are to the Animal Health Plant Inspection Service (APHIS) standard, both at the nucleotide level (96%, 97%) and at the amino acid level (94%, 97%). Both isolates had the glutamine to lysine shift in amino acid 249 which has been reported to be critical in binding the virus neutralizing Mab B69. Phenotypic studies showed that both isolates produced rapid atrophy of the bursae and weight loss, without the edematous bursal phase, in 2-wk-old commercial broilers having antibody against IBDV. A progeny challenge study showed both isolates produced more atrophy of the bursae (less percentage of protection) than the Del E isolate. Molecular and phenotypic data of these important IBDV isolates help in the improved detection and control of this continually changing and important viral pathogen of chickens. PMID:17626491

  5. Recombinant duck enteritis viruses expressing major structural proteins of the infectious bronchitis virus provide protection against infectious bronchitis in chickens.

    PubMed

    Li, Huixin; Wang, Yulong; Han, Zongxi; Wang, Yu; Liang, Shulin; Jiang, Lu; Hu, Yonghao; Kong, Xiangang; Liu, Shengwang

    2016-06-01

    To design an alternative vaccine for control of infectious bronchitis in chickens, three recombinant duck enteritis viruses (rDEVs) expressing the N, S, or S1 protein of infectious bronchitis virus (IBV) were constructed using conventional homologous recombination methods, and were designated as rDEV-N, rDEV-S, and rDEV-S1, respectively. Chickens were divided into five vaccinated groups, which were each immunized with one of the rDEVs, covalent vaccination with rDEV-N & rDEV-S, or covalent vaccination with rDEV-N & rDEV-S1, and a control group. An antibody response against IBV was detectable and the ratio of CD4(+)/CD8(+) T-lymphocytes decreased at 7 days post-vaccination in each vaccinated group, suggesting that humoral and cellular responses were elicited in each group as early as 7 days post-immunization. After challenge with a homologous virulent IBV strain at 21 days post-immunization, vaccinated groups showed significant differences in the percentage of birds with clinical signs, as compared to the control group (p < 0.01), as the two covalent-vaccination groups and the rDEV-S group provided better protection than the rDEV-N- or rDEV-S1-vaccinated group. There was less viral shedding in the rDEV-N & rDEV-S- (2/10) and rDEV-N & rDEV-S1- (2/10) vaccinated groups than the other three vaccinated groups. Based on the clinical signs, viral shedding, and mortality rates, rDEV-N & rDEV-S1 covalent vaccination conferred better protection than use of any of the single rDEVs. PMID:26946113

  6. 3D rotating wall vessel and 2D cell culture of four veterinary virus pathogens: A comparison of virus yields, portions of infectious particles and virus growth curves.

    PubMed

    Malenovská, Hana

    2016-02-01

    Only very few comparative studies have been performed that evaluate general trends of virus growth under 3D in comparison with 2D cell culture conditions. The aim of this study was to investigate differences when four animal viruses are cultured in 2D and 3D. Suid herpesvirus 1 (SuHV-1), Vesicular stomatitis virus (VSIV), Bovine adenovirus (BAdV) and Bovine parainfluenza 3 virus (BPIV-3) were cultivated in 3D rotating wall vessels (RWVs) and conventional 2D cultures. The production of virus particles, the portion of infectious particles, and the infectious growth curves were compared. For all viruses, the production of virus particles (related to cell density), including the non-infectious ones, was lower in 3D than in 2D culture. The production of only infectious particles was significantly lower in BAdV and BPIV-3 in 3D cultures in relation to cell density. The two cultivation approaches resulted in significantly different virus particle-to-TCID50 ratios in three of the four viruses: lower in SuHV-1 and BPIV-3 and higher in BAdV in 3D culture. The infectious virus growth rates were not significantly different in all viruses. Although 3D RWV culture resulted in lower production of virus particles compared to 2D systems, the portion of infectious particles was higher for some viruses. PMID:26562056

  7. Epstein-Barr virus, infectious mononucleosis, and posttransplant lymphoproliferative disorders.

    PubMed

    Nalesnik, M A; Starzl, T E

    1994-09-01

    PTLD may be considered as an "opportunistic cancer" in which the immunodeficiency state of the host plays a key role in fostering the environment necessary for abnormal lymphoproliferation. The following discussion reflects our own current thoughts regarding events which may result in PTLD and its sequelae. Many of the individual steps have not been rigorously proved or disproved at this point in time. Following transplantation and iatrogenic immunosuppression, the host:EBV equilibrium is shifted in favor of the virus. Most seronegative patients will become infected either via the graft or through natural means; seropositive patients will begin to shed higher levels of virus and may become secondarily superinfected via the graft. There is a "grace" period of approximately one month posttransplant before increased viral shedding begins. PTLD is almost never seen during this interval. In many cases infection continues to be silent whereas in rare individuals there is an overwhelming polyclonal proliferation of infected B lymphocytes. This is the parallel of infectious mononucleosis occurring in patients with a congenital defect in virus handling (X-linked lymphoproliferative disorder). It is possible that transplant patients with this presentation also suffer a defect in virus handling. In other cases excessive iatrogenic immunosuppression may paralyze their ability to respond to the infection. With CsA and FK506 regimens, individual tumors may occur within a matter of months following transplant. The short time of incubation suggests that these are less than fully developed malignancies. It may be that local events conspire to allow outgrowth of limited numbers of B-lymphocyte clones. A cytokine environment favoring B-lymphocyte growth may be one factor and differential inhibition by the immuno-suppressive drugs of calcium-dependent and -independent B-cell stimulation may be another. In addition, there is some evidence that CsA itself may inhibit apoptosis within B

  8. Prevalence of bovine virus diarrhoea and infectious bovine rhinotracheitis antibodies in Nigerian sheep and goats.

    PubMed

    Taylor, W P; Okeke, A N; Shidali, N N

    1977-08-01

    Neutralising antibodies to bovine virus diarrhoea virus were commoner in Nigerian sheep than goats while precipitating antibodies offered an alternative but less reliable indicator of previous infection. In contrast, neutralising antibodies to infectious bovine rhinotracheitis virus were more common in goats than sheep. These findings are discussed in relation to infectivity rates in cattle and general husbandry practices. PMID:410130

  9. Canarypox virus expressing infectious bursal disease VP2 protein as immunogen for chickens.

    PubMed

    Zanetti, Flavia Adriana; Grand, María Daniela Conte; Mitarotonda, Romina Cristina; Taboga, Oscar Alberto; Calamante, Gabriela

    2014-01-01

    Canarypox viruses (CNPV) carrying the coding sequence of VP2 protein from infectious bursal disease virus (IBDV) were obtained. These viruses were able to express VP2 protein in vitro and to induce IBDV-neutralizing antibodies when inoculated in specific pathogen-free chickens demonstrating that CNPV platform is usefulness to develop immunogens for chickens. PMID:24948937

  10. The Acyclic Retinoid Peretinoin Inhibits Hepatitis C Virus Replication and Infectious Virus Release in Vitro

    NASA Astrophysics Data System (ADS)

    Shimakami, Tetsuro; Honda, Masao; Shirasaki, Takayoshi; Takabatake, Riuta; Liu, Fanwei; Murai, Kazuhisa; Shiomoto, Takayuki; Funaki, Masaya; Yamane, Daisuke; Murakami, Seishi; Lemon, Stanley M.; Kaneko, Shuichi

    2014-04-01

    Clinical studies suggest that the oral acyclic retinoid Peretinoin may reduce the recurrence of hepatocellular carcinoma (HCC) following surgical ablation of primary tumours. Since hepatitis C virus (HCV) infection is a major cause of HCC, we assessed whether Peretinoin and other retinoids have any effect on HCV infection. For this purpose, we measured the effects of several retinoids on the replication of genotype 1a, 1b, and 2a HCV in vitro. Peretinoin inhibited RNA replication for all genotypes and showed the strongest antiviral effect among the retinoids tested. Furthermore, it reduced infectious virus release by 80-90% without affecting virus assembly. These effects could be due to reduced signalling from lipid droplets, triglyceride abundance, and the expression of mature sterol regulatory element-binding protein 1c and fatty acid synthase. These negative effects of Peretinoin on HCV infection may be beneficial in addition to its potential for HCC chemoprevention in HCV-infected patients.

  11. Efficacy of certain disinfectants against infectious pancreatic necrosis virus

    USGS Publications Warehouse

    Elliott, Diane G.; Amend, Donald F.

    1978-01-01

    The virucidal properties of iodophor, chlorine (sodium hypochlorite), formalin, thimerosal (organic mercurial compound), malachite green, and acriflavine were tested on infectious pancreatic necrosis virus (IPNV). Iodine and chlorine showed good activity, but efficacy depended on the concentration of virus, the presence of organic matter (calf serum), and water pH. Water hardness (0-300 mg 1−1 as CaCO3) did not affect virucidal activity. In a 5 min exposure, 4 mg 1−1available iodine inactivated 103.9 TCID50 m1−1 IPNV but 16 mg 1−1 iodine were needed for inactivation of 106.3TCID50m1−1. The addition of 0-5% calf serum significantly reduced the iodine concentration and the virucidal activity. In comparison, 4 mg 1−1 chlorine were needed to inactivate 1046 TCID50 m1−1 IPNV in 5 min. However, the addition of 0-07 % serum greatly reduced the chlorine concentration and extended the virucidal contact time to 30 min or more. IPNV at 106.3 TCID60 m1−1 was not inactivated by exposures for 60 min to 0-2% formalin, 10 min to 0-2% thimerosal, 60 min to 5 mg 1−1 malachite green, or 20 min to 500 mg 1−1 acriflavine. However, acriflavine at 0-5 mg 1−1 in cell culture media prevented the development of cytopathology caused by IPNV and may be useful in the treatment of the disease.

  12. Molecular Determinants of the Ratio of Inert to Infectious Virus Particles

    PubMed Central

    Klasse, P.J.

    2016-01-01

    The ratio of virus particles to infectious units is a classic measurement in virology and ranges widely from several million to below 10 for different viruses. Much evidence suggests a distinction be made between infectious and infecting particles or virions: out of many potentially infectious virions, few infect under regular experimental conditions, largely because of diffusion barriers. Still, some virions are inert from the start; others become defective through decay. And with increasing cell- and molecular-biological knowledge of each step in the replicative cycle for different viruses, it emerges that many processes entail considerable losses of potential viral infectivity. Furthermore, all-or-nothing assumptions about virion infectivity are flawed and should be replaced by descriptions that allow for spectra of infectious propensities. A more realistic understanding of the infectivity of individual virions has both practical and theoretical implications for virus neutralization, vaccine research, antiviral therapy, and the use of viral vectors. PMID:25595808

  13. Molecular determinants of the ratio of inert to infectious virus particles.

    PubMed

    Klasse, P J

    2015-01-01

    The ratio of virus particles to infectious units is a classic measurement in virology and ranges widely from several million to below 10 for different viruses. Much evidence suggests a distinction be made between infectious and infecting particles or virions: out of many potentially infectious virions, few infect under regular experimental conditions, largely because of diffusion barriers. Still, some virions are inert from the start; others become defective through decay. And with increasing cell- and molecular-biological knowledge of each step in the replicative cycle for different viruses, it emerges that many processes entail considerable losses of potential viral infectivity. Furthermore, all-or-nothing assumptions about virion infectivity are flawed and should be replaced by descriptions that allow for spectra of infectious propensities. A more realistic understanding of the infectivity of individual virions has both practical and theoretical implications for virus neutralization, vaccine research, antiviral therapy, and the use of viral vectors. PMID:25595808

  14. Variability Assessment of California Infectious Bronchitis Virus Variants.

    PubMed

    Gallardo, R A; Aleuy, O A; Pitesky, M; Sentíes-Cué, G; Abdelnabi, A; Woolcock, P R; Hauck, R; Toro, H

    2016-06-01

    On the basis of the data from the California Animal Health and Food Safety Laboratory System, 1444 infectious bronchitis (IB) cases were diagnosed between 1997 and 2012. Epidemiologic analyses demonstrated two major IB virus (IBV) outbreak peaks, affecting mainly 35-to-49-day-old broiler chickens. California variant 1737 (CA1737) and California variant 1999 (Cal 99) IBV types were the most prevalent genotypes during the analyzed period. To further understand the increased prevalence of these genotypes, we assessed and compared the variability of the S1 gene hypervariable region of CA1737 and Cal 99 with the variability of IBV strains belonging to the Massachusetts 41 (M41) and Arkansas (Ark) types during serial passages in embryonated chicken eggs. On the basis of the S1 nonsynonymous changes, seven different subpopulations were detected in M41. However, the predominant population of the field strain M41 before passages continued to be predominant throughout the experiment. In contrast, Ark passaging resulted in the detection of 13 different subpopulations, and the field sequence became extinct after the first passage. In IBV Cal 99, eight different subpopulations were detected; one of these became predominant after the second passage. In CA1737, 10 different subpopulations were detected. The field strain major sequence was not detected after the first passage but reappeared after the second passage and remained at low levels throughout the experiment. Compared with M41 and Ark, Cal 99 and CA1737 showed intermediate variability. PMID:27309282

  15. Soil-borne wheat mosaic virus infectious clone and manipulation for gene-carrying capacity.

    PubMed

    Jarugula, Sridhar; Charlesworth, Steven R; Qu, Feng; Stewart, Lucy R

    2016-08-01

    A full-length infectious cDNA clone of soil-borne wheat mosaic virus (SBWMV; genus Furovirus; family Virgaviridae) was developed for agrobacterium delivery. The cloned virus can be agroinfiltrated to Nicotiana benthamiana for subsequent infection of wheat (Triticum aestivum, L.). The utility of the virus as a vector for gene silencing and expression was assessed through sequence insertions in multiple sites of RNA2. Virus-induced photobleaching was observed in N. benthamiana but not in wheat, despite the stability of the inserts. The SBWMV infectious clone can be used for further studies to investigate the biology of SBWMV through mutagenesis. PMID:27236459

  16. Comparative nucleotide sequence analysis of three virulent strains of infectious laryngotracheitis virus (ILTV)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Infectious laryngotracheitis (ILT) is a very serious and widespread respiratory disease of chickens caused by gallid herpesvirus type 1, commonly named infectious laryngotracheitis virus (ILTV). For protection from ILT, chickens have traditionally been vaccinated with live-attenuated strains that ha...

  17. Understanding the role of ORF-C gene in the pathogenicity of infectious laryngotracheitis virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Infectious laryngotracheitis (ILT) is a very serious and widespread respiratory disease of chickens caused by infectious laryngotracheitis virus (ILTV). Conventional attenuated ILT vaccines, obtained by continuous passages in chicken embryos and tissue culture, had been the main tools utilized by th...

  18. Baculovirus virions displaying infectious bursal disease virus VP2 protein protect chickens against infectious bursal disease virus infection.

    PubMed

    Xu, Xin-Gang; Tong, De-Wen; Wang, Zhi Sheng; Zhang, Qi; Li, Zhao-Cai; Zhang, Kuan; Li, Wei; Liu, Hung-Jen

    2011-06-01

    Infectious bursal disease (IBD) is an acute and contagious viral infection of young chickens caused by IBD virus (IBDV). The VP2 protein of IBDV is the only antigen for inducing neutralizing antibodies and protective immunity in the natural host. In the current study, we have succeeded in construction of one recombinant baculovirus BacSC-VP2 expressing His6-tagged VP2 with the baculovirus envelope protein gp64 transmembrane domain (TM) and cytoplasmic domain (CTD). The His6-tagged recombinant VP2 was expressed and anchored on the plasma membrane of Sf-9 cells, as examined by western blot and confocal microscopy. Immunogold electron microscopy demonstrated that the VP2 protein of IBDV was successfully displayed on the viral surface. Vaccination of chickens with the VP2-pseudotyped baculovirus vaccine (BacSC-VP2) elicited significantly higher levels of VP2-specific enzyme-linked immunosorbent assay antibodies and neutralizing antibodies than the control groups. IBDV-specific proliferation of lymphocytes was observed in chickens immunized with the recombinant BacSC-VP2. An in vivo challenge study of the recombinant baculovirus BacSC-VP2 showed effective protection against a very virulent (vv) IBDV infection in chickens. In addition, mortality and gross and histopathological findings in the bursa demonstrated the efficacy of the vaccine in reducing virulence of the disease. These results indicate that the recombinant baculovirus BacSC-VP2 can be a potential vaccine against IBDV infections. PMID:21793437

  19. Immunogenicity of West Nile virus infectious DNA and its noninfectious derivatives

    SciTech Connect

    Seregin, Alexey; Nistler, Ryan; Borisevich, Victoria; Yamshchikov, Galina; Chaporgina, Elena; Kwok, Chun Wai; Yamshchikov, Vladimir . E-mail: yaximik@ku.edu

    2006-12-20

    The exceptionally high virulence of the West Nile NY99 strain makes its suitability in the development of a live WN vaccine uncertain. The aim of this study is to investigate the immunogenicity of noninfectious virus derivatives carrying pseudolethal mutations, which preclude virion formation without affecting preceding steps of the viral infectious cycle. When administered using DNA immunization, such constructs initiate an infectious cycle but cannot lead to a viremia. While the magnitude of the immune response to a noninfectious replication-competent construct was lower than that of virus or infectious DNA, its overall quality and the protective effect were similar. In contrast, a nonreplicating construct of similar length induced only a marginally detectable immune response in the dose range used. Thus, replication-competent noninfectious constructs derived from infectious DNA may offer an advantageous combination of the safety of noninfectious formulations with the quality of the immune response characteristic of infectious vaccines.

  20. Apoptosis transcriptional mechanism of feline infectious peritonitis virus infected cells.

    PubMed

    Shuid, Ahmad Naqib; Safi, Nikoo; Haghani, Amin; Mehrbod, Parvaneh; Haron, Mohd Syamsul Reza; Tan, Sheau Wei; Omar, Abdul Rahman

    2015-11-01

    Apoptosis has been postulated to play an important role during feline infectious peritonitis virus (FIPV) infection; however, its mechanism is not well characterized. This study is focused on apoptosis and transcriptional profiling of FIPV-infected cells following in vitro infection of CRFK cells with FIPV 79-1146 WSU. Flow cytometry was used to determine mode of cell death in first 42 h post infection (hpi). FIPV infected cells underwent early apoptosis at 9 hpi (p < 0.05) followed by late apoptosis at 12 hpi (p < 0.05) and necrosis from 24 hpi (p < 0.05). Then, next generation sequencing was performed on 9 hpi and control uninfected cells by Illumina analyzer. An aggregate of 4546 genes (2229 down-regulated and 2317 up-regulated) from 17 cellular process, 11 molecular functions and 130 possible biological pathways were affected by FIPV. 131 genes from apoptosis cluster (80 down-regulated and 51 up-regulated) along with increase of apoptosis, p53, p38 MAPK, VEGF and chemokines/cytokines signaling pathways were probably involved in apoptosis process. Six of the de-regulated genes expression (RASSF1, BATF2, MAGEB16, PDCD5, TNFα and TRAF2) and TNFα protein concentration were analyzed by RT-qPCR and ELISA, respectively, at different time-points. Up-regulations of both pro-apoptotic (i.e. PDCD5) and anti-apoptotic (i.e. TRAF2) were detected from first hpi and continuing to deregulate during apoptosis process in the infected cells. PMID:26386572

  1. First complete genome sequence of infectious laryngotracheitis virus

    PubMed Central

    2011-01-01

    Background Infectious laryngotracheitis virus (ILTV) is an alphaherpesvirus that causes acute respiratory disease in chickens worldwide. To date, only one complete genomic sequence of ILTV has been reported. This sequence was generated by concatenating partial sequences from six different ILTV strains. Thus, the full genomic sequence of a single (individual) strain of ILTV has not been determined previously. This study aimed to use high throughput sequencing technology to determine the complete genomic sequence of a live attenuated vaccine strain of ILTV. Results The complete genomic sequence of the Serva vaccine strain of ILTV was determined, annotated and compared to the concatenated ILTV reference sequence. The genome size of the Serva strain was 152,628 bp, with a G + C content of 48%. A total of 80 predicted open reading frames were identified. The Serva strain had 96.5% DNA sequence identity with the concatenated ILTV sequence. Notably, the concatenated ILTV sequence was found to lack four large regions of sequence, including 528 bp and 594 bp of sequence in the UL29 and UL36 genes, respectively, and two copies of a 1,563 bp sequence in the repeat regions. Considerable differences in the size of the predicted translation products of 4 other genes (UL54, UL30, UL37 and UL38) were also identified. More than 530 single-nucleotide polymorphisms (SNPs) were identified. Most SNPs were located within three genomic regions, corresponding to sequence from the SA-2 ILTV vaccine strain in the concatenated ILTV sequence. Conclusions This is the first complete genomic sequence of an individual ILTV strain. This sequence will facilitate future comparative genomic studies of ILTV by providing an appropriate reference sequence for the sequence analysis of other ILTV strains. PMID:21501528

  2. Scientists Try to Stop Another Deadly Virus

    MedlinePlus

    ... Try to Stop Another Deadly Virus Junin, an Ebola-like disease in Argentina, has a death rate ... companies that developed a similar treatment against the Ebola virus during the 2014-2015 outbreak. That drug, ...

  3. Detection of Persistent Chikungunya Virus RNA but not Infectious Virus in Experimental Vertical Transmission in Aedes aegypti from Malaysia.

    PubMed

    Wong, Hui Vern; Vythilingam, Indra; Sulaiman, Wan Yusof Wan; Lulla, Aleksei; Merits, Andres; Chan, Yoke Fun; Sam, I-Ching

    2016-01-01

    Vertical transmission may contribute to the maintenance of arthropod-borne viruses, but its existence in chikungunya virus (CHIKV) is unclear. Experimental vertical transmission of infectious clones of CHIKV in Aedes aegypti mosquitoes from Malaysia was investigated. Eggs and adult progeny from the second gonotrophic cycles of infected parental mosquitoes were tested. Using polymerase chain reaction (PCR), 56.3% of pooled eggs and 10% of adult progeny had detectable CHIKV RNA, but no samples had detectable infectious virus by plaque assay. Transfected CHIKV RNA from PCR-positive eggs did not yield infectious virus in BHK-21 cells. Thus, vertical transmission of viable CHIKV was not demonstrated. Noninfectious CHIKV RNA persists in eggs and progeny of infected Ae. aegypti, but the mechanism and significance are unknown. There is insufficient evidence to conclude that vertical transmission exists in CHIKV, as positive results reported in previous studies were almost exclusively based only on viral RNA detection. PMID:26598564

  4. Novel Cell Culture-Adapted Genotype 2a Hepatitis C Virus Infectious Clone

    PubMed Central

    Date, Tomoko; Kato, Takanobu; Kato, Junko; Takahashi, Hitoshi; Morikawa, Kenichi; Akazawa, Daisuke; Murayama, Asako; Tanaka-Kaneko, Keiko; Sata, Tetsutaro; Tanaka, Yasuhito; Mizokami, Masashi

    2012-01-01

    Although the recently developed infectious hepatitis C virus system that uses the JFH-1 clone enables the study of whole HCV viral life cycles, limited particular HCV strains have been available with the system. In this study, we isolated another genotype 2a HCV cDNA, the JFH-2 strain, from a patient with fulminant hepatitis. JFH-2 subgenomic replicons were constructed. HuH-7 cells transfected with in vitro transcribed replicon RNAs were cultured with G418, and selected colonies were isolated and expanded. From sequencing analysis of the replicon genome, several mutations were found. Some of the mutations enhanced JFH-2 replication; the 2217AS mutation in the NS5A interferon sensitivity-determining region exhibited the strongest adaptive effect. Interestingly, a full-length chimeric or wild-type JFH-2 genome with the adaptive mutation could replicate in Huh-7.5.1 cells and produce infectious virus after extensive passages of the virus genome-replicating cells. Virus infection efficiency was sufficient for autonomous virus propagation in cultured cells. Additional mutations were identified in the infectious virus genome. Interestingly, full-length viral RNA synthesized from the cDNA clone with these adaptive mutations was infectious for cultured cells. This approach may be applicable for the establishment of new infectious HCV clones. PMID:22787209

  5. Detection and identification of infectious bronchitis virus by RT-PCR in Iran.

    PubMed

    Homayounimehr, Alireza; Pakbin, Ahmad; Momayyez, Reza; Fatemi, Seyyedeh Mahsa Rastegar

    2016-06-01

    Infectious bronchitis virus (IBV) causes severe diseases in poultry with significant economic consequences to the poultry industry in Iran. The aim of this study was the detection and identification of IBV by reverse transcription(RT)-PCR in Iran. Ten IB virus strains were detected by testing trachea, cecal tonsil, and kidney tissues collected from broiler and layer farms in Iran. In order to detect infectious bronchitis virus, an optimized RT-PCR was used. Primers targeting the conserved region of known IBV serotypes were used in the RT-PCR assay. Primers selectively detecting Massachusetts and 793/B type IB viruses were designed to amplify the S1 gene of the virus and used in the nested PCR test. Our findings indicate the circulation of at least three genotypes of IB viruses (Massachusetts, 793/B, and variant 2) among poultry flocks. PMID:27010714

  6. Kinetics of transcription of infectious laryngotracheitis virus genes.

    PubMed

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

    2012-03-01

    The kinetics of expression of only a few genes of infectious laryngotracheitis virus (ILTV) have been determined, using northern blot analysis. We used quantitative reverse transcriptase PCR to examine the kinetics of expression of 74 ILTV genes in LMH cells. ICP4 was the only gene fully expressed in the presence of cycloheximide, and thus classified as immediate-early. The genes most highly expressed early in infection, and thus classified as early, included UL1 (gL), UL2, UL3, UL4, UL5, UL6, UL7, UL8, UL13, UL14, UL19, UL20, UL23 (TK), UL25, UL28, UL29, UL31, UL33, UL34, UL38, UL39, UL40, UL42, UL43, UL44 (gC), UL47, UL48 (α-TIF), UL49, UL54 (ICP27), US3 and US10. ORF A, ORF B, ORF C, ORF E, sORF 4/3, UL[-1], UL0, UL3.5, UL9, UL10 (gM), UL11, UL15a, UL15b, UL18, UL22 (gH), UL24, UL26, UL30, UL32, UL36, UL45, UL49.5 (gN), UL52, US2, US4 (gG), US5 (gJ) and US9 were most highly expressed late in infection and were thus considered late genes. Several genes, including ORF D, UL12, UL17, UL21, UL27 (gB), UL35, UL37, UL41, UL46, UL50, UL51, UL53 (gK), US8 (gE), US6 (gD) and US7 (gI), had features of both early and late genes and were classified as early/late. Our findings suggest transcription from most of ILTV genes is leaky or subject to more complex patterns of regulation than those classically described for herpesviruses. This is the first study examining global expression of ILTV genes and the data provide a basis for future investigations of the pathogenesis of infection with ILTV. PMID:22195977

  7. Effects of chicken anaemia virus and infectious bursal disease virus-induced immunodeficiency on infectious bronchitis virus replication and genotypic drift.

    PubMed

    Gallardo, Rodrigo A; van Santen, Vicky L; Toro, Haroldo

    2012-10-01

    We followed changes in a portion of the S1 gene sequence of the dominant populations of an infectious bronchitis virus (IBV) Arkansas (Ark) vaccine strain during serial passage in chickens infected with the immunosuppressive chicken anaemia virus (CAV) and/or infectious bursal disease virus (IBDV) as well as in immunocompetent chickens. The IBV-Ark vaccine was applied ocularly and tears were collected from infected chickens for subsequent ocular inoculation in later passages. The experiment was performed twice. In both experiments the dominant S1 genotype of the vaccine strain was rapidly and negatively selected in all chicken groups (CAV, IBDV, CAV+IBDV and immunocompetent). Based on the S1 genotype, the same IBV subpopulations previously reported in immunocompetent chickens and named component (C) 1 to C5 emerged both in immunocompetent and immunodeficient chickens. During the first passage different subpopulations emerged, followed by the establishment of one or two predominant populations after further passages. Only when the subpopulation designated C2 became established in either CAV-infected or IBDV-infected chickens was IBV maintained for more than four passages. These results indicate that selection does not cease in immunodeficient chickens and that phenotype C2 may show a distinct adaptation to this environment. Subpopulations C1 or C4 initially became established in immunocompetent birds but became extinct after only a few succeeding passages. A similar result was observed in chickens co-infected with CAV+IBDV. These results suggest that the generation of genetic diversity in IBV is constrained. This finding constitutes further evidence for phenotypic drift occurring mainly as a result of selection. PMID:22897690

  8. Intracellular proteins of feline immunodeficiency virus and their antigenic relationship with equine infectious anaemia virus proteins.

    PubMed

    Egberink, H F; Ederveen, J; Montelaro, R C; Pedersen, N C; Horzinek, M C; Koolen, M J

    1990-03-01

    Feline immunodeficiency virus (FIV) grown in cat lymphocyte and thymocyte cultures was labelled with L-[35S]methionine or [3H]glucosamine and virus-coded proteins were identified using immunoprecipitation. Polypeptides with apparent Mr values of 15K, 24K, 43K, 50K, 120K and 160K were detected. An additional polypeptide of 10K was detected by Western blot analysis. The two highest Mr species sometimes appeared as one band, of which only the 120K polypeptide was glycosylated. In the presence of tunicamycin gp120 was no longer detectable and a non-glycosylated precursor of 75K was found instead. Pulse-chase experiments suggested that the smaller polypeptides p24 and p15 are cleavage products of both p160 and p50. Western blot analysis using a rabbit serum directed against p26 of equine infectious anaemia virus (EIAV) and an anti-EIAV horse serum from a field case of infection revealed a cross-reactivity with p24 of FIV. Cat sera collected late after experimental FIV infection recognized p26 of EIAV, indicating a reciprocal cross-reactivity. PMID:1690264

  9. Protection induced by infectious laryngotracheitis virus vaccines alone and combined with Newcastle disease virus and/or infectious bronchitis virus vaccines.

    PubMed

    Vagnozzi, Ariel; García, Maricarmen; Riblet, Sylva M; Zavala, Guillermo

    2010-12-01

    Two types of live attenuated vaccines have been used worldwide for the control of infectious laryngotracheitis virus (ILTV): 1) chicken embryo origin (CEO) vaccines; and 2) tissue culture origin vaccines (TCO). However, the disease persists in spite of extensive use of vaccination, particularly in areas of intense broiler production. Among the factors that may influence the efficiency of ILTV live attenuated vaccines is a possible interference of Newcastle Disease virus (NDV) and infectious bronchitis virus (IBV) vaccines with the protection induced by ILTV vaccines. The protection induced by CEO and TCO vaccines was evaluated when administered at 14 days of age alone or in combination with the B1 type strain of NDV (B1) and/or the Arkansas (ARK) and Massachusetts (MASS) serotypes of IBV vaccines. Two weeks after vaccination (28 days of age), the chickens were challenged with a virulent ILTV field strain (63140 isolate, group V genotype). Protection was evaluated at 5 and 7 days postchallenge by scoring clinical signs and quantifying the challenge virus load in the trachea using real-time PCR (qPCR). In addition, the viral load of the vaccine viruses (ILTV, NDV, and IBV) was quantified 3 and 5 days postvaccination also using qPCR. The results of this study indicate that the NDV (B1) and IBV (ARK) vaccines and a multivalent vaccine constituted by NDV (B1) and IBV (ARK and MASS) did not interfere with the protection induced by the CEO ILTV vaccine. However, the NDV (BI) and the multivalent (B1/MASS/ARK) vaccines interfered with the protection induced by the TCO vaccine (P < 0.05). Either in combination or by themselves, the NDV and IBV vaccines decreased the tracheal replication of the TCO vaccine and the protection induced by this vaccine, since the ILTV-vaccinated and -challenged chickens displayed significantly more severe clinical signs and ILTV load (P < 0.05) than chickens vaccinated with the TCO vaccine alone. Although NDV and IBV challenges were not performed

  10. Detection and transmission of infectious hematopoietic necrosis virus in rainbow trout

    USGS Publications Warehouse

    Amend, Donald F.

    1975-01-01

    Detection and transmission of Infectious Hematopoietic Necrosis Virus in rainbow trout (Salmo gairdneri) was studied at a commercial trout hatchery. Transmission of virus was demonstrated via water, feed and contaminated eggs. If eggs from carrier females were incubated several weeks in virus-free water, the resulting fry did not become infected. However, if fry subsequently became infected they were lifetime carriers. Infectious virus was readily detectable in most tissues of moribund fish; in carriers it was detected in sex products of spawning fish, and in samples from the intestine of post-spawning fish, but not in samples from blood, feces, kidney, or liver. The carrier rate was not significantly different between sexes. It was concluded that adult carriers are the reservoir of infection and that transmission occurs primarily when carriers shed virus and expose susceptible fish or eggs.

  11. Plasmodium knowlesi Sporozoite Antigen: Expression by Infectious Recombinant Vaccinia Virus

    NASA Astrophysics Data System (ADS)

    Smith, Geoffrey L.; Godson, G. Nigel; Nussenzweig, Victor; Nussenzweig, Ruth S.; Barnwell, John; Moss, Bernard

    1984-04-01

    The gene coding for the circumsporozoite antigen of the malaria parasite Plasmodium knowlesi was inserted into the vaccinia virus genome under the control of a defined vaccinia virus promoter. Cells infected with the recombinant virus synthesized polypeptides of 53,000 to 56,000 daltons that reacted with monoclonal antibody against the repeating epitope of the malaria protein. Furthermore, rabbits vaccinated with the recombinant virus produced antibodies that bound specifically to sporozoites. These data provide evidence for expression of a cloned malaria gene in mammalian cells and illustrate the potential of vaccinia virus recombinants as live malaria vaccines.

  12. Full-length infectious clone of a low passage dengue virus serotype 2 from Brazil

    PubMed Central

    Santos, Jefferson José da Silva; Magalhães, Tereza; Silva, José Valter Joaquim; da Silva, Andréa Nazaré Monteiro Rangel; Cordeiro, Marli Tenório; Gil, Laura Helena Vega Gonzales

    2015-01-01

    Full-length dengue virus (DENV) cDNA clones are an invaluable tool for many studies, including those on the development of attenuated or chimeric vaccines and on host-virus interactions. Furthermore, the importance of low passage DENV infectious clones should be highlighted, as these may harbour critical and unique strain-specific viral components from field-circulating isolates. The successful construction of a functional Brazilian low passage DENV serotype 2 full-length clone through homologous recombination reported here supports the use of a strategy that has been shown to be highly useful by our group for the development of flavivirus infectious clones and replicons. PMID:26200712

  13. [Complete genomic analysis of a novel infectious bronchitis virus isolate].

    PubMed

    Hu, Bei-Xia; Yang, Shao-Hua; Zhang, Xiu-Mei; Zhang, Wei; Cao, San-Jie; Xu, Chuan-Tian; Huang, Qing-Hua; Zhang, Lin; Huang, Yan-Yan; Wen, Xin-Tian

    2014-07-01

    The genome of CK/CH/SD09/005, an isolate of infectious bronchitis virus (IBV), was characterized to enable the further understanding of the epidemiology and evolution of IBV in China. Twenty-five pairs of primers were designed to amplify the full-length genome of CK/CH/SD09/005. The nucleotide sequence of CK/CH/SD09/005 was compared with reference IBV strains retrieved from GenBank. The phylogenic relationship between CK/CH/SD09/005 and the reference strains was analyzed based on S1 gene sequences. The complete genome of CK/CH/SD09/005 consisted of 27691 nucleotides (nt), excluding the 5' cap and 3' poly A tail. The whole-genome of CK/CH/SD09/005 shared 97 - 99% nucleotide sequence homology with the GX-NN09032 strain, which was the only complete genome that was closely related to CK/CH/SD09/005. When compared with all reference strains except GX-NN09032, CK/CH/SD09/005 showed the highest similarity to ck/CH/LDL/091022 and SDIB821/2012 (QX-like) in the replicase gene (Gene 1) and 3'UTR, with a sequence identity rate of 97% and 98%, respectively. However, CK/CH/SD09/005 exhibited lower levels of similarity with ck/CH/LDL/091022 and SDIB821/2012 in S-3a-3b-3c/ E-M-5a-5b-N with a sequence identity of 72% - 90%. CK/CH/SD09/005 showed the highest level of nucleotide identity with Korean strain 1011, and Chinese strains CK/CH/LXJ/02I, DK/CH/HN/ZZ2004 and YX10, in ORF 3c/E (97%), 5a (96%), 5b (99%) and N (96%), respectively. ORFs 3a, 3b and M of CK/CH/SD09/005 exhibited no more than 90% homology with the reference strains, excluding GX-NN09032. The phylogenic analysis based on the S1 gene revealed that CK/CH/SD09/005 and 39 published strains were classified into seven clades (genotypes). CK/CH/SD09/005 was distributed in clade IV with several isolates collected between 2007 and 2012. CK/CH/SD09/005 showed 66% - 69% and 72% - 81% nucleotide identities with the IBV strains of other six clades in the S1 and S2 subunits, respectively. More over, multiple substitutions were

  14. Infectious Chikungunya Virus in the Saliva of Mice, Monkeys and Humans

    PubMed Central

    Gardner, Joy; Rudd, Penny A.; Prow, Natalie A.; Belarbi, Essia; Roques, Pierre; Larcher, Thibaut; Gresh, Lionel; Balmaseda, Angel; Harris, Eva; Schroder, Wayne A.; Suhrbier, Andreas

    2015-01-01

    Chikungunya virus (CHIKV) is a reemerging, ordinarily mosquito-transmitted, alphavirus that occasionally produces hemorrhagic manifestations, such as nose bleed and bleeding gums, in human patients. Interferon response factor 3 and 7 deficient (IRF3/7-/-) mice, which are deficient for interferon α/β responses, reliably develop hemorrhagic manifestations after CHIKV infection. Here we show that infectious virus was present in the oral cavity of CHIKV infected IRF3/7-/- mice, likely due to hemorrhagic lesions in the olfactory epithelium that allow egress of infected blood into the nasal, and subsequently, oral cavities. In addition, IRF3/7-/- mice were more susceptible to infection with CHIKV via intranasal and oral routes, with IRF3/7-/- mice also able to transmit virus mouse-to-mouse without an arthropod vector. Cynomolgus macaques often show bleeding gums after CHIKV infection, and analysis of saliva from several infected monkeys also revealed the presence of viral RNA and infectious virus. Furthermore, saliva samples collected from several acute CHIKV patients with hemorrhagic manifestations were found to contain viral RNA and infectious virus. Oral fluids can therefore be infectious during acute CHIKV infections, likely due to hemorrhagic manifestations in the oral/nasal cavities. PMID:26447467

  15. Expert-novice differences in mental models of viruses, vaccines, and the causes of infectious disease.

    PubMed

    Jee, Benjamin D; Uttal, David H; Spiegel, Amy; Diamond, Judy

    2015-02-01

    Humans are exposed to viruses everywhere they live, play, and work. Yet people's beliefs about viruses may be confused or inaccurate, potentially impairing their understanding of scientific information. This study used semi-structured interviews to examine people's beliefs about viruses, vaccines, and the causes of infectious disease. We compared people at different levels of science expertise: middle school students, teachers, and professional virologists. The virologists described more entities involved in microbiological processes, how these entities behaved, and why. Quantitative and qualitative analyses revealed distinctions in the cognitive organization of several concepts, including infection and vaccination. For example, some students and teachers described viral replication in terms of cell division, independent of a host. Interestingly, most students held a mental model for vaccination in which the vaccine directly attacks a virus that is present in the body. Our findings have immediate implications for how to communicate about infectious disease to young people. PMID:23959975

  16. Expert-Novice Differences in Mental Models of Viruses, Vaccines, and the Causes of Infectious Disease

    PubMed Central

    Jee, Benjamin D.; Uttal, David H.; Spiegel, Amy; Diamond, Judy

    2014-01-01

    Humans are exposed to viruses everywhere they live, play, and work. Yet people’s beliefs about viruses may be confused or inaccurate, potentially impairing their understanding of scientific information. This study used semi-structured interviews to examine people’s beliefs about viruses, vaccines, and the causes of infectious disease. We compared people at different levels of science expertise: middle school students, teachers, and professional virologists. The virologists described more entities involved in microbiological processes, how these entities behaved, and why. Quantitative and qualitative analyses revealed distinctions in the cognitive organization of several concepts, including infection and vaccination. For example, some students and teachers described viral replication in terms of cell division, independent of a host. Interestingly, most students held a mental model for vaccination in which the vaccine directly attacks a virus that is present in the body. Our findings have immediate implications for how to communicate about infectious disease to young people. PMID:23959975

  17. S1 gene-based phylogeny of infectious bronchitis virus: An attempt to harmonize virus classification.

    PubMed

    Valastro, Viviana; Holmes, Edward C; Britton, Paul; Fusaro, Alice; Jackwood, Mark W; Cattoli, Giovanni; Monne, Isabella

    2016-04-01

    Infectious bronchitis virus (IBV) is the causative agent of a highly contagious disease that results in severe economic losses to the global poultry industry. The virus exists in a wide variety of genetically distinct viral types, and both phylogenetic analysis and measures of pairwise similarity among nucleotide or amino acid sequences have been used to classify IBV strains. However, there is currently no consensus on the method by which IBV sequences should be compared, and heterogeneous genetic group designations that are inconsistent with phylogenetic history have been adopted, leading to the confusing coexistence of multiple genotyping schemes. Herein, we propose a simple and repeatable phylogeny-based classification system combined with an unambiguous and rationale lineage nomenclature for the assignment of IBV strains. By using complete nucleotide sequences of the S1 gene we determined the phylogenetic structure of IBV, which in turn allowed us to define 6 genotypes that together comprise 32 distinct viral lineages and a number of inter-lineage recombinants. Because of extensive rate variation among IBVs, we suggest that the inference of phylogenetic relationships alone represents a more appropriate criterion for sequence classification than pairwise sequence comparisons. The adoption of an internationally accepted viral nomenclature is crucial for future studies of IBV epidemiology and evolution, and the classification scheme presented here can be updated and revised novel S1 sequences should become available. PMID:26883378

  18. Topoisomerase I activity associated with human immunodeficiency virus (HIV) particles and equine infectious anemia virus core.

    PubMed Central

    Priel, E; Showalter, S D; Roberts, M; Oroszlan, S; Segal, S; Aboud, M; Blair, D G

    1990-01-01

    In the present study, we found a topoisomerase I (topo I) activity in two strains of human immunodeficiency virus type 1 (HIV-1) and equine infectious anemia virus (EIAV) particles. The topo I activity was located in the EIAV cores and differed from the cellular topo I in its ionic requirements and response to ATP, indicating that these were two distinct forms of this enzyme. Topo I activity was removed from the viral lysates and viral cores by anti-topo I antiserum. The only protein recognized by this antiserum was an 11.5 kd protein in HIV lysate and 11 kd in EIAV lysate. We showed that the 11 kd protein recognized by the anti-topo I antiserum is the EIAV p11 nucleocapsid protein. Furthermore, purified topo I protein blocked the binding of the antibodies to the p11 protein and vice versa, purified p11 protein blocked the binding of these antibodies to the cellular topo I. These results suggest that the EIAV p11 nucleocapsid protein and the cellular topo I share similar epitopes. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. PMID:2174357

  19. Infectious Entry by Amphotropic as well as Ecotropic Murine Leukemia Viruses Occurs through an Endocytic Pathway

    PubMed Central

    Katen, Louis J.; Januszeski, Michael M.; Anderson, W. French; Hasenkrug, Kim J.; Evans, Leonard H.

    2001-01-01

    Infectious entry of enveloped viruses is thought to proceed by one of two mechanisms. pH-dependent viruses enter the cells by receptor-mediated endocytosis and are inhibited by transient treatment with agents that prevent acidification of vesicles in the endocytic pathway, while pH-independent viruses are not inhibited by such agents and are thought to enter the cell by direct fusion with the plasma membrane. Nearly all retroviruses, including amphotropic murine leukemia virus (MuLV) and human immunodeficiency virus type 1, are classified as pH independent. However, ecotropic MuLV is considered to be a pH-dependent virus. We have examined the infectious entry of ecotropic and amphotropic MuLVs and found that they were equally inhibited by NH4Cl and bafilomycin A. These agents inhibited both viruses only partially over the course of the experiments. Agents that block the acidification of endocytic vesicles also arrest vesicular trafficking. Thus, partial inhibition of the MuLVs could be the result of virus inactivation during arrest in this pathway. In support of this contention, we found that that the loss of infectivity of the MuLVs during treatment of target cells with the drugs closely corresponded to the loss of activity due to spontaneous inactivation at 37°C in the same period of time. Furthermore, the drugs had no effect on the efficiency of infection under conditions in which the duration of infection was held to a very short period to minimize the effects of spontaneous inactivation. These results indicate that the infectious processes of both ecotropic and amphotropic MuLVs were arrested rather than aborted by transient treatment of the cells with the drugs. We also found that infectious viruses were efficiently internalized during treatment. This indicated that the arrest occurred in an intracellular compartment and that the infectious process of both the amphotropic and ecotropic MuLVs very likely involved endocytosis. An important aspect of this study

  20. Multiplex nested RT-PCR for detecting avian influenza virus, infectious bronchitis virus and Newcastle disease virus.

    PubMed

    Nguyen, Thanh Trung; Kwon, Hyuk-Joon; Kim, Il-Hwan; Hong, Seung-Min; Seong, Won-Jin; Jang, Jin-Wook; Kim, Jae-Hong

    2013-03-01

    In this study, multiplex nested RT-PCR (mnRT-PCR) was applied to simultaneous detect multiplex PCR with the higher sensitivity of nested PCR that is required for avian influenza, infectious bronchitis and Newcastle disease virus using two steps of amplification. For the first PCR, primers that were specific for each virus were newly designed from the nucleoprotein gene of AIV, the nucleocapsid protein gene of IBV and the fusion protein gene of NDV to amplify products of 665, 386 and 236 nucleotides, respectively. The multiplex PCR step provides mass amplification using common primers, which increased markedly the sensitivity of the test. Non-specific reactions were not observed when other viruses and bacteria were used for evaluating the mnRT-PCR. As a field application, 172 samples were tested by RT-PCR and mnRT-PCR. Among these samples, the concordance rates for mnRT-PCR and the single conventional RT-PCR showed 98.9% (kappa=0.98) and 98.8% (kappa=0.96) similarity for IBV and AIV, respectively. As a result, it is recommended the multiplex nested PCR as an effective tool for detecting and studying the molecular epidemiology of various mixed infections of one or more of these viruses in poultry. PMID:23261801

  1. Persistence of antibodies and anamnestic response in calves vaccinated with inactivated infectious bovine rhinotracheitis virus and parainfluenza-3 virus vaccines.

    PubMed

    Sweat, R L

    1983-04-15

    Persistence of antibodies in calves vaccinated with 2 types of inactivated infectious bovine rhinotracheitis (IBR) virus and parainfluenza-3 (PI-3) virus vaccines were determined. Calves seronegative for IBR and PI-3 viruses were inoculated with 2 doses of inactivated IBR virus-PI-3 virus vaccines administered 2 weeks apart. Blood samples were obtained from the calves for serum at 2 weeks, 6 months, and 1 year after vaccination. The serums were tested by serum-neutralization tests. Antibody response to the vaccines persisted on a declining scale for 1 year. The anamnestic responses to the vaccines were determined by inoculating the same calves with a booster dose of vaccine 1 year after the original 2 doses were given. Blood samples were obtained from the calves for serum 2 weeks later. The serums were tested by serum-neutralization tests. The single booster dose of vaccine elicited an anamnestic response to both IBR and PI-3 viruses. PMID:6303996

  2. Presence of infectious bursal disease virus in chicken meat and effect of vaccination in decreasing the virus titers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Infectious bursal disease virus (IBDV) causes economic losses to the poultry industry worldwide and impacts chicken meat importation in countries with self-declared freedom. This study sought to determine the presence of IBDV in chicken meat and the role of vaccination as a mitigation strategy. In...

  3. Efficient recovery of infectious vesicular stomatitis virus entirely from cDNA clones.

    PubMed

    Whelan, S P; Ball, L A; Barr, J N; Wertz, G T

    1995-08-29

    Infectious vesicular stomatitis virus (VSV), the prototypic nonsegmented negative-strand RNA virus, was recovered from a full-length cDNA clone of the viral genome. Bacteriophage T7 RNA polymerase expressed from a recombinant vaccinia virus was used to drive the synthesis of a genome-length positive-sense transcript of VSV from a cDNA clone in baby hamster kidney cells that were simultaneously expressing the VSV nucleocapsid protein, phosphoprotein, and polymerase from separate plasmids. Up to 10(5) infectious virus particles were obtained from transfection of 10(6) cells, as determined by plaque assays. This virus was amplified on passage, neutralized by VSV-specific antiserum, and shown to possess specific nucleotide sequence markers characteristic of the cDNA. This achievement renders the biology of VSV fully accessible to genetic manipulation of the viral genome. In contrast to the success with positive-sense RNA, attempts to recover infectious virus from negative-sense T7 transcripts were uniformly unsuccessful, because T7 RNA polymerase terminated transcription at or near the VSV intergenic junctions. PMID:7667300

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  5. Development and applications of single-cycle infectious influenza A virus (sciIAV).

    PubMed

    Nogales, Aitor; Baker, Steven F; Domm, William; Martínez-Sobrido, Luis

    2016-05-01

    The diverse host range, high transmissibility, and rapid evolution of influenza A viruses justify the importance of containing pathogenic viruses studied in the laboratory. Other than physically or mechanically changing influenza A virus containment procedures, modifying the virus to only replicate for a single round of infection similarly ensures safety and consequently decreases the level of biosafety containment required to study highly pathogenic members in the virus family. This biological containment is more ideal because it is less apt to computer, machine, or human error. With many necessary proteins that can be deleted, generation of single-cycle infectious influenza A viruses (sciIAV) can be achieved using a variety of approaches. Here, we review the recent burst in sciIAV generation and summarize the applications and findings on this important human pathogen using biocontained viral mimics. PMID:26220478

  6. Infectious hematopoietic necrosis virus detected by separation and incubation of cells from salmonid cavity fluid.

    USGS Publications Warehouse

    Mulcahy, D.; Batts, W.N.

    1987-01-01

    Infectious hematopoietic necrosis (IHN) virus is usually detected by inoculating susceptible cell cultures with cavity ("ovarian") fluid (CF) from spawning females. We identified additional adult carriers of virus in spawning populations of steelhead trout (Salmo gairdneri) and sockeye salmon (Oncorhynchus nerka) by collecting nonerythrocytic cells from CF samples by low-speed centrifugation, culturing the cells for at least 7 d at 15 °C, and then testing the culture medium for virus. Virus appeared in the cultured cells from some samples of CF that remained negative during incubation. In additional samples of CF from these species, the virus titer increased in cultured cells compared with the titer in the original CF sample. With chinook salmon (O.tshawytscha), no negative samples converted to positive during incubation, but the virus titer was retained in incubated CF cells, but not in cell-free CF.

  7. Haggling over viruses: the downside risks of securitizing infectious disease.

    PubMed

    Elbe, Stefan

    2010-11-01

    This article analyses how the 'securitization' of highly pathogenic avian influenza (H5N1) contributed to the rise of a protracted international virus-sharing dispute between developing and developed countries. As fear about the threat of a possible human H5N1 pandemic spread across the world, many governments scrambled to stockpile anti-viral medications and vaccines, albeit in a context where there was insufficient global supply to meet such a rapid surge in demand. Realizing that they were the likely 'losers' in this international race, some developing countries began to openly question the benefits of maintaining existing forms of international health cooperation, especially the common practice of sharing national virus samples with the rest of the international community. Given that such virus samples were also crucial to the high-level pandemic preparedness efforts of the West, the Indonesian government in particular felt emboldened to use international access to its H5N1 virus samples as a diplomatic 'bargaining chip' for negotiating better access to vaccines and other benefits for developing countries. The securitized global response to H5N1 thus ended up unexpectedly entangling the long-standing international virus-sharing mechanism within a wider set of political disputes, as well as prompting governments to subject existing virus-sharing arrangements to much narrower calculations of national interest. In the years ahead, those risks to international health cooperation must be balanced with the policy attractions of the global health security agenda. PMID:20961948

  8. Increased susceptibility to infectious salmon anemia virus (ISAv) in Lepeophtheirus salmonis – infected Atlantic salmon

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The salmon louse and infectious salmon anemia virus (ISAv) are the two most significant pathogens of concern to the Atlantic salmon (Salmo salar) aquaculture industry. However, the interactions between sea lice and ISAv, as well as the impact of a prior sea lice infection on the susceptibility of th...

  9. Evaluation of endocrine and immune disruption of steers challenged with infectious bovine rhinotracheitis virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Objective: To evaluate the endocrine response of steers administered an immune challenge utilizing infectious bovine rhinotracheitis virus (IBRV). Animals: Twelve crossbred steers (228.82 ± 22.15 kg BW) Procedures: Steers fitted with indwelling rectal probes and randomly assigned to a Control (CON) ...

  10. Transcriptomic analysis of responses to infectious salmon anemia virus infection in macrophage-like cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The aquatic orthomyxovirus infectious salmon anemia virus (ISAV) is an important pathogen for salmonid aquaculture, however little is known about protective and pathological host responses to infection. We have investigated intracellular responses during cytopathic ISAV infection in the macrophage-l...

  11. Establishment and characterization of a chimeric infectious cDNA clone of classical swine fever virus.

    PubMed

    Zhao, T S; Xia, Y H

    2016-01-01

    Classical swine fever virus (CSFV) causes a highly contagious disease among swine that has an important economic impact worldwide. There are two important CSFV strains in China, Shimen and hog cholera lapinized virus (HCLV). Shimen strain is highly virulent while HCLV, also referred to as C-strain, is a live attenuated vaccine strain considered to be one of the most effective and safest live vaccines. In this study, a chimeric infectious cDNA clone of CSFV named pT7SM-c was engineered by replacing the Erns genomic region of an infectious clone of CSFV Shimen strain, pT7SM, with the same region obtained from HCLV. RNA transcripts of pT7SM-c containing an engineered EcoRI site that served as a genetic marker were directly infectious in PK15 cells. The rescued virus vT7SM-c showed similar growth kinetics and cytopathic effect with the parental virus vT7SM in the cells. The chimeric infectious cDNA clone can be used as a practical tool for further studying of the virulence, protein function and pathogenesis of CSFV through genetic manipulation. PMID:27265471

  12. LETTUCE INFECTIOUS YELLOWS VIRUS-ENCODED P26 INDUCES PLASMALEMMA DEPOSIT CYTOPATHOLOGY

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lettuce infectious yellows virus (LIYV) encodes a 26 kDa protein (P26) previously shown to associate with plasmalemma deposits (PLDs), unique LIYV-induced cytopathologies located at the plasmalemma over plasmodesmata pit fields in companion cells and phloem parenchyma. To further characterize the re...

  13. Interaction of Hepatitis C Virus Core Protein with Janus Kinase Is Required for Efficient Production of Infectious Viruses

    PubMed Central

    Lee, Choongho

    2013-01-01

    Chronic hepatitis C virus (HCV) infection is responsible for the development of liver cirrhosis and hepatocellular carcinoma. HCV core protein plays not only a structural role in the virion morphogenesis by encapsidating a virus RNA genome but also a non-structural role in HCV-induced pathogenesis by blocking innate immunity. Especially, it has been shown to regulate JAK-STAT signaling pathway through its direct interaction with Janus kinase (JAK) via its proline-rich JAK-binding motif (79PGYPWP84). However, little is known about the physiological significance of this HCV core-JAK association in the context of the virus life cycle. In order to gain an insight, a mutant HCV genome (J6/JFH1-79A82A) was constructed to express the mutant core with a defective JAK-binding motif (79AGYAWP84) using an HCV genotype 2a infectious clone (J6/JFH1). When this mutant HCV genome was introduced into hepatocarcinoma cells, it was found to be severely impaired in its ability to produce infectious viruses in spite of its robust RNA genome replication. Taken together, all these results suggest an essential requirement of HCV core-JAK protein interaction for efficient production of infectious viruses and the potential of using core-JAK blockers as a new anti-HCV therapy. PMID:24009866

  14. Multivalent virus-like-particle vaccine protects against classic and variant infectious bursal disease viruses.

    PubMed

    Jackwood, Daral J

    2013-03-01

    Nucleotide sequences that encode the pVP2 proteins from a variant infectious bursal disease virus (IBDV) strain designated USA08MD34p and a classic IBDV strain designated Mo195 were produced with the use of reverse-transcriptase-polymerase chain reaction (RT-PCR) and cloned into a pGEM-T Easy vector. A nucleotide sequence that encodes the VP3 protein was also produced from the USA08MD34p viral genome with the use of RT-PCR and cloned into a pGEM-T Easy vector. The VP3 and pVP2 clones were inserted into the pVL1393 baculovirus transfer vector and sequenced to confirm their orientation to the promoter and to ensure they contained uninterrupted open reading frames. Recombinant baculoviruses were constructed by transfection in Sf9 cells. Three recombinant baculoviruses were produced and contained the USA08MD34p-VP3, USA08MD34p-pVP2, or Mo195-pVP2 genomic sequences. Virus-like particles (VLPs) were observed with the use of transmission electron microscopy when the USA08MD34p-VP3 baculovirus was co-inoculated into Sf9 cells with either of the pVP2 constructs. VLPs were also observed when the USA08MD34p-pVP2 and Mo195-pVP2 were coexpressed with USA08MD34p-VP3. These multivalent VLPs contained both classic and variant pVP2 molecules. Stability tests demonstrated the VLPs were stable at 4 and 24 C for 8 wk. The USA08MD34p, Mo195, and multivalent VLPs were used to vaccinate chickens. They induced an IBDV-specific antibody response that was detected by enzyme-linked immunosorbent assay (ELISA), and virus-neutralizing antibodies were detected in vitro. Chickens vaccinated with the multivalent VLPs were protected from a virulent variant IBDV strain (V1) and a virulent classic IBDV strain (STC). The results indicate the multivalent VLPs maintained the antigenic integrity of the variant and classic viruses and have the potential to serve as a multivalent vaccine for use in breeder-flock vaccination programs. PMID:23678728

  15. 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.

  16. Detection of infectious laryngotracheitis virus by real-time PCR in naturally and experimentally infected chickens.

    PubMed

    Zhao, Yan; Kong, Congcong; Cui, Xianlan; Cui, Hongyu; Shi, Xingming; Zhang, Xiaomin; Hu, Shunlei; Hao, Lianwei; Wang, Yunfeng

    2013-01-01

    Infectious laryngotracheitis (ILT) is an acute, highly contagious upper-respiratory infectious disease of chickens. In this study, a real-time PCR method was developed for fast and accurate detection and quantitation of ILTV DNA of chickens experimentally infected with ILTV strain LJS09 and naturally infected chickens. The detection lower limit of the assay was 10 copies of DNA. There were no cross reactions with the DNA and RNA of infectious bursal disease virus, chicken anemia virus, reticuloendotheliosis virus, avian reovirus, Newcastle disease virus, and Marek's disease virus. The real-time PCR was reproducible as the coefficients of variation of reproducibility of the intra-assay and the inter-assay were less than 2%. The real-time PCR was used to detect the levels of the ILTV DNA in the tissues of specific pathogen free (SPF) chickens infected with ILTV at different times post infection. ILTV DNA was detected by real-time PCR in the heart, liver, spleen, lung, kidney, larynx, tongue, thymus, glandular stomach, duodenum, pancreatic gland, small intestine, large intestine, cecum, cecal tonsil, bursa of Fabricius, and brain of chickens in the infection group and the contact-exposure group. The sensitivity, specificity, and reproducibility of the ILTV real-time PCR assay revealed its suitability for detection and quantitation of ILTV in the samples from clinically and experimentally ILTV infected chickens. PMID:23840745

  17. Detection of Infectious Laryngotracheitis Virus by Real-Time PCR in Naturally and Experimentally Infected Chickens

    PubMed Central

    Zhao, Yan; Kong, Congcong; Cui, Xianlan; Cui, Hongyu; Shi, Xingming; Zhang, Xiaomin; Hu, Shunlei; Hao, Lianwei; Wang, Yunfeng

    2013-01-01

    Infectious laryngotracheitis (ILT) is an acute, highly contagious upper-respiratory infectious disease of chickens. In this study, a real-time PCR method was developed for fast and accurate detection and quantitation of ILTV DNA of chickens experimentally infected with ILTV strain LJS09 and naturally infected chickens. The detection lower limit of the assay was 10 copies of DNA. There were no cross reactions with the DNA and RNA of infectious bursal disease virus, chicken anemia virus, reticuloendotheliosis virus, avian reovirus, Newcastle disease virus, and Marek's disease virus. The real-time PCR was reproducible as the coefficients of variation of reproducibility of the intra-assay and the inter-assay were less than 2%. The real-time PCR was used to detect the levels of the ILTV DNA in the tissues of specific pathogen free (SPF) chickens infected with ILTV at different times post infection. ILTV DNA was detected by real-time PCR in the heart, liver, spleen, lung, kidney, larynx, tongue, thymus, glandular stomach, duodenum, pancreatic gland, small intestine, large intestine, cecum, cecal tonsil, bursa of Fabricius, and brain of chickens in the infection group and the contact-exposure group. The sensitivity, specificity, and reproducibility of the ILTV real-time PCR assay revealed its suitability for detection and quantitation of ILTV in the samples from clinically and experimentally ILTV infected chickens. PMID:23840745

  18. The nucleotide sequence of an infectious clone of the geminivirus beet curly top virus.

    PubMed

    Stanley, J; Markham, P G; Callis, R J; Pinner, M S

    1986-08-01

    A number of infectious clones of a Californian isolate of the leafhopper-transmitted geminivirus beet curly top virus (BCTV) have been constructed from virus-specific double-stranded DNA isolated from infected Beta vulgaris and used to demonstrate a single component genome. The nucleotide sequence of one infectious clone has been determined (2993 nucleotides). Comparison with other geminiviruses has shown that the organisation of the genome closely resembles DNA 1 of the whitefly-transmitted members. The four conserved coding regions of DNA 1 have highly homologous counterparts in BCTV with the exception of the putative coat protein which is more closely related to those of the leafhopper-transmitted geminiviruses suggesting a strong interrelationship between coat protein and insect vector. A BCTV component equivalent to DNA 2 is not required for virus infection or transmission and has not been isolated from infected plants. PMID:16453696

  19. An infectious disease of ducks caused by a newly emerged Tembusu virus strain in mainland China.

    PubMed

    Yan, Pixi; Zhao, Youshu; Zhang, Xu; Xu, Dawei; Dai, Xiaoguang; Teng, Qiaoyang; Yan, Liping; Zhou, Jiewen; Ji, Xiwen; Zhang, Shumei; Liu, Guangqing; Zhou, Yanjun; Kawaoka, Yoshihiro; Tong, Guangzhi; Li, Zejun

    2011-08-15

    During investigations into an outbreak of egg production decline, retarded growth, and even death among ducks in Southeast China, a novel Tembusu virus strain named Tembusu virus Fengxian 2010 (FX2010) was isolated. This virus replicated in embryonated chicken eggs and caused embryo death. In cross-neutralization tests, antiserum to the partial E protein of Tembusu virus Mm1775 strain neutralized FX2010, whereas antiserum to Japanese encephalitis virus did not. FX2010 is an enveloped RNA virus of approximately 45-50 nm in diameter. Sequence analysis of its E and NS5 genes showed that both genes share up to 99.6% nucleotide sequence identity with Baiyangdian virus, and up to 88% nucleotide sequence identity with their counterparts in Tembusu virus. FX2010 was transmitted without mosquito, and caused systemic infection and lesions in experimentally infected ducks. These results indicate that FX2010 and BYD virus are newly emerged Tembusu virus strains that cause an infectious disease in ducks. PMID:21722935

  20. Occurrence of different types of infectious hematopoietic necrosis virus in fish.

    PubMed Central

    Hsu, Y L; Engelking, H M; Leong, J C

    1986-01-01

    The virion protein patterns of 71 isolates of infectious hematopoietic necrosis virus (IHNV) from the Pacific Northwest were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [35S]-methionine-labeled virus. This analysis led to the classification of these virus isolates into four or more types. Type 1 virus was characterized by a nucleocapsid protein with an approximate molecular weight of 40,500. Type 2 and type 3 viruses have nucleocapsid proteins with molecular weights of 42,800 and 43,250, respectively. Type 2 virus was responsible for the recent epizootics of IHNV among fish in the lower Columbia River. The California IHNV isolates were type 3 with the exception of some of those isolated from fish at the Coleman Hatchery on the Sacramento River. These Coleman Hatchery isolates belonged to a type 4 virus group characterized by a larger glycoprotein of approximately 70,000 molecular weight. All other viruses examined had glycoproteins of 67,000 molecular weight. The "type 5" virus isolates were grouped together because they were not sufficiently distinct to warrant classification into a separate type. These findings have been useful in determining that a particular virus type is characteristic for a geographic area and will infect many different salmonid species in that area and the same type isolated from parental fish is responsible for the subsequent outbreak of the diseases in progeny. Images PMID:3789723

  1. Production of Infectious Dengue Virus in Aedes aegypti Is Dependent on the Ubiquitin Proteasome Pathway.

    PubMed

    Choy, Milly M; Sessions, October M; Gubler, Duane J; Ooi, Eng Eong

    2015-11-01

    Dengue virus (DENV) relies on host factors to complete its life cycle in its mosquito host for subsequent transmission to humans. DENV first establishes infection in the midgut of Aedes aegypti and spreads to various mosquito organs for lifelong infection. Curiously, studies have shown that infectious DENV titers peak and decrease thereafter in the midgut despite relatively stable viral genome levels. However, the mechanisms that regulate this decoupling of infectious virion production from viral RNA replication have never been determined. We show here that the ubiquitin proteasome pathway (UPP) plays an important role in regulating infectious DENV production. Using RNA interference studies, we show in vivo that knockdown of selected UPP components reduced infectious virus production without altering viral RNA replication in the midgut. Furthermore, this decoupling effect could also be observed after RNAi knockdown in the head/thorax of the mosquito, which otherwise showed direct correlation between infectious DENV titer and viral RNA levels. The dependence on the UPP for successful DENV production is further reinforced by the observed up-regulation of key UPP molecules upon DENV infection that overcome the relatively low expression of these genes after a blood meal. Collectively, our findings indicate an important role for the UPP in regulating DENV production in the mosquito vector. PMID:26566123

  2. Production of Infectious Dengue Virus in Aedes aegypti Is Dependent on the Ubiquitin Proteasome Pathway

    PubMed Central

    Choy, Milly M.; Sessions, October M.; Gubler, Duane J.; Ooi, Eng Eong

    2015-01-01

    Dengue virus (DENV) relies on host factors to complete its life cycle in its mosquito host for subsequent transmission to humans. DENV first establishes infection in the midgut of Aedes aegypti and spreads to various mosquito organs for lifelong infection. Curiously, studies have shown that infectious DENV titers peak and decrease thereafter in the midgut despite relatively stable viral genome levels. However, the mechanisms that regulate this decoupling of infectious virion production from viral RNA replication have never been determined. We show here that the ubiquitin proteasome pathway (UPP) plays an important role in regulating infectious DENV production. Using RNA interference studies, we show in vivo that knockdown of selected UPP components reduced infectious virus production without altering viral RNA replication in the midgut. Furthermore, this decoupling effect could also be observed after RNAi knockdown in the head/thorax of the mosquito, which otherwise showed direct correlation between infectious DENV titer and viral RNA levels. The dependence on the UPP for successful DENV production is further reinforced by the observed up-regulation of key UPP molecules upon DENV infection that overcome the relatively low expression of these genes after a blood meal. Collectively, our findings indicate an important role for the UPP in regulating DENV production in the mosquito vector. PMID:26566123

  3. Detection of infectious haematopoietic necrosis virus in river water and demonstration of waterborne transmission

    USGS Publications Warehouse

    Mulcahy, D.; Pascho, R.J.; Jenes, C.K.

    1983-01-01

    In a study of the possible role of waterborne infectious haematopoietic necrosis virus in transmission of the disease among spawning sockeye salmon, Oncorhynchus nerka (Walbaum), both infection rates and virus titres were higher in fish held at high density in a side channel than in fish in the adjacent river. Virus was never isolated from river water, but was found in water from the side channel at levels ranging from 32.5 to 1600 plaque-forming units (p.f.u.)/ml. Uninfected yearling sockeye salmon held in a box in the side channel developed localized gill infections with IHN virus. The disease did not progress to the viscera until a threshold titre of about 105 p.f.u./g was reached in the gill. The effectiveness of the gill as a barrier limiting development of systemic infections means that waterborne IHN virus probably does not greatly increase the infection rate in a sockeye salmon population during spawning.

  4. Amino Acid Polymorphisms in Hepatitis C Virus Core Affect Infectious Virus Production and Major Histocompatibility Complex Class I Molecule Expression.

    PubMed

    Tasaka-Fujita, Megumi; Sugiyama, Nao; Kang, Wonseok; Masaki, Takahiro; Masaski, Takahiro; Murayama, Asako; Yamada, Norie; Sugiyama, Ryuichi; Tsukuda, Senko; Watashi, Koichi; Asahina, Yasuhiro; Sakamoto, Naoya; Wakita, Takaji; Shin, Eui-Cheol; Kato, Takanobu

    2015-01-01

    Amino acid (aa) polymorphisms in the hepatitis C virus (HCV) genotype 1b core protein have been reported to be a potent predictor for poor response to interferon (IFN)-based therapy and a risk factor for hepatocarcinogenesis. We investigated the effects of these polymorphisms with genotype 1b/2a chimeric viruses that contained polymorphisms of Arg/Gln at aa 70 and Leu/Met at aa 91. We found that infectious virus production was reduced in cells transfected with chimeric virus RNA that had Gln at aa 70 (aa70Q) compared with RNA with Arg at aa 70 (aa70R). Using flow cytometry analysis, we confirmed that HCV core protein accumulated in aa70Q clone transfected cells, and it caused a reduction in cell-surface expression of major histocompatibility complex (MHC) class I molecules induced by IFN treatment through enhanced protein kinase R phosphorylation. We could not detect any effects due to the polymorphism at aa 91. In conclusion, the polymorphism at aa 70 was associated with efficiency of infectious virus production, and this deteriorated virus production in strains with aa70Q resulted in the intracellular accumulation of HCV proteins and attenuation of MHC class I molecule expression. These observations may explain the strain-associated resistance to IFN-based therapy and hepatocarcinogenesis of HCV. PMID:26365522

  5. Comparison of the pathogenicity of the USDA challenge virus strain to a field strain of infectious laryngotracheitis virus.

    PubMed

    Koski, Danielle M; Predgen, Ann S; Trampel, Darrell W; Conrad, Sandra K; Narwold, Debra R; Hermann, Joseph R

    2015-07-01

    Infectious laryngotracheitis virus (ILTV) causes respiratory disease in chickens. This alphaherpesvirus infects laryngeal tracheal epithelial cells and causes outbreaks culminating in decreases in egg production, respiratory distress in chickens and mortality. There are several different vaccines to combat symptoms of the virus, including chicken embryo origin, tissue culture origin and recombinant vaccines. All vaccines licensed for use in the U.S. are tested for efficacy and potency according to U.S. federal regulation using a vaccine challenge assay involving the use of an ILT challenge virus. This challenge virus is provided to biologics companies by the Center for Veterinary Biologics (CVB), United States Department of Agriculture (USDA). The current USDA challenge virus originated from a vaccine strain and has been subjected to multiple passages in eggs, and may not represent what is currently circulating in the field. The objective of this study was to evaluate and compare the pathogenicity of USDA's challenge virus strain to the pathogenicity of a recent ILT field isolate. Using the challenge virus and various dilutions of the field isolate, clinical signs, mortality and pathology were evaluated in chickens. Results indicate that the field isolate at a 1:20 dilution is comparable in pathogenicity to the USDA challenge virus at a 1:4 dilution, and that the ILTV field isolate is a viable candidate that could be used as a challenge virus when evaluating vaccine efficacy. PMID:26050912

  6. Understanding foot-and-mouth disease virus transmission biology: identification of the indicators of infectiousness

    PubMed Central

    2013-01-01

    The control of foot-and-mouth disease virus (FMDV) outbreaks in non-endemic countries relies on the rapid detection and removal of infected animals. In this paper we use the observed relationship between the onset of clinical signs and direct contact transmission of FMDV to identify predictors for the onset of clinical signs and identify possible approaches to preclinical screening in the field. Threshold levels for various virological and immunological variables were determined using Receiver Operating Characteristic (ROC) curve analysis and then tested using generalized linear mixed models to determine their ability to predict the onset of clinical signs. In addition, concordance statistics between qualitative real time PCR test results and virus isolation results were evaluated. For the majority of animals (71%), the onset of clinical signs occurred 3–4 days post infection. The onset of clinical signs was associated with high levels of virus in the blood, oropharyngeal fluid and nasal fluid. Virus is first detectable in the oropharyngeal fluid, but detection of virus in the blood and nasal fluid may also be good candidates for preclinical indicators. Detection of virus in the air was also significantly associated with transmission. This study is the first to identify statistically significant indicators of infectiousness for FMDV at defined time periods during disease progression in a natural host species. Identifying factors associated with infectiousness will advance our understanding of transmission mechanisms and refine intra-herd and inter-herd disease transmission models. PMID:23822567

  7. First evidence of infectious hematopoietic necrosis virus (IHNV) in the Netherlands.

    PubMed

    Haenen, O L M; Schuetze, H; Cieslak, M; Oldenburg, S; Spierenburg, M A H; Roozenburg-Hengst, I; Voorbergen-Laarman, M; Engelsma, M Y; Olesen, N J

    2016-08-01

    In spring 2008, infectious hematopoietic necrosis virus (IHNV) was detected for the first time in the Netherlands. The virus was isolated from rainbow trout, Oncorhynchus mykiss (Walbaum), from a put-and-take fishery with angling ponds. IHNV is the causative agent of a serious fish disease, infectious hematopoietic necrosis (IHN). From 2008 to 2011, we diagnosed eight IHNV infections in rainbow trout originating from six put-and-take fisheries (symptomatic and asymptomatic fish), and four IHNV infections from three rainbow trout farms (of which two were co-infected by infectious pancreatic necrosis virus, IPNV), at water temperatures between 5 and 15 °C. At least one farm delivered trout to four of these eight IHNV-positive farms. Mortalities related to IHNV were mostly <40%, but increased to nearly 100% in case of IHNV and IPNV co-infection. Subsequent phylogenetic analysis revealed that these 12 isolates clustered into two different monophyletic groups within the European IHNV genogroup E. One of these two groups indicates a virus-introduction event by a German trout import, whereas the second group indicates that IHNV was already (several years) in the Netherlands before its discovery in 2008. PMID:26763082

  8. Growth Kinetics and Transmission Potential of Existing and Emerging Field Strains of Infectious Laryngotracheitis Virus

    PubMed Central

    Coppo, Mauricio J. C.; Vaz, Paola K.; Legione, Alistair R.; Quinteros, José A.; Noormohammadi, Amir H.; Markham, Phillip F.; Browning, Glenn F.; Devlin, Joanne M.

    2015-01-01

    Attenuated live infectious laryngotracheitis virus (ILTV) vaccines are widely used in the poultry industry to control outbreaks of disease. Natural recombination between commercial ILTV vaccines has resulted in virulent recombinant viruses that cause severe disease, and that have now emerged as the dominant field strains in important poultry producing regions in Australia. Genotype analysis using PCR—restriction fragment length polymorphism has shown one recombinant virus (class 9) has largely replaced the previously dominant class 2 field strain. To examine potential reasons for this displacement we compared the growth kinetics and transmission potential of class 2 and class 9 viruses. The class 9 ILTV grew to higher titres in cell culture and embryonated eggs, but no differences were observed in entry kinetics or egress into the allantoic fluid from the chorioallantoic membrane. In vivo studies showed that birds inoculated with class 9 ILTV had more severe tracheal pathology and greater weight loss than those inoculated with the class 2 virus. Consistent with the predominance of class 9 field strains, birds inoculated with 102 or 103 plaque forming units of class 9 ILTV consistently transmitted virus to in-contact birds, whereas this could only be seen in birds inoculated with 104 PFU of the class 2 virus. Taken together, the improved growth kinetics and transmission potential of the class 9 virus is consistent with improved fitness of the recombinant virus over the previously dominant field strain. PMID:25785629

  9. Growth kinetics and transmission potential of existing and emerging field strains of infectious laryngotracheitis virus.

    PubMed

    Lee, Sang-Won; Hartley, Carol A; Coppo, Mauricio J C; Vaz, Paola K; Legione, Alistair R; Quinteros, José A; Noormohammadi, Amir H; Markham, Phillip F; Browning, Glenn F; Devlin, Joanne M

    2015-01-01

    Attenuated live infectious laryngotracheitis virus (ILTV) vaccines are widely used in the poultry industry to control outbreaks of disease. Natural recombination between commercial ILTV vaccines has resulted in virulent recombinant viruses that cause severe disease, and that have now emerged as the dominant field strains in important poultry producing regions in Australia. Genotype analysis using PCR-restriction fragment length polymorphism has shown one recombinant virus (class 9) has largely replaced the previously dominant class 2 field strain. To examine potential reasons for this displacement we compared the growth kinetics and transmission potential of class 2 and class 9 viruses. The class 9 ILTV grew to higher titres in cell culture and embryonated eggs, but no differences were observed in entry kinetics or egress into the allantoic fluid from the chorioallantoic membrane. In vivo studies showed that birds inoculated with class 9 ILTV had more severe tracheal pathology and greater weight loss than those inoculated with the class 2 virus. Consistent with the predominance of class 9 field strains, birds inoculated with 10(2) or 10(3) plaque forming units of class 9 ILTV consistently transmitted virus to in-contact birds, whereas this could only be seen in birds inoculated with 10(4) PFU of the class 2 virus. Taken together, the improved growth kinetics and transmission potential of the class 9 virus is consistent with improved fitness of the recombinant virus over the previously dominant field strain. PMID:25785629

  10. Infectious vaccinia virus recombinants that express hepatitis B virus surface antigen

    NASA Astrophysics Data System (ADS)

    Smith, Geoffrey L.; Mackett, Michael; Moss, Bernard

    1983-04-01

    Potential live vaccines against hepatitis B virus have been produced. The coding sequence for hepatitis B virus surface antigen (HBsAg) has been inserted into the vaccinia virus genome under control of vaccinia virus early promoters. Cells infected with these vaccinia virus recombinants synthesize and excrete HBsAg and vaccinated rabbits rapidly produce antibodies to HBsAg.

  11. Full-Length GB Virus C (Hepatitis G Virus) RNA Transcripts Are Infectious in Primary CD4-Positive T Cells

    PubMed Central

    Xiang, Jinhua; Wünschmann, Sabina; Schmidt, Warren; Shao, Jianqiang; Stapleton, Jack T.

    2000-01-01

    GB virus C (GBV-C or hepatitis G virus) is a recently described flavivirus which frequently leads to chronic viremia in humans. Although GBV-C is associated with acute posttransfusion hepatitis, it is not clear if the virus is pathogenic for humans. We constructed a full-length cDNA from the plasma of a person with chronic GBV-C viremia. Peripheral blood mononuclear cells (PBMCs) transfected with full-length RNA transcripts from this GBV-C clone resulted in viral replication. This was demonstrated by serial passage of virus from cell culture supernatants, detection of increasing concentrations of positive- and negative-sense GBV-C RNA over time, and the detection of the GBV-C E2 antigen by confocal microscopy. In addition, two types of GBV-C particles were identified in cell lysates; these particles had buoyant densities of 1.06 and 1.12 to 1.17 g/ml in sucrose gradients. PBMCs sorted for expression of CD4 contained 100-fold-more GBV-C RNA than CD4-negative cells. Taken together, these data demonstrate that RNA transcripts from GBV-C full-length cDNA are infectious in primary CD4-positive T cells. In contrast, RNA transcripts from an infectious hepatitis C virus clone did not replicate in the same cell culture system. Infectious RNA transcripts from GBV-C cDNA should prove useful for studying viral replication and may allow identification of differences between GBV-C and hepatitis C virus cultivation in vitro. PMID:10982359

  12. Infectious hepatitis B virus from cloned DNA of known nucleotide sequence.

    PubMed Central

    Will, H; Cattaneo, R; Darai, G; Deinhardt, F; Schellekens, H; Schaller, H

    1985-01-01

    The infectivity of cloned hepatitis B viral DNA (HBV) has been tested in chimpanzees to identify a fully functional HBV genome and to assess the risk associated with its handling. Only one of two HBV DNA sequence variants tested was shown to be infectious. "Clone purified" virus of predicted nucleotide sequence was produced from the infectious HBV DNA, and the cloned viral genome was identical in structure with naturally occurring HBV. Infection could be initiated independent of whether circular monomeric or plasmid integrated dimeric forms of the viral genome were inoculated, but the infectivity of the DNA depended on liver cell transfection or intrahepatic injection. Intravenous injection of high doses of infectious HBV DNA did not induce hepatitis, suggesting that there is virtually no risk associated with routine laboratory handling of cloned HBV DNA. Images PMID:2983320

  13. Genetics and pathogenesis of feline infectious peritonitis virus.

    PubMed

    Brown, Meredith A; Troyer, Jennifer L; Pecon-Slattery, Jill; Roelke, Melody E; O'Brien, Stephen J

    2009-09-01

    Feline coronavirus (FCoV) is endemic in feral cat populations and cat colonies, frequently preceding outbreaks of fatal feline infectious peritonitis (FIP). FCoV exhibits 2 biotypes: the pathogenic disease and a benign infection with feline enteric coronavirus (FECV). Uncertainty remains regarding whether genetically distinctive avirulent and virulent forms coexist or whether an avirulent form mutates in vivo, causing FIP. To resolve these alternative hypotheses, we isolated viral sequences from FCoV-infected clinically healthy and sick cats (8 FIP cases and 48 FECV-asymptomatic animals); 735 sequences from 4 gene segments were generated and subjected to phylogenetic analyses. Viral sequences from healthy cats were distinct from sick cats on the basis of genetic distances observed in the membrane and nonstructural protein 7b genes. These data demonstrate distinctive circulating virulent and avirulent strains in natural populations. In addition, 5 membrane protein amino acid residues with functional potential differentiated healthy cats from cats with FIP. These findings may have potential as diagnostic markers for virulent FIP-associated FCoV. PMID:19788813

  14. Genetics and Pathogenesis of Feline Infectious Peritonitis Virus

    PubMed Central

    Troyer, Jennifer L.; Pecon-Slattery, Jill; Roelke, Melody E.; O’Brien, Stephen J.

    2009-01-01

    Feline coronavirus (FCoV) is endemic in feral cat populations and cat colonies, frequently preceding outbreaks of fatal feline infectious peritonitis (FIP). FCoV exhibits 2 biotypes: the pathogenic disease and a benign infection with feline enteric coronavirus (FECV). Uncertainty remains regarding whether genetically distinctive avirulent and virulent forms coexist or whether an avirulent form mutates in vivo, causing FIP. To resolve these alternative hypotheses, we isolated viral sequences from FCoV-infected clinically healthy and sick cats (8 FIP cases and 48 FECV-asymptomatic animals); 735 sequences from 4 gene segments were generated and subjected to phylogenetic analyses. Viral sequences from healthy cats were distinct from sick cats on the basis of genetic distances observed in the membrane and nonstructural protein 7b genes. These data demonstrate distinctive circulating virulent and avirulent strains in natural populations. In addition, 5 membrane protein amino acid residues with functional potential differentiated healthy cats from cats with FIP. These findings may have potential as diagnostic markers for virulent FIP-associated FCoV. PMID:19788813

  15. Relative resistance of Pacific salmon to infectious salmon anaemia virus

    USGS Publications Warehouse

    Rolland, J.B.; Winton, J.R.

    2003-01-01

    Infectious salmon anaemia (ISA) is a major disease of Atlantic salmon, Salmo salar, caused by an orthomyxovirus (ISAV). Increases in global aqua culture and the international movement of fish made it important to determine if Pacific salmon are at risk. Steelhead trout, Oncorhynchus mykiss, and chum, O. keta, Chinook, O. tshawytscha, coho, O. kisutch, and Atlantic salmon were injected intraperitoneally with a high, medium, or low dose of a Norwegian strain of ISAV. In a second challenge, the same species, except chum salmon, were injected with a high dose of either a Canadian or the Norwegian strain. Average cumulative mortality of Atlantic salmon in trial 1 was 12% in the high dose group, 20% in the medium dose group and 16% in the low dose group. The average cumulative mortality of Atlantic salmon in trial 2 was 98%. No signs typical of ISA and no ISAV-related mortality occurred among any of the groups of Oncorhynchus spp. in either experiment, although ISAV was reisolated from some fish sampled at intervals post-challenge. The results indicate that while Oncorhynchus spp. are quite resistant to ISAV relative to Atlantic salmon, the potential for ISAV to adapt to Oncorhynchus spp. should not be ignored.

  16. An in vivo experimental model to determine antigenic variations among infectious bursal disease viruses.

    PubMed

    Durairaj, Vijay; Linnemann, Erich; Icard, Alan H; Williams, Susan M; Sellers, Holly S; Mundt, Egbert

    2013-08-01

    Infectious bursal disease virus (IBDV) is a double-stranded RNA virus causing infectious bursal disease in chickens. IBDV undergoes antigenic drift, so characterizing the antigenicity of IBDV plays an important role for identification and selection of vaccine candidates. In this study, an in vivo experimental model was developed to differentiate a new antigenic variant of IBDV. To this end, a hyper-immune serum to IBDV E/Del-type virus was generated in specific pathogen-free chickens and a standard volume of the hyper-immune serum was serially diluted and injected in specific pathogen-free birds via intravenous, subcutaneous, or intramuscular routes. The chickens were bled at different time points in order to evaluate the dynamics of virus neutralization titres. Based on the results, chickens were injected with different serum dilutions by the subcutaneous route. Twenty-four hours later, chickens were bled and then challenged with 100 median chicken infectious doses of the E/Del virus and a new IBDV variant. Chickens were euthanized at 7 days post infection and the bursa of Fabricius was removed for microscopic evaluation to determine the bursal lesion score. The determined virus neutralization titre along with the bursal lesion score was used to determine the breakthrough titre in the in vivo chicken model. Based on the data obtained, an antigenic subtype of IBDV was identified and determined to be different from E/Del. This model is a sensitive model for determination of IBDV antigenicity of non-tissue culture adapted IBDV. PMID:23662946

  17. Molecular evidence for a geographically restricted population of infectious bursal disease viruses.

    PubMed

    Jackwood, Daral J; Stoute, Simone T

    2013-03-01

    A population of infectious bursal disease virus (IBDV) in northeast Ohio that appears to be geographically restricted was identified. Thirteen broiler farms containing a total of 36 houses were examined for the presence of IBDV. Twenty-four of the 36 houses were positive for IBDV, and of those viruses, 15 viruses from six different broiler farms formed a unique phylogenetic group. Nucleotide sequence analysis identified glutamic acid (E) at position 253 in all 15 viruses. Only one other virus in the GenBank database contained this mutation, and it was also from northeast Ohio. All 15 viruses from this study and the one identified in GenBank also had a unique VP1 sequence. The amino acids located at position 253 in VP2 are typically histidine (attenuated viruses) and glutamine (pathogenic viruses). Because amino acid 253 has been linked to pathogenicity in IBDV, two viruses from the E253 population were selected for pathogenicity studies. They were observed to be pathogenic in 4-wk-old specific-pathogen-free layer chicks. When these two viruses were used to challenge broilers from the parent flock that supplies the birds to all 13 broiler farms examined in this study, the viruses were able to break through the maternal immunity at 14 and 21 days of age but not at 7 days of age. A similar scenario was observed on the six broiler farms that had these viruses. The phylogeographic data suggest this population of IBDV has been restricted for more than 14 yr to northeast Ohio. Because commercially available classic and variant vaccines do not effectively control this population of IBDV, other alternatives are needed. PMID:23678730

  18. Viral fitness does not correlate with three genotype displacement events involving infectious hematopoietic necrosis virus.

    PubMed

    Kell, Alison M; Wargo, Andrew R; Kurath, Gael

    2014-09-01

    Viral genotype displacement events are characterized by the replacement of a previously dominant virus genotype by a novel genotype of the same virus species in a given geographic region. We examine here the fitness of three pairs of infectious hematopoietic necrosis virus (IHNV) genotypes involved in three major genotype displacement events in Washington state over the last 30 years to determine whether increased virus fitness correlates with displacement. Fitness was assessed using in vivo assays to measure viral replication in single infection, simultaneous co-infection, and sequential superinfection in the natural host, steelhead trout. In addition, virion stability of each genotype was measured in freshwater and seawater environments at various temperatures. By these methods, we found no correlation between increased viral fitness and displacement in the field. These results suggest that other pressures likely exist in the field with important consequences for IHNV evolution. PMID:25068402

  19. Viral fitness does not correlate with three genotype displacement events involving infectious hematopoietic necrosis virus

    PubMed Central

    Kell, Alison M; Wargo, Andrew R; Kurath, Gael

    2014-01-01

    Viral genotype displacement events are characterized by the replacement of a previously dominant virus genotype by a novel genotype of the same virus species in a given geographic region. We examine here the fitness of three pairs of infectious hematopoietic necrosis virus (IHNV) genotypes involved in three major genotype displacement events in Washington state over the last 30 years to determine whether increased virus fitness correlates with displacement. Fitness was assessed using in vivo assays to measure viral replication in single infection, simultaneous co-infection, and sequential superinfection in the natural host, steelhead trout. In addition, virion stability of each genotype was measured in freshwater and seawater environments at various temperatures. By these methods, we found no correlation between increased viral fitness and displacement in the field. These results suggest that other pressures likely exist in the field with important consequences for IHNV evolution. PMID:25068402

  20. Viral fitness does not correlate with three genotype displacement events involving infectious hematopoietic necrosis virus

    USGS Publications Warehouse

    Kell, Alison M.; Wargo, Andrew R.; Kurath, Gael

    2014-01-01

    Viral genotype displacement events are characterized by the replacement of a previously dominant virus genotype by a novel genotype of the same virus species in a given geographic region. We examine here the fitness of three pairs of infectious hematopoietic necrosis virus (IHNV) genotypes involved in three major genotype displacement events in Washington state over the last 30 years to determine whether increased virus fitness correlates with displacement. Fitness was assessed using in vivo assays to measure viral replication in single infection, simultaneous co-infection, and sequential superinfection in the natural host, steelhead trout. In addition, virion stability of each genotype was measured in freshwater and seawater environments at various temperatures. By these methods, we found no correlation between increased viral fitness and displacement in the field. These results suggest that other pressures likely exist in the field with important consequences for IHNV evolution.

  1. Experimental infection of eastern cottontail rabbits Sylvilagus floridanus) with infectious bovine rhinotracheitis virus.

    PubMed

    Lupton, H W; Reed, D E

    1979-09-01

    Experimental infection of eastern cottontail rabbits (Sylvilagus floridanus) with infectious bovine rhinotracheitis virus caused acute keratoconjunctivitis and a fatal systemic infection. The clinical syndrome was characterized initially by blepharospasm and ocular discharge. The rabbits were markedly depressed on post-exposure day (PED) 5 and were dead or moribund on PED 6. The virus was readily recovered from liver and adrenal gland tissue on PED 6 and from conjunctival swabs on PED 1 to 6. Histopathologic studies revealed a few necrotic foci in the liver and multiple focal to diffuse necrosis of the adrenal glands. Viral isolation and immunofluorescence tests were used to demonstrate a direct association between infectious bovine rhinotracheitis viral antigens and the lesions. PMID:230773

  2. Pathogenicity of virulent infectious bronchitis virus isolate YN on hen ovary and oviduct.

    PubMed

    Zhong, Qi; Hu, Yan-Xin; Jin, Ji-Hui; Zhao, Ye; Zhao, Jing; Zhang, Guo-Zhong

    2016-09-25

    Avian infectious bronchitis is an economically important poultry disease caused by avian infectious bronchitis virus (IBV). IBV isolate YN is a virulent strain, which is genetically similar to most of the prevalent strains in China. In this study, 21-day-old commercial laying hens were infected with IBV strain YN. The damaging effects of the virus on the reproductive organs were evaluated with clinical observations, gross autopsy and histopathological examinations during the 100-day monitoring period post infection. IBV strain YN infection caused a death rate of 40.5%. Microscopic lesions were observed on the ovary post-infection, but were restricted to the acute infection period. The pathological damage to the cystic oviducts were observed throughout the surveillance period. This study provides detailed information on the pathological changes in the hen ovary and oviduct after challenge with IBV strain YN, which could provide a better understanding about the pathogenicity of IBV. PMID:27599936

  3. Analysis of infectious laryngotracheitis virus isolates from Ontario and New Brunswick by the polymerase chain reaction.

    PubMed Central

    Alexander, H S; Key, D W; Nagy, E

    1998-01-01

    The polymerase chain reaction (PCR) was used to amplify DNA of infectious laryngotracheitis virus (ILTV) isolates obtained from field specimens. The examined 47 samples included 37 isolates representing 35 cases of infectious laryngotracheitis from Ontario and 10 isolates originating from 10 field cases in New Brunswick. The viruses were grown in either embryonated chicken eggs or cell culture, the DNA extracted and amplified using primers designed from the sequence information of a 1.1 kb BamHI fragment of the Ontario 1598 ILTV strain. Thirty-four of the Ontario isolates and all of the New Brunswick isolates were amplified successfully. This suggests that the selected primers would be useful for the majority of the isolates encountered in outbreaks of ILTV. Images Figure 1. Figure 2. PMID:9442943

  4. Isolation of novel variants of infectious bursal disease virus from different outbreaks in Northeast India.

    PubMed

    Morla, Sudhir; Deka, Pankaj; Kumar, Sachin

    2016-04-01

    Infectious bursal disease virus (IBDV) is a highly infectious disease of young chicken that predominantly affects the immune system. In the present study, we are reporting first comprehensive study of IBDV outbreaks from the Northeastern part of India. Northeast India shares a porous border with four different countries; and as a rule any outbreak in the neighboring countries substantially affects the poultry population in the adjoining states. Nucleotide sequence analysis of the VP2 gene of the IBDV isolates from the Northeastern part of India suggested the extreme virulent nature of the virus. The virulent marker amino acids (A222, I242, Q253, I256 and S299) in the hypervariable region of the Northeastern isolates were found identical with the reported very virulent strains of IBDV. A unique insertion of I/L294V was recorded in all the isolates of the Northeastern India. The study will be useful in understanding the circulating pathotypes of IBDV in India. PMID:26854869

  5. Neutralizing monoclonal antibodies recognize antigenic variants among isolates of infectious hematopoietic necrosis virus

    USGS Publications Warehouse

    Winton, J.R.; Arakawa, C.N.; Lannan, C.N.; Fryer, J.L.

    1988-01-01

    eutralizing monoclonal antibodies were developed against strains of infectious hematopoietic necrosis virus (IHNV) from steelhead trout Salmo gairdneri in the Deschutes River of Oregon, chinook salmon Oncorhynchus tshawytscha in the Sacramento River of California, and rainbow trout Salmo gairdneri reared in the Hagerman Valley of Idaho, USA. These antibodies were tested for neutralization of 12 IHNV isolates obtained from salmonids in Japan, Alaska, Washington, Oregon, California, and Idaho. The antibodies recognized antigenic variants among the isolates and could be used to separate the viruses into 4 groups. The members of each group tended to be related by geographic area rather than by source host species, virulence, or date of isolation.

  6. Malignant transformation of hamster cells following infection with bovine herpesvirus (infectious bovine rhinotracheitis virus.

    PubMed

    Michalski, F; Hsiung, G D

    1975-03-01

    Hamster embryo cells, following infection with IBR virus, showed malignant transformation. Hamsters of all ages, inbred or random bred, inoculated with two of the transformed cell lines developed solid tumors. Preliminary characterization of the tumors induced by one of the cell lines has indicated undifferentiated sarcomas. Viral specific antigen was detected in about 5% of the transformed cells and 10% of primary tumor cells in culture. Viral specific antibody was detected in the serum of tumor-bearing hamsters by the indirect immunofluorescent method, but no neutralizing antibodies were found. Infectious virus has not been recovered from either the transformed or tumor cells by cocultivation with bovine embryonic kidney cells. PMID:165538

  7. [IDENTIFICATION OF THE INFECTIOUS PANCREATIC NECROSIS VIRUS (IPNV) USING THE ENZYME IMMUNOASSAY].

    PubMed

    Zavyalova, E A; Gulyukin, M I; Carpova, M A; Bogdanova, P D; Droshnev, A E

    2016-01-01

    The infectious pancreatic necrosis (IPN) caused by a non-enveloped virus of the Birnaviridae family is one of the most important loss factors in the salmonid aquaculture. Virus isolation in the sensitive cell cultures has been approved in the Russian Federation as the diagnostic method for determination of IPNV antigen. This work gives the results of the development of the diagnostic test to reveal IPNV using the antigen-bound ELISA (sandwich ELISA). The developed test supplements a new diagnostic method and verifies some disputable results obtained with classical methods. PMID:27145601

  8. Development of a high throughput TaqMan assay for the detection of infectious laryngotracheitis virus in vector vaccinated chickens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Infectious laryngotracheitis virus (ILTV) causes an acute, highly contagious upper-respiratory disease of chickens. Sensitive detection of the causative alphaherpesvirus is important in clinical investigations and experimental studies. In particular, it is essential to quantify the viral genome co...

  9. Comparative full genome analysis of four infectious laryngotracheitis virus (gallid herpesvirus-1) virulent isolates from the United States

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Gallid herpesvirus type 1 (GaHV-1), commonly named infectious laryngotracheitis virus causes the respiratory disease in chickens known as infectious laryngotracheitis (ILT). Molecular determinants associated with differences in pathogenicity of GaHV-1 strains are not completely understood. Comparis...

  10. Prototypical Recombinant Multi-Protease Inhibitor Resistant Infectious Molecular Clones of Human Immunodeficiency Virus Type-1.

    PubMed

    Varghese, Vici; Mitsuya, Yumi; Fessel, W Jeffrey; Liu, Tommy F; Melikian, George L; Katzenstein, David A; Schiffer, Celia A; Holmes, Susan P; Shafer, Robert W

    2013-06-24

    The many genetic manifestations of HIV-1 protease inhibitor (PI) resistance present challenges to research into the mechanisms of PI-resistance and the assessment of new PIs. To address these challenges, we created a panel of recombinant multi-PI resistant infectious molecular clones designed to represent the spectrum of clinically relevant multi-PI resistant viruses. To assess the representativeness of this panel, we examined the sequences of the panel's viruses in the context of a correlation network of PI-resistance amino acid substitutions in sequences from more than 10,000 patients. The panel of recombinant infectious molecular clones comprised 29 of 41 study-defined PI-resistance amino acid substitutions and 23 of the 27 tightest amino acid substitution clusters. Based on their phenotypic properties, the clones were classified into four groups with increasing cross-resistance to the PIs most commonly used for salvage therapy: lopinavir (LPV), tipranavir (TPV), and darunavir (DRV). The panel of recombinant infectious molecular clones has been made available without restriction through the NIH AIDS Research and Reference Reagent Program. The public availability of the panel makes it possible to compare the inhibitory activity of different PIs with one another. The diversity of the panel and the high-level PI resistance of its clones suggest that investigational PIs active against the clones in this panel will retain antiviral activity against most, if not all clinically relevant PI-resistant viruses. PMID:23796938

  11. Complete Genome Sequence of a Chicken Embryo Fibroblast-Adapted Attenuated Infectious Bursal Disease Virus Isolate from India

    PubMed Central

    Priyadharsini, C. V.; Raja, P.; Kumanan, K.

    2016-01-01

    Infectious bursal disease virus is an avian pathogen that causes huge morbidity and mortality in the poultry sector all over the world. Here, we report the full-length genome sequence of an Indian strain, MB11/ABT/MVC/2016, isolated from a commercial broiler flock. This is a first report of a complete genome sequence of infectious bursal disease virus from India. PMID:27174268

  12. Complete Genome Sequence of a Chicken Embryo Fibroblast-Adapted Attenuated Infectious Bursal Disease Virus Isolate from India.

    PubMed

    Senthilkumar, T M A; Priyadharsini, C V; Raja, P; Kumanan, K

    2016-01-01

    Infectious bursal disease virus is an avian pathogen that causes huge morbidity and mortality in the poultry sector all over the world. Here, we report the full-length genome sequence of an Indian strain, MB11/ABT/MVC/2016, isolated from a commercial broiler flock. This is a first report of a complete genome sequence of infectious bursal disease virus from India. PMID:27174268

  13. Equine Endothelial Cells Support Productive Infection of Equine Infectious Anemia Virus

    PubMed Central

    Maury, Wendy; Oaks, J. Lindsay; Bradley, Sarahann

    1998-01-01

    Previous cell infectivity studies have demonstrated that the lentivirus equine infectious anemia virus (EIAV) infects tissue macrophages in vivo and in vitro. In addition, some strains of EIAV replicate to high titer in vitro in equine fibroblasts and fibroblast cell lines. Here we report a new cell type, macrovascular endothelial cells, that is infectible with EIAV. We tested the ability of EIAV to infect purified endothelial cells isolated from equine umbilical cords and renal arteries. Infectivity was detected by cell supernatant reverse transcriptase positivity, EIAV antigen positivity within individual cells, and the detection of viral RNA by in situ hybridization. Virus could rapidly spread through the endothelial cultures, and the supernatants of infected cultures contained high titers of infectious virus. There was no demonstrable cell killing in infected cultures. Three of four strains of EIAV that were tested replicated in these cultures, including MA-1, a fibroblast-tropic strain, Th.1, a macrophage-tropic strain, and WSU5, a strain that is fibroblast tropic and can cause disease. Finally, upon necropsy of a WSU5-infected horse 4 years postinfection, EIAV-positive endothelial cells were detected in outgrowths of renal artery cultures. These findings identify a new cell type that is infectible with EIAV. The role of endothelial cell infection in the course of equine infectious anemia is currently unknown, but endothelial cell infection may be involved in the edema that can be associated with infection. Furthermore, the ability of EIAV to persistently infect endothelial cultures and the presence of virus in endothelial cells from a long-term carrier suggest that this cell type can serve as a reservoir for the virus during subclinical phases of infection. PMID:9765477

  14. Establishment of reverse genetics system for infectious bronchitis virus attenuated vaccine strain H120.

    PubMed

    Zhou, Ying Shun; Zhang, Yi; Wang, Hong Ning; Fan, Wen Qiao; Yang, Xin; Zhang, An Yun; Zeng, Fan Ya; Zhang, Zhi Kun; Cao, Hai Peng; Zeng, Cheng

    2013-02-22

    Infectious bronchitis virus (IBV) strain H120 was successfully rescued as infectious clone by reverse genetics. Thirteen 1.5-2.8 kb fragments contiguously spanning the virus genome were amplified and cloned into pMD19-T. Transcription grade complete length cDNA was acquired by a modified "No See'm" ligation strategy, which employed restriction enzyme Bsa I and BsmB I and ligated more than two fragments in one T4 ligase reaction. The full-length genomic cDNA was transcribed and its transcript was transfected by electroporation into BHK-21 together with the transcript of nucleocapsid gene. At 48 h post transfection, the medium to culture the transfected BHK-21 cells was harvested and inoculated into 10-days old SPF embryonated chicken eggs (ECE) to replicate the rescued virus. After passage of the virus in ECE five times, the rescued H120 virus (R-H120) was successfully recovered. R-H120 was subsequently identified to possess the introduced silent mutation site in its genome. Some biological characteristics of R-H120 such as growth curve, EID50 and HA titers, were tested and all of them were very similar to its parent strain H120. In addition, both R-H120 and H120 induced a comparable titer of HA inhibition (HI) antibody in immunized chickens and also provided up to 85% of immune protection to the chickens that were challenged with Mass41 IBV strain. The present study demonstrated that construction of infectious clone from IBV vaccine strain H120 is possible and IBV-H120 can be use as a vaccine vector for the development of novel vaccines through molecular recombination and the modified reverse genetics approach. PMID:22999521

  15. Survival of the salmonid viruses infectious hematopoietic necrosis (IHNV) and infectious pancreatic necrosis (IPNV) in ozonated, chlorinated, and Untreated waters

    USGS Publications Warehouse

    Wedemeyer, Gary A.; Nelson, Nancy C.; Smith, Cathy A.

    1978-01-01

    Ozone and chlorine inactivation curves were determined in three water types at 10 °C for the fish pathogenic viruses infectious hematopoietic necrosis (IHNV) and infectious pancreatic necrosis (IPNV). In phosphate-buffered, distilled water (PBDW) an ozone dose of 0.01 mg/L for 30 or 60 s inactivated IHNV or IPNV, respectively, suspended at a tissue culture 50% infective dose (TCID50) of 104–105/mL. In hard (120 mg/L as CaCO3) and soft water (30 mg/L) lake waters, an ozone application rate of 70 mg∙h−1∙L−1 for 10 min destroyed IHNV. IPNV inactivation in hard water required 90 mg∙O3∙h−1∙L−1 for 10 min but only a 30-s contact time in soft water. The IPNV was also somewhat more resistant to chlorine. In PBDW, a residual of 0.1 mg/L with contact times of 30 and 60 s, respectively, destroyed IHNV and IPNV. In soft lake water IHNV was destroyed within 5 min at 0.5 mg/L, while in hard water a 10-min contact time was required. For IPNV disinfection in soft water, 0.2 mg/L for 10 min was sufficient but this chlorine residual had essentially no effect on IPNV in hard water. Increasing this dose to 0.7 mg/L destroyed IPNV in hard water within 2 min. In untreated waters, IPNV was stable for at least 8 wk in either distilled, soft, or hard lake waters. However, IHNV survived only about 2 wk in distilled and 7 wk in the soft or hard lake waters. We suggest the serious consideration of ozone as a fish disease control agent. Key words: ozone, chlorine disinfection, fish pathogens, viruses

  16. Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons.

    PubMed

    Contreras, Deisy; Arumugaswami, Vaithilingaraja

    2016-01-01

    Zika Virus (ZIKV) is an emerging pathogen that is linked to fetal developmental abnormalities such as microcephaly, eye defects, and impaired growth. ZIKV is an RNA virus of the Flaviviridae family. ZIKV is mainly transmitted by mosquitoes, but can also be spread by maternal to fetal vertical transmission as well as sexual contact. To date, there are no reliable treatment or vaccine options available to protect those infected by the virus. The development of a reproducible, effective Zika virus infectious cell culture system is critical for studying the molecular mechanisms of ZIKV replication as well as drug and vaccine development. In this regard, a protocol describing a mammalian cell-based in vitro Zika virus culture system for viral production and growth analysis is reported here. Details on the formation of plaques by Zika virus on a cell monolayer and plaque assay for measuring viral titer are presented. Viral genome replication kinetics and double-stranded RNA genome replicatory intermediates are determined. This culture platform was utilized to screen against a library of a small set of cytokines resulting in the identification of interferon-α (IFN-α), IFN-β and IFN-γ as potent inhibitors of Zika viral growth. In summary, an in vitro infectious Zika viral culture system and various virological assays are demonstrated in this study, which has the potential to greatly benefit the research community in elucidating further the mechanisms of viral pathogenesis and the evolution of viral virulence. Antiviral IFN-alpha can further be evaluated as a prophylactic, post-exposure prophylactic, and treatment option for Zika virus infections in high-risk populations, including infected pregnant women. PMID:27584546

  17. The cloned genome of ground squirrel hepatitis virus is infectious in the animal.

    PubMed Central

    Seeger, C; Ganem, D; Varmus, H E

    1984-01-01

    The lack of an in vitro infectivity assay for hepatitis B viruses has impeded the analysis of their genetic organization. To examine the feasibility of generating mutant and recombinant viruses after manipulation of cloned viral DNA in vitro, we have tested the infectivity of the cloned genome of ground squirrel hepatitis virus (GSHV) in virus-free Beechey ground squirrels. We demonstrate that cloned GSHV DNA is infectious when injected directly into the liver in the form of trimeric, head-to-tail recombinant clones and recircularized monomeric molecules but not when injected into the portal vein. Infections established in all four recipients of intrahepatic injections of cloned GSHV DNA exhibited the characteristics observed after administration of virus: GSHV surface antigen and viral DNA appeared in the serum 14-22 weeks after inoculation, and both circular and heterogeneous protein-linked forms of viral DNA were found in liver biopsy samples. Furthermore, virus present in the sera of these animals can be transmitted to other ground squirrels. These findings imply that any function of virion proteins in the initiation of infection by hepatitis B viruses can be bypassed with the use of cloned viral DNA and that this animal model is suitable for testing mutant genomes. Images PMID:6091114

  18. The Complete Nucleotide Sequence and Genome Organization of Tomato Cholrosis Virus: A Distinct Crinivirus most Closely Related to Lettuce Infectious Yellow Virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tomato infectious chlorosis virus (TICV), affects tomato production in many temperate to subtropical parts of the world where production is impacted by the presence of the greenhouse whitefly (Trialeurodes vaporariorum). Symptoms include interveinal yellowing, and leaves become thickened and crispy,...

  19. Evaluation of modified vaccinia virus Ankara expressing VP2 protein of infectious bursal disease virus as an immunogen in chickens.

    PubMed

    Zanetti, Flavia Adriana; Del Médico Zajac, María Paula; Taboga, Oscar Alberto; Calamante, Gabriela

    2012-06-01

    A recombinant modified vaccinia Ankara (MVA) virus expressing mature viral protein 2 (VP2) of the infectious bursal disease virus (IBDV) was constructed to develop MVA-based vaccines for poultry. We demonstrated that this recombinant virus was able to induce a specific immune response by observing the production of anti-IBDV-seroneutralizing antibodies in specific pathogen-free chickens. Besides, as the epitopes of VP2 responsible to induce IBDV-neutralizing antibodies are discontinuous, our results suggest that VP2 protein expressed from MVA-VP2 maintained the correct conformational structure. To our knowledge, this is the first report on the usefulness of MVA-based vectors for developing recombinant vaccines for poultry. PMID:22705743

  20. Evaluation of modified vaccinia virus Ankara expressing VP2 protein of infectious bursal disease virus as an immunogen in chickens

    PubMed Central

    Zajac, María Paula Del Médico; Taboga, Oscar Alberto; Calamante, Gabriela

    2012-01-01

    A recombinant modified vaccinia Ankara (MVA) virus expressing mature viral protein 2 (VP2) of the infectious bursal disease virus (IBDV) was constructed to develop MVA-based vaccines for poultry. We demonstrated that this recombinant virus was able to induce a specific immune response by observing the production of anti-IBDV-seroneutralizing antibodies in specific pathogen-free chickens. Besides, as the epitopes of VP2 responsible to induce IBDV-neutralizing antibodies are discontinuous, our results suggest that VP2 protein expressed from MVA-VP2 maintained the correct conformational structure. To our knowledge, this is the first report on the usefulness of MVA-based vectors for developing recombinant vaccines for poultry. PMID:22705743

  1. Generation of Newcastle diease virus (NDV) recombinants expressing the infectious laryngotracheitis virus (ILTV) glycoprotein gB or gD as dual vaccines

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Infectious laryngotracheitis (ILT) is a highly contagious acute respiratory disease of chickens caused by infection of infectious laryngotracheitis virus (ILTV). The current commercial ILT vaccines are either not safe or less effective. Therefore, there is a pressing need to develop safer and more...

  2. Crystal Structure of Feline Infectious Peritonitis Virus Main Protease in Complex with Synergetic Dual Inhibitors

    PubMed Central

    Wang, Fenghua; Chen, Cheng; Liu, Xuemeng; Yang, Kailin

    2015-01-01

    ABSTRACT Coronaviruses (CoVs) can cause highly prevalent diseases in humans and animals. Feline infectious peritonitis virus (FIPV) belongs to the genus Alphacoronavirus, resulting in a lethal systemic granulomatous disease called feline infectious peritonitis (FIP), which is one of the most important fatal infectious diseases of cats worldwide. No specific vaccines or drugs have been approved to treat FIP. CoV main proteases (Mpros) play a pivotal role in viral transcription and replication, making them an ideal target for drug development. Here, we report the crystal structure of FIPV Mpro in complex with dual inhibitors, a zinc ion and a Michael acceptor. The complex structure elaborates a unique mechanism of two distinct inhibitors synergizing to inactivate the protease, providing a structural basis to design novel antivirals and suggesting the potential to take advantage of zinc as an adjunct therapy against CoV-associated diseases. IMPORTANCE Coronaviruses (CoVs) have the largest genome size among all RNA viruses. CoV infection causes various diseases in humans and animals, including severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS). No approved specific drugs or vaccinations are available to treat their infections. Here, we report a novel dual inhibition mechanism targeting CoV main protease (Mpro) from feline infectious peritonitis virus (FIPV), which leads to lethal systemic granulomatous disease in cats. Mpro, conserved across all CoV genomes, is essential for viral replication and transcription. We demonstrated that zinc ion and a Michael acceptor-based peptidomimetic inhibitor synergistically inactivate FIPV Mpro. We also solved the structure of FIPV Mpro complexed with two inhibitors, delineating the structural view of a dual inhibition mechanism. Our study provides new insight into the pharmaceutical strategy against CoV Mpro through using zinc as an adjuvant therapy to enhance the efficacy of an irreversible

  3. Diagnosis of a naturally occurring dual infection of layer chickens with fowlpox virus and gallid herpesvirus 1 (infectious laryngotracheitis virus).

    PubMed

    Diallo, Ibrahim S; Taylor, Jim; Gibson, John; Hoad, John; De Jong, Amanda; Hewitson, Glen; Corney, Bruce G; Rodwell, Barry J

    2010-02-01

    An outbreak of acute respiratory disease in layers was diagnosed as being of dual nature due to fowlpox and infectious laryngotracheitis using a multidisciplinary approach including virus isolation, histopathology, electron microscopy and polymerase chain reaction (PCR). The diagnosis was based on virus isolation of gallid herpesvirus 1 (GaHV-1) in chicken kidney cells and fowlpox virus (FWPV) in 9-day-old chicken embryonated eggs inoculated via the chorioallantoic membrane. The histopathology of tracheas from dead birds revealed intra-cytoplasmic and intra-nuclear inclusions suggestive of poxvirus and herpesvirus involvement. The presence of FWPV was further confirmed by electron microscopy, PCR and histology. All FWPV isolates contained the long terminal repeats of reticuloendotheliosis virus as demonstrated by PCR. GaHV-1 isolates were detected by PCR and were shown to have a different restriction fragment length polymorphism pattern when compared with the chicken embryo origin SA2 vaccine strain; however, they shared the same pattern with the Intervet chicken embryo origin vaccine strain. This is a first report of dual infection of chickens with GaHV-1 and naturally occurring FWPV with reticuloendotheliosis virus insertions. Further characterization of the viruses was carried out and the results are reported here. PMID:20390533

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

    PubMed

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

    2010-02-01

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

  5. An Infectious cDNA Clone of Zika Virus to Study Viral Virulence, Mosquito Transmission, and Antiviral Inhibitors.

    PubMed

    Shan, Chao; Xie, Xuping; Muruato, Antonio E; Rossi, Shannan L; Roundy, Christopher M; Azar, Sasha R; Yang, Yujiao; Tesh, Robert B; Bourne, Nigel; Barrett, Alan D; Vasilakis, Nikos; Weaver, Scott C; Shi, Pei-Yong

    2016-06-01

    The Asian lineage of Zika virus (ZIKV) has recently caused epidemics and severe disease. Unraveling the mechanisms causing increased viral transmissibility and disease severity requires experimental systems. We report an infectious cDNA clone of ZIKV that was generated using a clinical isolate of the Asian lineage. The cDNA clone-derived RNA is infectious in cells, generating recombinant ZIKV. The recombinant virus is virulent in established ZIKV mouse models, leading to neurological signs relevant to human disease. Additionally, recombinant ZIKV is infectious for Aedes aegypti and thus provides a means to examine virus transmission. The infectious cDNA clone was further used to generate a luciferase ZIKV that exhibited sensitivity to a panflavivirus inhibitor, highlighting its potential utility for antiviral screening. This ZIKV reverse genetic system, together with mouse and mosquito infection models, may help identify viral determinants of human virulence and mosquito transmission as well as inform vaccine and therapeutic strategies. PMID:27198478

  6. Release of infectious human enteric viruses by full-scale wastewater utilities.

    PubMed

    Simmons, Fredrick James; Xagoraraki, Irene

    2011-06-01

    In the United States, infectious human enteric viruses are introduced daily into the environment through the discharge of treated water and the digested sludge (biosolids). In this study, a total of 30 wastewater and 6 biosolids samples were analyzed over five months (May-September 2008-2009) from five full-scale wastewater treatment plants (WWTPs) in Michigan using real-time PCR and cell culture assays. Samples were collected from four different locations at each WWTP (influent, pre-disinfection, post-disinfection and biosolids) using the 1MDS electropositive cartridge filter. Adenovirus (HAdV), enterovirus (EV) and norovirus genogroup II (NoV GGII) were detected in 100%, 67% and 10%, respectively of the wastewater samples using real-time PCR. Cytopathic effect (CPE) was present in 100% of the cell culture samples for influent, pre- and post-disinfection and biosolids with an average log concentration of 4.1 (2.9-4.7, range) 1.1 (0.0-2.3, range) and 0.5 (0.0-1.6, range) MPN/100 L and 2.1 (0.5-4.1) viruses/g, respectively. A significant log reduction in infectious viruses throughout the wastewater treatment process was observed at an average 4.2 (1.9-5.0, range) log units. A significant difference (p-value <0.05) was observed using real-time PCR data for HAdV but not for EV (p-value >0.05) removal in MBR as compared to conventional treatment. MBR treatment was able to achieve an additional 2 and 0.5 log reduction of HAdV and EV, respectively. This study has demonstrated the release of infectious enteric viruses in the final effluent and biosolids of wastewater treatment into the environment. PMID:21570703

  7. Engineering the largest RNA virus genome as an infectious bacterial artificial chromosome

    PubMed Central

    Almazán, Fernando; González, José M.; Pénzes, Zoltan; Izeta, Ander; Calvo, Enrique; Plana-Durán, Juan; Enjuanes, Luis

    2000-01-01

    The construction of cDNA clones encoding large-size RNA molecules of biological interest, like coronavirus genomes, which are among the largest mature RNA molecules known to biology, has been hampered by the instability of those cDNAs in bacteria. Herein, we show that the application of two strategies, cloning of the cDNAs into a bacterial artificial chromosome and nuclear expression of RNAs that are typically produced within the cytoplasm, is useful for the engineering of large RNA molecules. A cDNA encoding an infectious coronavirus RNA genome has been cloned as a bacterial artificial chromosome. The rescued coronavirus conserved all of the genetic markers introduced throughout the sequence and showed a standard mRNA pattern and the antigenic characteristics expected for the synthetic virus. The cDNA was transcribed within the nucleus, and the RNA translocated to the cytoplasm. Interestingly, the recovered virus had essentially the same sequence as the original one, and no splicing was observed. The cDNA was derived from an attenuated isolate that replicates exclusively in the respiratory tract of swine. During the engineering of the infectious cDNA, the spike gene of the virus was replaced by the spike gene of an enteric isolate. The synthetic virus replicated abundantly in the enteric tract and was fully virulent, demonstrating that the tropism and virulence of the recovered coronavirus can be modified. This demonstration opens up the possibility of employing this infectious cDNA as a vector for vaccine development in human, porcine, canine, and feline species susceptible to group 1 coronaviruses. PMID:10805807

  8. Emergence of novel nephropathogenic infectious bronchitis viruses currently circulating in Chinese chicken flocks.

    PubMed

    Xu, Qianqian; Han, Zongxi; Wang, Qiuling; Zhang, Tingting; Gao, Mengying; Zhao, Yan; Shao, Yuhao; Li, Huixin; Kong, Xiangang; Liu, Shengwang

    2016-01-01

    The emergence of novel infectious bronchitis viruses (IBVs) has been reported worldwide. Between 2011 and 2014, eight IBV isolates were identified from disease outbreaks in northeast China. In the current study we analysed the S1 gene of these eight IBV isolates in addition to the complete genome of five of them. We confirmed that these isolates emerged through the recombination of LX4 and Taiwan group 1 (TW1) viruses at two switch sites, one was in the Nsp 16 region and the other in the spike protein gene. The S1 gene in these viruses exhibited high nucleotide similarity with TW1-like viruses; the TW1 genotype was found to be present in southern China from 2009. Pathogenicity experiments in chickens using three of the eight virus isolates revealed that they were nephropathogenic and had similar pathogenicity to the parental viruses. The results of our study demonstrate that recombination, coupled with mutations, is responsible for the emergence of novel IBVs. PMID:26551660

  9. Responses of cloned rainbow trout Oncorhynchus mykiss to an attenuated strain of infectious hematopoietic necrosis virus.

    PubMed

    Ristow, S S; LaPatra, S E; Dixon, R; Pedrow, C R; Shewmaker, W D; Park, J W; Thorgaard, G H

    2000-09-28

    The objective of this work was to examine the response of homozygous clones of rainbow trout to vaccination by an attenuated strain (Nan Scott Lake; NSL) of infectious hematopoietic necrosis virus (IHNV). Adult rainbow trout of the Hot Creek Strain (YY males maintained in a recirculating system at 12 degrees C) were injected 3 times with 10(5) to 10(7) plaque forming units (pfu) of NSL. Intraperitoneal injections were given at Day 0 and at 2 and 4 mo post-infection. All fish were nonlethally bled at monthly intervals for 18 mo. Serum from each fish was analyzed by the complement-dependent neutralization assay and by western blot against purified NSL virus. The highest virus neutralization titers were detected 4 mo after the first injection, and peaked at 1280. When sera were analyzed by western blot, the predominating responses of the serum from immunized fish on the reduced western blot were against M1, a matrix protein of the virus and to a 90 kDa stress protein. The 90 kDa protein was identified by a monoclonal antibody as a stress protein derived from the CHSE-214 cells in which the purified IHN virus was grown and which associates with the virus during purification. PMID:11104067

  10. Genomics, Molecular Epidemiology and Diagnostics of Infectious hypodermal and hematopoietic necrosis virus.

    PubMed

    Rai, Praveen; Safeena, Muhammed P; Krabsetsve, Kjersti; La Fauce, Kathy; Owens, Leigh; Karunasagar, Indrani

    2012-09-01

    Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is one of the major viral pathogens of penaeid shrimps worldwide, which has resulted in severe mortalities of up to 90 % in cultured Penaeus (Litopenaeus) stylirostris from Hawaii and hence designated Penaeus stylirostris densovirus (PstDNV). IHHNV is distributed in shrimp culture facilities worldwide. It causes large economic loss to the shrimp farming industry. Our knowledge about the natural reservoirs of IHHNV is still scarce. Recent studies suggest that there is sufficient sequence variation among the isolates from different locations in Asia, suggesting multiple geographical strains of the virus. Four complete genomes and several partial sequences of the virus are available in the GenBank. Complete genome information would be useful for assessing the specificity of diagnostics for viruses from different geographical areas. Comparisons of complete genome sequences will help us gain insights into point mutations that can affect virulence of the virus. In addition, because of unavailability of shrimp cell lines for culturing IHHNV in vitro, quantification of virus is difficult. The recent progress in research regarding clinical signs, geographical distribution, complete genome sequence and genetic variation, transmission has made it possible to obtain information on IHHNV. A comprehensive understanding of IHHNV infection process, pathogenesis, structural proteins and replication is essential for developing prevention measures. To date, no effective prophylactic measure for IHHNV infection is available for shrimp to reduce its impact. This review provides an overview of key issues regarding IHHNV infection and disease in commercially important shrimp species. PMID:23997444

  11. Shedding of Infectious Borna Disease Virus-1 in Living Bicolored White-Toothed Shrews

    PubMed Central

    Nobach, Daniel; Bourg, Manon; Herzog, Sibylle; Lange-Herbst, Hildburg; Encarnação, Jorge A.; Eickmann, Markus; Herden, Christiane

    2015-01-01

    Background Many RNA viruses arise from animal reservoirs, namely bats, rodents and insectivores but mechanisms of virus maintenance and transmission still need to be addressed. The bicolored white-toothed shrew (Crocidura leucodon) has recently been identified as reservoir of the neurotropic Borna disease virus 1 (BoDV-1). Principal Findings Six out of eleven wild living bicoloured white-toothed shrews were trapped and revealed to be naturally infected with BoDV-1. All shrews were monitored in captivity in a long-term study over a time period up to 600 days that differed between the individual shrews. Interestingly, all six animals showed an asymptomatic course of infection despite virus shedding via various routes indicating a highly adapted host-pathogen interaction. Infectious virus and viral RNA were demonstrated in saliva, urine, skin swabs, lacrimal fluid and faeces, both during the first 8 weeks of the investigation period and for long time shedding after more than 250 days in captivity. Conclusions The various ways of shedding ensure successful virus maintenance in the reservoir population but also transmission to accidental hosts such as horses and sheep. Naturally BoDV-1-infected living shrews serve as excellent tool to unravel host and pathogen factors responsible for persistent viral co-existence in reservoir species while maintaining their physiological integrity despite high viral load in many organ systems. PMID:26313904

  12. Evidence for a carrier state of infectious hematopoietic necrosis virus in chinook salmon Oncorhynchus tshawytscha.

    PubMed

    St Hilaire, S; Ribble, C; Traxler, G; Davies, T; Kent, M L

    2001-10-01

    In British Columbia, Canada, infectious hematopoietic necrosis virus (IHNV) is prevalent in wild sockeye salmon Oncorhynchus nerka and has caused disease in seawater net-pen reared Atlantic salmon Salmo salar. In this study, chinook salmon Oncorhynchus tshawytscha experimentally exposed to an isolate of IHNV found in British Columbia became carriers of the virus. When Atlantic salmon were cohabited with these virus-exposed chinook salmon, IHNV was isolated from the Atlantic salmon. Identification of chinook salmon populations that have been exposed to IHNV may be difficult, as virus isolation was successful only in fish that were concurrently infected with either Renibacterium salmoninarum or Piscirickettisia salmonis. Also, IHNV-specific antibodies were detected in only 2 of the 70 fish experimentally exposed to the virus. Two samples collected from chinook salmon exposed to IHNV while at a salt water net-pen site had a seroprevalence of 19 and 22%; however, the inconsistencies between our laboratory and field data suggest that further research is required before we can rely on serological analysis for identifying potential carrier populations. Because of the difficulty in determining the exposure status of populations of chinook salmon, especially if there is no concurrent disease, it may be prudent not to cohabit Atlantic salmon with chinook salmon on a farm if there is any possibility that the latter have been exposed to the virus. PMID:11710551

  13. Use of propidium monoazide in reverse transcriptase PCR to distinguish between infectious and noninfectious enteric viruses in water samples

    EPA Science Inventory

    Human enteric viruses can be present in untreated and inadequately treated drinking water. Molecular methods, such as the reverse transcriptase PCR (RT-PCR), can detect viral genomes in a few hours, but they cannot distinguish between infectious and noninfectious viruses. Since o...

  14. Free-virus and cell-to-cell transmission in models of equine infectious anemia virus infection.

    PubMed

    Allen, Linda J S; Schwartz, Elissa J

    2015-12-01

    Equine infectious anemia virus (EIAV) is a lentivirus in the retrovirus family that infects horses and ponies. Two strains, referred to as the sensitive strain and the resistant strain, have been isolated from an experimentally-infected pony. The sensitive strain is vulnerable to neutralization by antibodies whereas the resistant strain is neutralization-insensitive. The sensitive strain mutates to the resistant strain. EIAV may infect healthy target cells via free virus or alternatively, directly from an infected target cell through cell-to-cell transfer. The proportion of transmission from free-virus or from cell-to-cell transmission is unknown. A system of ordinary differential equations (ODEs) is formulated for the virus-cell dynamics of EIAV. In addition, a Markov chain model and a branching process approximation near the infection-free equilibrium (IFE) are formulated. The basic reproduction number R0 is defined as the maximum of two reproduction numbers, R0s and R0r, one for the sensitive strain and one for the resistant strain. The IFE is shown to be globally asymptotically stable for the ODE model in a special case when the basic reproduction number is less than one. In addition, two endemic equilibria exist, a coexistence equilibrium and a resistant strain equilibrium. It is shown that if R0>1, the infection persists with at least one of the two strains. However, for small infectious doses, the sensitive strain and the resistant strain may not persist in the Markov chain model. Parameter values applicable to EIAV are used to illustrate the dynamics of the ODE and the Markov chain models. The examples highlight the importance of the proportion of cell-to-cell versus free-virus transmission that either leads to infection clearance or to infection persistence with either coexistence of both strains or to dominance by the resistant strain. PMID:25865935

  15. Infectious Progeny of 2009 A (H1N1) Influenza Virus Replicated in and Released from Human Neutrophils

    PubMed Central

    Zhang, Zhang; Huang, Tao; Yu, Feiyuan; Liu, Xingmu; Zhao, Conghui; Chen, Xueling; Kelvin, David J.; Gu, Jiang

    2015-01-01

    Various reports have indicated that a number of viruses could infect neutrophils, but the multiplication of viruses in neutrophils was abortive. Based on our previous finding that avian influenza viral RNA and proteins were present in the nucleus of infected human neutrophils in vivo, we investigated the possibility of 2009 A (H1N1) influenza viral synthesis in infected neutrophils and possible release of infectious progeny from host cells. In this study we found that human neutrophils in vitro without detectable level of sialic acid expression could be infected by this virus strain. We also show that the infected neutrophils can not only synthesize 2009 A (H1N1) viral mRNA and proteins, but also produce infectious progeny. These findings suggest that infectious progeny of 2009 A (H1N1) influenza virus could be replicated in and released from human neutrophils with possible clinical implications. PMID:26639836

  16. Infectious Progeny of 2009 A (H1N1) Influenza Virus Replicated in and Released from Human Neutrophils.

    PubMed

    Zhang, Zhang; Huang, Tao; Yu, Feiyuan; Liu, Xingmu; Zhao, Conghui; Chen, Xueling; Kelvin, David J; Gu, Jiang

    2015-01-01

    Various reports have indicated that a number of viruses could infect neutrophils, but the multiplication of viruses in neutrophils was abortive. Based on our previous finding that avian influenza viral RNA and proteins were present in the nucleus of infected human neutrophils in vivo, we investigated the possibility of 2009 A (H1N1) influenza viral synthesis in infected neutrophils and possible release of infectious progeny from host cells. In this study we found that human neutrophils in vitro without detectable level of sialic acid expression could be infected by this virus strain. We also show that the infected neutrophils can not only synthesize 2009 A (H1N1) viral mRNA and proteins, but also produce infectious progeny. These findings suggest that infectious progeny of 2009 A (H1N1) influenza virus could be replicated in and released from human neutrophils with possible clinical implications. PMID:26639836

  17. Optimization of a duplex real-time PCR method for relative quantitation of infectious laryngotracheitis virus.

    PubMed

    Vagnozzi, Ariel; Riblet, Sylva M; Zavala, Guillermo; García, Maricarmen

    2012-06-01

    Infectious laryngotracheitis is a highly contagious respiratory disease of chickens controlled by biosecurity and vaccination with live attenuated or recombinant vaccines. Infectious laryngotracheitis virus (ILTV) infections are characterized by a peak of viral replication in the trachea followed by a steady decrease in replication that results in the establishment of latency. Estimation of viral load is an important tool to determine the stage of ILTV infection. Here, a multiplex real-time PCR was optimized for the quantification of ILTV genomes. Quantification of viral genomes was based on the amplification of the ILTV UL44 gene, and sample variability was normalized using the chicken (Gallusgallus domesticus) alpha2-collagen gene as an endogenous control in a duplex reaction. PMID:22856202

  18. In vitro and in vivo effects of Houttuynia cordata on infectious bronchitis virus.

    PubMed

    Yin, Jiechao; Li, Guangxing; Li, Jing; Yang, Qing; Ren, Xiaofeng

    2011-10-01

    Avian infectious bronchitis virus (IBV), a coronavirus, causes infectious bronchitis leading to enormous economic loss in the poultry industry worldwide. Houttuynia cordata (Saururaceae) (HC) is a traditional Chinese medicine used in China. In the present study, the effect of HC on cell infection by IBV was determined using plaque assay and reverse transcription-polymerase chain reaction. The inhibitory effect of HC on IBV infection in ovo and in vivo was analysed using specific pathogen free (SPF) chicken embryos and chickens. Moreover, the effect of HC on cell apoptosis induced by IBV was investigated. Results showed that HC had more than 90% inhibition rate against IBV infection in Vero cells and chicken embryo kidney cells, and decreased more than 90% apoptotic cells caused by IBV. HC fully protected the SPF embryos, and had more than 50% protection rate in SPF chickens, against IBV challenge. PMID:21848486

  19. Construction of Poxviruses as Cloning Vectors: Insertion of the Thymidine Kinase Gene from Herpes Simplex Virus into the DNA of Infectious Vaccinia Virus

    NASA Astrophysics Data System (ADS)

    Panicali, Dennis; Paoletti, Enzo

    1982-08-01

    We have constructed recombinant vaccinia viruses containing the thymidine kinase gene from herpes simplex virus. The gene was inserted into the genome of a variant of vaccinia virus that had undergone spontaneous deletion as well as into the 120-megadalton genome of the large prototypic vaccinia variant. This was accomplished via in vivo recombination by contransfection of eukaryotic tissue culture cells with cloned BamHI-digested thymidine kinase gene from herpes simplex virus containing flanking vaccinia virus DNA sequences and infectious rescuing vaccinia virus. Pure populations of the recombinant viruses were obtained by replica filter techniques or by growth of the recombinant virus in biochemically selective medium. The herpes simplex virus thymidine kinase gene, as an insert in vaccinia virus, is transcribed in vivo and in vitro, and the fidelity of in vivo transcription into a functional gene product was detected by the phosphorylation of 5-[125I]iodo-2'-deoxycytidine.

  20. Inactivation of Infectious Bursal Disease Virus Through Composting of Litter from Poultry Houses.

    PubMed

    Crespo, Rocio; Badcoe, Lyndon M; Williams, Cheryl; Bary, Andrew I

    2016-06-01

    Very virulent infectious bursal disease virus (vvIBDV) was diagnosed in a pullet farm in Washington in 2014. Infectious bursal disease virus is resistant to many environmental stresses and often persists on farms for months. There have been conflicting reports as to whether composting can destroy vvIBDV in the manure. This project investigated the composting of litter from the affected house using an aerated static pile to inactivate the virus. Two weeks before the affected pullet flocks were moved to the layer house, specific-pathogen-free (SPF) birds were placed in the barns. Ten days after they were placed, three SPF birds died and were positive for vvIBDV. Thirty percent of the SPF birds were positive for vvIBDV. After the pullets were moved, at 20 wk of age, the litter in the house was composted using the aerated static pile method. The pile was maintained at above 55 C for 4 wk. After this time, 30 additional SPF birds were placed on the composted material. Two weeks later, the birds were healthy and there was no evidence of vvIBDV. The subsequent pullet flock did not break with vvIBDV. These results demonstrate that this composting method can be used to decontaminate the litter from vvIBDV and help prevent the spread of vvIBDV. PMID:27309296

  1. Dynamic distribution and tissue tropism of infectious laryngotracheitis virus in experimentally infected chickens.

    PubMed

    Wang, Lin-Guo; Ma, Jun; Xue, Chun-Yi; Wang, Wei; Guo, Chao; Chen, Feng; Qin, Jian-Ping; Huang, Ning-Hai; Bi, Ying-Zuo; Cao, Yong-Chang

    2013-03-01

    Infectious laryngotracheitis (ILT), caused by infectious laryngotracheitis virus (ILTV), is an Office International des Epizooties (OIE) notifiable disease. However, we have not clearly understood the dynamic distribution, tissue tropism, pathogenesis, and replication of ILTV in chickens. In this report, we investigated the dynamic distribution and tissue tropism of the virus in internal organs of experimentally infected chickens using quantitative real-time polymerase chain reaction (qPCR) and a histopathological test. The study showed that ILTV could be clearly detected in eight internal organs (throat, trachea, lung, cecum, kidney, pancreas, thymus and esophagus) of infected chickens, whereas the virus was difficult to detect in heart, spleen, proventriculus, liver, brain and bursa. Meanwhile, the thymidine kinase (TK) gene levels in eight internal organs increased from 3 days to 5 days postinfection, and then decreased from 6 days to 8 days postinfection. The log copy number of ILTV progressively increased over 3 days, which corresponds to the clinical score and the result of the histopathological test. The results provide a foundation for further clarification of the pathogenic mechanism of ILTV in internal organs and indicate that throat, lung, trachea, cecum, kidney, pancreas and esophagus may be preferred sites of acute infection, suggesting that the tissue tropism and distribution of ILTV is very broad. PMID:23392630

  2. Clinical and laboratory characteristics of infectious mononucleosis by Epstein-Barr virus in Mexican children

    PubMed Central

    2012-01-01

    Background Infectious mononucleosis (IM) or Mononucleosis syndrome is caused by an acute infection of Epstein-Barr virus. In Latin American countries, there are little information pertaining to the clinical manifestations and complications of this disease. For this reason, the purpose of this work was to describe the clinical and laboratory characteristics of infection by Epstein-Barr virus in Mexican children with infectious mononucleosis. Methods A descriptive study was carried out by reviewing the clinical files of patients less than 18 years old with clinical and serological diagnosis of IM by Epstein-Barr virus from November, 1970 to July, 2011 in a third level pediatric hospital in Mexico City. Results One hundred and sixty three cases of IM were found. The most frequent clinical signs were lymphadenopathy (89.5%), fever (79.7%), general body pain (69.3%), pharyngitis (55.2%), hepatomegaly (47.2%). The laboratory findings were lymphocytosis (41.7%), atypic lymphocytes (24.5%), and increased transaminases (30.9%), there were no rupture of the spleen and no deaths among the 163 cases. Conclusions Our results revealed that IM appeared in earlier ages compared with that reported in industrialized countries, where adolescents are the most affected group. Also, the order and frequency of the clinical manifestations were different in our country than in industrialized ones. PMID:22818256

  3. The proteome of the infectious bronchitis virus Beau-R virion.

    PubMed

    Dent, Stuart D; Xia, Dong; Wastling, Jonathan M; Neuman, Benjamin W; Britton, Paul; Maier, Helena J

    2015-12-01

    Infectious bronchitis is a highly contagious respiratory disease of poultry caused by the coronavirus infectious bronchitis virus (IBV). It was thought that coronavirus virions were composed of three major viral structural proteins until investigations of other coronaviruses showed that the virions also include viral non-structural and genus-specific accessory proteins as well as host-cell proteins. To study the proteome of IBV virions, virus was grown in embryonated chicken eggs, purified by sucrose-gradient ultracentrifugation and analysed by mass spectrometry. Analysis of three preparations of purified IBV yielded the three expected structural proteins plus 35 additional virion-associated host proteins. The virion-associated host proteins had a diverse range of functional attributions, being involved in cytoskeleton formation, RNA binding and protein folding pathways. Some of these proteins were unique to this study, while others were found to be orthologous to proteins identified in severe acute respiratory syndrome coronavirus virions and also virions from a number of other RNA and DNA viruses. PMID:27257648

  4. Structure of equine infectious anemia virus proteinase complexed with an inhibitor.

    PubMed Central

    Gustchina, A.; Kervinen, J.; Powell, D. J.; Zdanov, A.; Kay, J.; Wlodawer, A.

    1996-01-01

    Equine infectious anemia virus (EIAV), the causative agent of infectious anemia in horses, is a member of the lentiviral family. The virus-encoded proteinase (PR) processes viral polyproteins into functional molecules during replication and it also cleaves viral nucleocapsid protein during infection. The X-ray structure of a complex of the 154G mutant of EIAV PR with the inhibitor HBY-793 was solved at 1.8 A resolution and refined to a crystallographic R-factor of 0.136. The molecule is a dimer in which the monomers are related by a crystallographic twofold axis. Although both the enzyme and the inhibitor are symmetric, the interactions between the central part of the inhibitor and the active site aspartates are asymmetric, and the inhibitor and the two flaps are partially disordered. The overall fold of EIAV PR is very similar to that of other retroviral proteinases. However, a novel feature of the EIAV PR structure is the appearance of the second alpha-helix in the monomer in a position predicted by the structural template for the family of aspartic proteinases. The parts of the EIAV PR with the highest resemblance to human immunodeficiency virus type 1 PR include the substrate-binding sites; thus, the differences in the specificity of both enzymes have to be explained by enzyme-ligand interactions at the periphery of the active site as well. PMID:8844837

  5. Development of an infectious surrogate hepatitis C virus based on a recombinant vesicular stomatitis virus expressing hepatitis C virus envelope glycoproteins and green fluorescent protein.

    PubMed

    Okuma, Kazu; Fukagawa, Koji; Tateyama, Seiji; Kohma, Takuya; Mochida, Keiko; Hiyoshi, Masateru; Takahama, Youichi; Hamaguchi, Yukio; Hirose, Kunitaka; Buonocore, Linda; Rose, John K; Mizuochi, Toshiaki; Hamaguchi, Isao

    2015-01-01

    To develop surrogate viruses for hepatitis C virus (HCV), we previously produced recombinant vesicular stomatitis viruses (rVSVs) lacking glycoprotein G but instead expressing chimeric HCV E1/E2 fused to G. These rVSVs were not infectious in HCV-susceptible hepatoma cells. In this study, to develop an infectious surrogate HCV based on an rVSV (vesicular stomatitis virus [VSV]/HCV), we generated a novel rVSV encoding the native E1/E2 (H77 strain) and green fluorescent protein (GFP) instead of G. Here, we showed that this VSV/HCV efficiently infected human hepatoma cells, including Huh7 human hepatoma cells, expressed GFP in these cells, and propagated, but did not do so in nonsusceptible BHK-21 cells. The infectivity of VSV/HCV, measured as the number of foci of GFP-positive cells, was specifically reduced by the addition of chimpanzee anti-HCV serum, anti-E2 antibody, or anti-CD81 antibody to the cultures. When sera obtained from HCV-infected or uninfected patients were added, infection was selectively inhibited only by the sera of HCV-infected patients. These data together suggest that this infectious GFP-expressing VSV/HCV could be a useful tool for studying the mechanisms of HCV entry into cells and for assessing potential inhibitors of viral entry, including neutralizing antibodies. PMID:25672345

  6. Use of Propidium Monoazide in Reverse Transcriptase PCR To Distinguish between Infectious and Noninfectious Enteric Viruses in Water Samples▿

    PubMed Central

    Parshionikar, Sandhya; Laseke, Ian; Fout, G. Shay

    2010-01-01

    Human enteric viruses can be present in untreated and inadequately treated drinking water. Molecular methods, such as the reverse transcriptase PCR (RT-PCR), can detect viral genomes in a few hours, but they cannot distinguish between infectious and noninfectious viruses. Since only infectious viruses are a public health concern, methods that not only are rapid but also provide information on the infectivity of viruses are of interest. The intercalating dye propidium monoazide (PMA) has been used for distinguishing between viable and nonviable bacteria with DNA genomes, but it has not been used to distinguish between infectious and noninfectious enteric viruses with RNA genomes. In this study, PMA in conjunction with RT-PCR (PMA-RT-PCR) was used to determine the infectivity of enteric RNA viruses in water. Coxsackievirus, poliovirus, echovirus, and Norwalk virus were rendered noninfectious or inactivated by treatment with heat (72°C, 37°C, and 19°C) or hypochlorite. Infectious or native and noninfectious or inactivated viruses were treated with PMA. This was followed by RNA extraction and RT-PCR or quantitative RT-PCR (qRT-PCR) analysis. The PMA-RT-PCR results indicated that PMA treatment did not interfere with detection of infectious or native viruses but prevented detection of noninfectious or inactivated viruses that were rendered noninfectious or inactivated by treatment at 72°C and 37°C and by hypochlorite treatment. However, PMA-RT-PCR was unable to prevent detection of enteroviruses that were rendered noninfectious by treatment at 19°C. After PMA treatment poliovirus that was rendered noninfectious by treatment at 37°C was undetectable by qRT-PCR, but PMA treatment did not affect detection of Norwalk virus. PMA-RT-PCR was also shown to be effective for detecting infectious poliovirus in the presence of noninfectious virus and in an environmental matrix. We concluded that PMA can be used to differentiate between potentially infectious and noninfectious

  7. Use of propidium monoazide in reverse transcriptase PCR to distinguish between infectious and noninfectious enteric viruses in water samples.

    PubMed

    Parshionikar, Sandhya; Laseke, Ian; Fout, G Shay

    2010-07-01

    Human enteric viruses can be present in untreated and inadequately treated drinking water. Molecular methods, such as the reverse transcriptase PCR (RT-PCR), can detect viral genomes in a few hours, but they cannot distinguish between infectious and noninfectious viruses. Since only infectious viruses are a public health concern, methods that not only are rapid but also provide information on the infectivity of viruses are of interest. The intercalating dye propidium monoazide (PMA) has been used for distinguishing between viable and nonviable bacteria with DNA genomes, but it has not been used to distinguish between infectious and noninfectious enteric viruses with RNA genomes. In this study, PMA in conjunction with RT-PCR (PMA-RT-PCR) was used to determine the infectivity of enteric RNA viruses in water. Coxsackievirus, poliovirus, echovirus, and Norwalk virus were rendered noninfectious or inactivated by treatment with heat (72 degrees C, 37 degrees C, and 19 degrees C) or hypochlorite. Infectious or native and noninfectious or inactivated viruses were treated with PMA. This was followed by RNA extraction and RT-PCR or quantitative RT-PCR (qRT-PCR) analysis. The PMA-RT-PCR results indicated that PMA treatment did not interfere with detection of infectious or native viruses but prevented detection of noninfectious or inactivated viruses that were rendered noninfectious or inactivated by treatment at 72 degrees C and 37 degrees C and by hypochlorite treatment. However, PMA-RT-PCR was unable to prevent detection of enteroviruses that were rendered noninfectious by treatment at 19 degrees C. After PMA treatment poliovirus that was rendered noninfectious by treatment at 37 degrees C was undetectable by qRT-PCR, but PMA treatment did not affect detection of Norwalk virus. PMA-RT-PCR was also shown to be effective for detecting infectious poliovirus in the presence of noninfectious virus and in an environmental matrix. We concluded that PMA can be used to differentiate

  8. Susceptibility of Koi and Yellow Perch to infectious hematopoietic necrosis virus by experimental exposure

    USGS Publications Warehouse

    Palmer, Alexander D.; Emmenegger, Eveline J.

    2014-01-01

    Infectious hematopoietic necrosis virus (IHNV) is a novirhabdoviral pathogen that originated in western North America among anadromous Pacific salmonids. Severe disease epidemics in the late 1970s resulting from IHNV's invasion into farmed Rainbow Trout Oncorhynchus mykiss in North America, Asia, and Europe emphasized IHNV's ability to adapt to new hosts under varying rearing conditions. Yellow Perch Perca flavescens and Koi Carp Cyprinus carpio (hereafter, “Koi”) are aquaculture-reared fish that are highly valued in sport fisheries and the ornamental fish trade, respectively, but it is unknown whether these fish species are vulnerable to IHNV infection. In this study, we exposed Yellow Perch, Koi, and steelhead (anadromous Rainbow Trout) to IHNV by intraperitoneal injection (106 PFU/fish) and by immersion (5.7×105 PFU/mL) for 7 h, and monitored fish for 28 d. The extended immersion exposure and high virus concentrations used in the challenges were to determine if the tested fish had any level of susceptibility. After experimental exposure, Yellow Perch and Koi experienced low mortality (35%). Virus was found in dead fish of all species tested and in surviving Yellow Perch by plaque assay and quantitative reverse transcription polymerase chain reaction (qPCR), with a higher prevalence in Yellow Perch than Koi. Infectious virus was also detected in Yellow Perch out to 5 d after bath challenge. These findings indicate that Yellow Perch and Koi are highly resistant to IHNV disease under the conditions tested, but Yellow Perch are susceptible to infection and may serve as possible virus carriers.

  9. Recovery of Infectious Pariacoto Virus from cDNA Clones and Identification of Susceptible Cell Lines

    PubMed Central

    Johnson, Karyn N.; Ball, L. Andrew

    2001-01-01

    Pariacoto virus (PaV) is a nodavirus that was recently isolated in Peru from the Southern armyworm, Spodoptera eridania. Virus particles are non enveloped and about 30 nm in diameter and have T=3 icosahedral symmetry. The 3.0-Å crystal structure shows that about 35% of the genomic RNA is icosahedrally ordered, with the RNA forming a dodecahedral cage of 25-nucleotide (nt) duplexes that underlie the inner surface of the capsid. The PaV genome comprises two single-stranded, positive-sense RNAs: RNA1 (3,011 nt), which encodes the 108-kDa catalytic subunit of the RNA-dependent RNA polymerase, and RNA2 (1,311 nt), which encodes the 43-kDa capsid protein precursor α. In order to apply molecular genetics to the structure and assembly of PaV, we identified susceptible cell lines and developed a reverse genetic system for this virus. Cell lines that were susceptible to infection by PaV included those from Spodoptera exigua, Helicoverpa zea and Aedes albopictus, whereas cells from Drosophila melanogaster and Spodoptera frugiperda were refractory to infection. To recover virus from molecular clones, full-length cDNAs of PaV RNAs 1 and 2 were cotranscribed by T7 RNA polymerase in baby hamster kidney cells that expressed T7 RNA polymerase. Lysates of these cells were infectious both for cultured cells from Helicoverpa zea (corn earworm) and for larvae of Galleria mellonella (greater wax moth). The combination of infectious cDNA clones, cell culture infectivity, and the ability to produce milligram amounts of virus allows the application of DNA-based genetic methods to the study of PaV structure and assembly. PMID:11711613

  10. Recovery of infectious pariacoto virus from cDNA clones and identification of susceptible cell lines.

    PubMed

    Johnson, K N; Ball, L A

    2001-12-01

    Pariacoto virus (PaV) is a nodavirus that was recently isolated in Peru from the Southern armyworm, Spodoptera eridania. Virus particles are non enveloped and about 30 nm in diameter and have T=3 icosahedral symmetry. The 3.0-A crystal structure shows that about 35% of the genomic RNA is icosahedrally ordered, with the RNA forming a dodecahedral cage of 25-nucleotide (nt) duplexes that underlie the inner surface of the capsid. The PaV genome comprises two single-stranded, positive-sense RNAs: RNA1 (3,011 nt), which encodes the 108-kDa catalytic subunit of the RNA-dependent RNA polymerase, and RNA2 (1,311 nt), which encodes the 43-kDa capsid protein precursor alpha. In order to apply molecular genetics to the structure and assembly of PaV, we identified susceptible cell lines and developed a reverse genetic system for this virus. Cell lines that were susceptible to infection by PaV included those from Spodoptera exigua, Helicoverpa zea and Aedes albopictus, whereas cells from Drosophila melanogaster and Spodoptera frugiperda were refractory to infection. To recover virus from molecular clones, full-length cDNAs of PaV RNAs 1 and 2 were cotranscribed by T7 RNA polymerase in baby hamster kidney cells that expressed T7 RNA polymerase. Lysates of these cells were infectious both for cultured cells from Helicoverpa zea (corn earworm) and for larvae of Galleria mellonella (greater wax moth). The combination of infectious cDNA clones, cell culture infectivity, and the ability to produce milligram amounts of virus allows the application of DNA-based genetic methods to the study of PaV structure and assembly. PMID:11711613

  11. Characterization of an infectious molecular clone of human T-cell leukemia virus type I.

    PubMed Central

    Zhao, T M; Robinson, M A; Bowers, F S; Kindt, T J

    1995-01-01

    An infectious molecular clone of human T-cell leukemia virus type I (HTLV-I) was derived from an HTLV-I-transformed rabbit T-cell line, RH/K30, obtained by coculture of rabbit peripheral blood mononuclear cells (PBMC) with the human HTLV-I-transformed cell line MT-2. The RH/K30 cell line contained two integrated proviruses, an intact HTLV-I genome and an apparently defective provirus with an in-frame stop codon in the env gene. A genomic DNA fragment containing the intact HTLV-I provirus was cloned into bacteriophage lambda (K30 phi) and subcloned into a plasmid vector (K30p). HTLV-I p24gag protein was detected in culture supernatants of human and rabbit T-cell and fibroblast lines transfected with these clones, at levels comparable to those of the parental cell line RH/K30. Persistent expression of virus was observed in one of these lines, RL-5/K30p, for more than 24 months. Biologic characterization of this cell line revealed the presence of integrated HTLV-I provirus, spliced and unspliced mRNA transcripts, and typical extracellular type C retrovirus particles. As expected, these virus particles contained HTLV-I RNA and reverse transcriptase activity. The transfected cells also expressed surface major histocompatibility complex class II, whereas no expression of this molecule was detected in the parental RL-5 cell line. Virus was passaged by cocultivation of irradiated RL-5/K30p cells with either rabbit PBMC or human cord blood mononuclear cells, demonstrating in vitro infectivity. The virus produced in these cells was also infectious in vivo, since rabbits injected with RL-5/K30p cells became productively infected, as evidenced by seroconversion, amplification of HTLV-I-specific sequences by PCR from PBMC DNA, and virus isolation from PBMC. Availability of infectious molecular clones will facilitate functional studies of HTLV-I genes and gene products. PMID:7884847

  12. Mosquito Cellular Factors and Functions in Mediating the Infectious entry of Chikungunya Virus

    PubMed Central

    Lee, Regina Ching Hua; Hapuarachchi, Hapuarachchige Chanditha; Chen, Karen Caiyun; Hussain, Khairunnisa' Mohamed; Chen, Huixin; Low, Swee Ling; Ng, Lee Ching; Lin, Raymond; Ng, Mary Mah-Lee; Chu, Justin Jang Hann

    2013-01-01

    Chikungunya virus (CHIKV) is an arthropod-borne virus responsible for recent epidemics in the Asia Pacific regions. A customized gene expression microarray of 18,760 transcripts known to target Aedes mosquito genome was used to identify host genes that are differentially regulated during the infectious entry process of CHIKV infection on C6/36 mosquito cells. Several genes such as epsin I (EPN1), epidermal growth factor receptor pathway substrate 15 (EPS15) and Huntingtin interacting protein I (HIP1) were identified to be differentially expressed during CHIKV infection and known to be involved in clathrin-mediated endocytosis (CME). Transmission electron microscopy analyses further revealed the presence of CHIKV particles within invaginations of the plasma membrane, resembling clathrin-coated pits. Characterization of vesicles involved in the endocytic trafficking processes of CHIKV revealed the translocation of the virus particles to the early endosomes and subsequently to the late endosomes and lysosomes. Treatment with receptor-mediated endocytosis inhibitor, monodansylcadaverine and clathrin-associated drug inhibitors, chlorpromazine and dynasore inhibited CHIKV entry, whereas no inhibition was observed with caveolin-related drug inhibitors. Inhibition of CHIKV entry upon treatment with low-endosomal pH inhibitors indicated that low pH is essential for viral entry processes. CHIKV entry by clathrin-mediated endocytosis was validated via overexpression of a dominant-negative mutant of Eps15, in which infectious entry was reduced, while siRNA-based knockdown of genes associated with CME, low endosomal pH and RAB trafficking proteins exhibited significant levels of CHIKV inhibition. This study revealed, for the first time, that the infectious entry of CHIKV into mosquito cells is mediated by the clathrin-dependent endocytic pathway. PMID:23409203

  13. Neutralization-resistant variants of infectious hematopoietic necrosis virus have altered virulence and tissue tropism.

    PubMed Central

    Kim, C H; Winton, J R; Leong, J C

    1994-01-01

    Infectious hematopoietic necrosis virus (IHNV) is a rhabdovirus that causes an acute disease in salmon and trout. In this study, a correlation between changes in tissue tropism and specific changes in the virus genome appeared to be made by examining four IHNV neutralization-resistant variants (RB-1, RB-2, RB-3, and RB-4) that had been selected with the glycoprotein (G)-specific monoclonal antibody RB/B5. These variants were compared with the parental strain (RB-76) for their virulence and pathogenicity in rainbow trout after waterborne challenge. Variants RB-2, RB-3, and RB-4 were only slightly attenuated and showed distributions of viral antigen in the livers and hematopoietic tissues of infected fish similar to those of the parental strain. Variant RB-1, however, was highly attenuated and the tissue distribution of viral antigen in RB-1-infected fish was markedly different, with more viral antigen in brain tissue. The sequences of the G genes of all four variants and RB-76 were determined. No significant changes were found for the slightly attenuated variants, but RB-1 G had two changes at amino acids 78 and 218 that dramatically altered its predicted secondary structure. These changes are thought to be responsible for the altered tissue tropism of the virus. Thus, IHNV G, like that of rabies virus and vesicular stomatitis virus, plays an integral part in the pathogenesis of viral infection. Images PMID:7525991

  14. [The Isolation and Identification of Infectious Bronchitis Virus PTFY Strain in Muscovy Ducks].

    PubMed

    Wu, Xiaoping; Pan, Shulei; Zhou, Wuduo; Wu, Yijiang; Huang, Yifan; Wu, Baocheng

    2016-03-01

    In July 2009, some farms of breeding Muscovy ducks on the peak of egg laying suffered the decrease of hatching rate and the quality of the eggs showing low mortality and no evident respiratory symptoms. The swelling and congestive ovary was visible after autopsy. This study was brought out for the diagnosis of these cases. The virus was isolated and identified by the methods of virus culture in chicken embryo, physical and chemical properties test, hemagglutinin test, NDV (Newcastle diseases Virus) interference test, electron microscope observation, pathogenicity test and the gene sequence analysis. The results indicated the virus showed the characters of inducing dwarf embryo after inocubation, the sensibility to lipid solvent and the hemagglutination capacity after pancreatic enzyme treatment, the typical morphology of coronavirus, the interference to NDV replication and the homology among 84.7% - 99% of the particial N gene sequences to the reference IBV (Avian infectious bronchitis virus) strains. The strain was identified as IBV isolate and this study confirmed the pathogenicity of IBV to Muscovy ducks. PMID:27396165

  15. Inhibition of Cellular DNA Synthesis in Cells Infected with Infectious Pancreatic Necrosis Virus

    PubMed Central

    Lothrop, David; Nicholson, Bruce L.

    1974-01-01

    In asynchronous RTG-2 cell cultures infected with infectious pancreatic necrosis (IPN) virus, inhibition of cellular DNA synthesis, but not protein synthesis, was detected 5 to 6 h postinfection and was 80 to 90% complete by 7 to 8 h. Inhibition of DNA synthesis was largely abolished by UV irradiation of the virus. Sedimentation analyses of phenol-extracted DNA indicated that native cellular DNA was not degraded during infection. Sedimentation on alkaline sucrose gradients of DNA from cells pulsed with radioactive thymidine for varying periods indicated that elongation of nascent DNA chains proceeded normally in infected cells. These and previous results suggest that IPN virus infection results in a reduction of the number of chromosomal sites active in DNA synthesis but does not affect the rate of polymerization at active sites. Cells synchronized with excess thymidine and hydroxyurea and infected with virus at the time of release from the block demonstrated an inhibition of DNA synthesis 3 h postinfection. Cells infected 4 h prior to release continued to synthesize normal amounts of DNA for 1 to 2 h after release. These results indicated that DNA synthesis in early synthetic phase is relatively insensitive to inhibition by IPN virus. PMID:4852469

  16. In vitro RNA synthesis by infectious pancreatic necrosis virus-associated RNA polymerase.

    PubMed

    Mertens, P P; Jamieson, P B; Dobos, P

    1982-03-01

    The presence of an RNA-dependent RNA polymerase was demonstrated in purified infectious pancreatic necrosis virus (IPNV). The enzyme was active in vitro without any pretreatment of the virus. Optimum activity was shown at 30 degrees C, pH 8 and in the presence of 6 mM-magnesium ions. Approx. 50% of the polymerase product remained associated with the dsRNA template of the virions. The remainder was found as extravirion ssRNA broken down to 5S to 7S fragments by virus-associated RNase(s). Although the addition of bentonite considerably reduced the amount of RNA synthesized, it protected the ssRNA product from degradation. This, in turn, permitted the synthesis of small amounts of ssRNA, which when analysed by sucrose gradient centrifugation or polyacrylamide gel electrophoresis behaved identically to the 24S single-stranded virus mRNA produced in infected cells. The virion polymerase was not stimulated by S-adenosyl-L-methionine or the addition of cellular or capped reovirus ssRNA. Several other modifications of the assay system were tried in an attempt to increase 24S RNA synthesis, but with little success. When [3H]uridine-labelled virus was used in the polymerase reaction, some labelled 24S ssRNA was obtained, indicating that in vitro transcription may proceed by a semi-conservative (displacement) mechanism. PMID:6175731

  17. Cross-protective immune responses between genotypically distinct lineages of infectious laryngotracheitis viruses.

    PubMed

    Lee, Sang-Won; Markham, Philip F; Coppo, Mauricio J C; Legione, Alistair R; Shil, Niraj K; Quinteros, José A; Noormohammadi, Amir H; Browning, Glenn F; Hartley, Carol A; Devlin, Joanne M

    2014-03-01

    Recent phylogenetic studies have identified different genotypic lineages of infectious laryngotracheitis virus (ILTV), and these lineages can recombine in the field. The emergence of virulent recombinant field strains of ILTV by natural recombination between commercial vaccines belonging to different genotypic lineages has been reported recently. Despite the use of attenuated ILTV vaccines, these recombinant viruses were able to spread and cause disease in commercial poultry flocks, raising the question of whether the different lineages of ILTV can induce cross-protective immune responses. This study examined the capacity of the Australian-origin A20 ILTV vaccine to protect against challenge with the class 8 ILTV recombinant virus, the genome of which is predominantly derived from a heterologous genotypic lineage. Following challenge, birds vaccinated via eyedrop were protected from clinical signs of disease and pathological changes in the tracheal mucosa, although they were not completely protected from viral infection or replication. In contrast, the challenge virus induced severe clinical signs and tracheal pathology in unvaccinated birds. This is the first study to examine the ability of a vaccine from the Australian lineage to protect against challenge with a virus from a heterologous lineage. These results suggest that the two distinct genotypic lineages of ILTV can both induce cross-protection, indicating that current commercial vaccines are still likely to assist in control of ILTV in the poultry industry, in spite of the emergence of novel recombinants derived from different genotypic lineages. PMID:24758128

  18. Occurrence and genetic typing of infectious hematopoietic necrosis virus in Kamchatka, Russia

    USGS Publications Warehouse

    Rudakova, S.L.; Kurath, G.; Bochkova, E.V.

    2007-01-01

    Infectious hematopoietic necrosis virus (IHNV) is a well known rhabdoviral pathogen of salmonid fish in North America that has become established in Asia and Europe. On the Pacific coast of Russia, IHNV was first detected in hatchery sockeye from the Kamchatka Peninsula in 2001. Results of virological examinations of over 10 000 wild and cultured salmonid fish from Kamchatka during 1996 to 2005 revealed IHNV in several sockeye salmon Oncorhynchus nerka populations. The virus was isolated from spawning adults and from juveniles undergoing epidemics in both hatchery and wild sockeye populations from the Bolshaya watershed. No virus was detected in 2 other water-sheds, or in species other than sockeye salmon. Genetic typing of 8 virus isolates by seguence analysis of partial glycoprotein and nucleocapsid genes revealed that they were genetically homogeneous and fell within the U genogroup of IHNV. In phylogenetic analyses, the Russian IHNV sequences were indistinguishable from the sequences of North American U genogroup isolates that occur throughout Alaska, British Columbia, Washington, and Oregon. The high similarity, and in some cases identity, between Russian and North American IHNV isolates suggests virus transmission or exposure to a common viral reservoir in the North Pacific Ocean. ?? Inter-Research 2007.

  19. Heterologous live infectious bronchitis virus vaccination in day-old commercial broiler chicks: clinical signs, ciliary health, immune responses and protection against variant infectious bronchitis viruses.

    PubMed

    Awad, Faez; Hutton, Sally; Forrester, Anne; Baylis, Matthew; Ganapathy, Kannan

    2016-04-01

    Groups of one-day-old broiler chicks were vaccinated via the oculo-nasal route with different live infectious bronchitis virus (IBV) vaccines: Massachusetts (Mass), 793B, D274 or Arkansas (Ark). Clinical signs and gross lesions were evaluated. Five chicks from each group were humanely killed at intervals and their tracheas collected for ciliary activity assessment and for the detection of CD4+, CD8+ and IgA-bearing B cells by immunohistochemistry (IHC). Blood samples were collected at intervals for the detection of anti-IBV antibodies. At 21 days post-vaccination (dpv), protection conferred by different vaccination regimes against virulent M41, QX and 793B was assessed. All vaccination programmes were able to induce high levels of CD4+, CD8+ and IgA-bearing B cells in the trachea. Significantly higher levels of CD4+ and CD8+ expression were observed in the Mass2 + 793B2-vaccinated group compared to the other groups (subscripts indicate different manufacturers). Protection studies showed that the group of chicks vaccinated with Mass2 + 793B2 produced 92% ciliary protection against QX challenge; compared to 53%, 68% and 73% ciliary protection against the same challenge virus by Mass1 + D274, Mass1 + 793B1 and Mass3 + Ark, respectively. All vaccination programmes produced more than 85% ciliary protection against M41 and 793B challenges. It appears that the variable levels of protection provided by different heterologous live IBV vaccinations are dependent on the levels of local tracheal immunity induced by the respective vaccine combination. The Mass2 + 793B2 group showed the worst clinical signs, higher mortality and severe lesions following vaccination, but had the highest tracheal immune responses and demonstrated the best protection against all three challenge viruses. PMID:26743315

  20. The incidence of antibody to infectious bovine rhinotracheitis virus in Kenyan cattle.

    PubMed

    Jessett, D M; Rampton, C S

    1975-03-01

    A total of 3204 cattle sera, collected between 1966 and 1974, from all seven provinces and 34 of the 42 districts of Kenya were screened in a neutralisation test for antibody to infectious bovine rhinotracheitis virus (IBRV). Antibody to IBRV was found in some sera from all the districts, with cattle over two years old showing the highest incidence. A small number of goat sera also showed some antibody. From the results obtained it is concluded that IBRV infection is widespread in Kenyan cattle. PMID:165572

  1. Detection of infectious bronchitis virus strains similar to Japan in Taiwan

    PubMed Central

    TSAI, Cheng-Ta; TSAI, Hsin-Fu; WANG, Ching-Ho

    2016-01-01

    A total of 1,320 tracheal samples from 66 broiler flocks sent to slaughterhouses and 42 tracheal samples from 42 flocks of local chickens in the field were collected for infectious bronchitis virus (IBV) gene detection by reverse transcription polymerase chain reaction using nucleocapsid-specific primers and spike-specific primers. Prevalence in broiler flocks was 39.4% (26/66) and in local chicken flocks was 11.9% (5/42). Several IBVs similar to Japan were detected in Taiwan. One-direction neutralization revealed that the reference antisera did not offer protection against the IBVs similar to those from Japan. PMID:26822119

  2. Control of infectious Hematopoietic Necrosis virus disease by elevating the water temperature

    USGS Publications Warehouse

    Amend, Donald F.

    1970-01-01

    Studies were performed to determine if increasing water temperatures could control infectious hematopoietic necrosis virus (IHN) disease in sockeye salmon (Oncorhynchus nerka). Mortalities could be prevented if the water temperature was raised to at least 18 C within the first 24 hr after infection of the fish and if the fish were maintained at this temperature for 4–6 days. The disease did not recur after the elevated temperature treatment, but the fish would still contract the disease if they were reinfected. Reasons for the protecting action are discussed.

  3. Genetic relatedness of infectious hematopoietic necrosis virus (IHNV) from cultured salmonids in Korea.

    PubMed

    Kim, Kwang Il; Cha, Seung Joo; Lee, Chu; Baek, Harim; Hwang, Seong Don; Cho, Mi Young; Jee, Bo Young; Park, Myoung-Ae

    2016-08-01

    Infectious hematopoietic necrosis virus (IHNV; n = 18) was identified in the Korean national surveillance program between February 2013 and April 2015, suggesting that IHNV is a major viral pathogen in cultured salmonids. By phylogeny analysis, we found that the JRt-Nagano and JRt-Shizuoka groups could each be further subdivided into three distinct subtypes. The Korean strains were genetically similar to Japanese isolates, suggesting introduction from Japan. Interestingly, the amino acid sequences of the middle glycoprotein gene show that distinct Korean subtypes have circulated, indicating that the settled IHNVs might be evolved stably in cultured salmonid farm environments. PMID:27255747

  4. The genome of infectious bursal disease virus consists of two segments of double-stranded RNA.

    PubMed Central

    Müller, H; Scholtissek, C; Becht, H

    1979-01-01

    The RNA of infectious bursal disease virus was reexamined in a detailed analysis. It could be established that its genome consists of two segments of double-stranded RNA. The RNA is RNase resistant and has a sedimentation coefficient of 14S and a buoyant density of 1.62 g/ml. The purine/pyrimidine ratio is nearly 1; the guanine plus cytosine content is 55.3%; the Tm is 95.5 degrees C. The molecular weights of the two double-stranded segments were determined to be 2.2 x 10(6) and 2.5 x 10(6). Images PMID:229240

  5. Complete Genome Sequence Analysis of a Naturally Reassorted Infectious Bursal Disease Virus from India.

    PubMed

    Raja, P; Senthilkumar, T M A; Parthiban, M; Thangavelu, A; Gowri, A Mangala; Palanisammi, A; Kumanan, K

    2016-01-01

    The novel infectious bursal disease virus (IBDV) isolate BGE14/ABT1/MVC/India is a very virulent IBDV that was isolated from broiler flocks in southern parts of India during 2014. Here, we report, for the first time in India, the complete genome sequence of BGE14/ABT1/MVC/India, a reassortment strain with segments A and B derived from a very virulent IBDV strain and an attenuated IBDV, respectively. The findings from this study provide additional insight into the genetic exchange between attenuated and very virulent strains of IBDV circulating in the field. PMID:27445389

  6. Detection of infectious bronchitis virus strains similar to Japan in Taiwan.

    PubMed

    Tsai, Cheng-Ta; Tsai, Hsin-Fu; Wang, Ching-Ho

    2016-06-01

    A total of 1,320 tracheal samples from 66 broiler flocks sent to slaughterhouses and 42 tracheal samples from 42 flocks of local chickens in the field were collected for infectious bronchitis virus (IBV) gene detection by reverse transcription polymerase chain reaction using nucleocapsid-specific primers and spike-specific primers. Prevalence in broiler flocks was 39.4% (26/66) and in local chicken flocks was 11.9% (5/42). Several IBVs similar to Japan were detected in Taiwan. One-direction neutralization revealed that the reference antisera did not offer protection against the IBVs similar to those from Japan. PMID:26822119

  7. Development and characterization of an in vivo pathogenic molecular clone of equine infectious anemia virus.

    PubMed

    Cook, R F; Leroux, C; Cook, S J; Berger, S L; Lichtenstein, D L; Ghabrial, N N; Montelaro, R C; Issel, C J

    1998-02-01

    An infectious nonpathogenic molecular clone (19-2-6A) of equine infectious anemia virus (EIAV) was modified by substitution of a 3.3-kbp fragment amplified by PCR techniques from a pathogenic variant (EIAV(PV)) of the cell culture-adapted strain of EIAV (EIAV(PR)). This substitution consisted of coding sequences for 77 amino acids at the carboxyl terminus of the integrase, the S1 (encoding the second exon of tat), S2, and S3 (encoding the second exon of rev) open reading frames, the complete env gene (including the first exon of rev), and the 3' long terminal repeat (LTR). Modified 19-2-6A molecular clones were designated EIAV(PV3.3), and infection of a single pony (678) with viruses derived from a mixture of five of these molecular clones induced clinical signs of acute equine infectious anemia (EIA) at 23 days postinfection (dpi). As a consequence of this initial study, a single molecular clone, EIAV(PV3.3#3) (redesignated EIAV(UK)), was selected for further study and inoculated into two ponies (613 and 614) and two horses (700 and 764). Pony 614 and the two horses developed febrile responses by 12 dpi, which was accompanied by a 48 to 64% reduction in platelet number, whereas pony 613 did not develop fever (40.6 degrees C) until 76 dpi. EIAV could be isolated from the plasma of these animals by 5 to 7 dpi, and all became seropositive for antibodies to this virus by 21 dpi. Analysis of the complete nucleotide sequence demonstrated that the 3.3-kbp 3' fragment of EIAV(UK) differed from the consensus sequence of EIAV(PV) by just a single amino acid residue in the second exon of the rev gene. Complete homology with the EIAV(PV) consensus sequence was observed in the hypervariable region of the LTR. However, EIAV(UK) was found to contain an unusual 68-bp nucleotide insertion/duplication in a normally conserved region of the LTR sequence. These results demonstrate that substitution of a 3.3-kbp fragment from the EIAV(PV) strain into the infectious nonpathogenic

  8. Complete Genome Sequence Analysis of a Naturally Reassorted Infectious Bursal Disease Virus from India

    PubMed Central

    Raja, P.; Parthiban, M.; Thangavelu, A.; Gowri, A. Mangala; Palanisammi, A.; Kumanan, K.

    2016-01-01

    The novel infectious bursal disease virus (IBDV) isolate BGE14/ABT1/MVC/India is a very virulent IBDV that was isolated from broiler flocks in southern parts of India during 2014. Here, we report, for the first time in India, the complete genome sequence of BGE14/ABT1/MVC/India, a reassortment strain with segments A and B derived from a very virulent IBDV strain and an attenuated IBDV, respectively. The findings from this study provide additional insight into the genetic exchange between attenuated and very virulent strains of IBDV circulating in the field. PMID:27445389

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

    PubMed

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

    2016-01-01

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

  10. Infectious bronchitis virus S2 expressed from recombinant virus confers broad protection against challenge

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We developed recombinant Newcastle disease virus (NDV) LaSota (rLS) expressing the IBV S2 gene (rLS/IBV.S2). The recombinant virus showed reduced pathogenicity compared to the parental LaSota strain but effectively elicited hemagglutination inhibition antibodies and protected chickens against lethal...

  11. The effect of infectious bursal disease virus induced immunosuppression on avian influenza virus vaccine efficacy

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In the field, poultry are exposed to a variety of infectious agents, many of which are immunosuppressive. Co-infections between these agents are common, and these co-infections have effects on disease, immune response, and vaccine efficacy. The effect of co-infections in poultry between immunosupp...

  12. Infectious bursal disease virus antibodies in eider ducks and Herring Gulls

    USGS Publications Warehouse

    Hollmen, T.; Franson, J.C.; Docherty, D.E.; Kilpi, Mikael; Hario, Martti; Creekmore, L.H.; Petersen, M.R.

    2000-01-01

    We measured antibodies to infectious bursal disease virus (IBDV) in blood of nesting Common Eider (Somateria mollissima) females and immature Herring Gulls (Larus argentatus) in the Baltic Sea, and in blood of Spectacled Eider (Somateria fischeri) females nesting in a remote area of western Alaska. Positive (??? 1:16) IBDV titers occurred in 75% of the eiders and 45% of the Herring Gull chicks. In eiders, the prevalence of positive titers differed among locations. We found no evidence that IBDV exposure impaired the immune function of Herring Gull chicks, based on their response to inoculation of sheep red blood cells. We suggest that eider ducks and Herring Gulls have been exposed to IBDV, even in locations where contact with poultry is unlikely. The presence of this virus in wild bird populations is of concern because it causes mortality of up to 30% in susceptible poultry.

  13. Infectious bursal disease DNA vaccination conferring protection by delayed appearance and rapid clearance of invading viruses.

    PubMed

    Chen, Yung-Yi; Hsieh, Ming Kun; Tung, Chun-Yu; Wu, Ching Ching; Lin, Tsang Long

    2011-12-01

    The present study was undertaken to determine the kinetics of viral load and immune response in protection against infectious bursal disease virus (IBDV) by DNA vaccination. Chickens were DNA-vaccinated and challenged with IBDV one week after the third vaccination. Tissues were collected at 12 hours postinfection (HPI), 1 day postinfection (DPI), 3, 5, 7 and 10 DPI. The vaccinated chickens had less viral RNA, with delayed appearance and shorter duration in the bursa of Fabricius, spleen, and cecal tonsil than the challenged control chickens. Their ELISA and neutralizing antibody titers were decreased at 12 HPI and significantly lower (P < 0.05) than those in the challenged control chickens at later time points. Their spleen IFNγ expression was up-regulated compared to that in the DNA-vaccinated chickens without IBDV challenge. These results indicate that DNA vaccination confers protection against IBDV challenge by delayed appearance and rapid clearance of the invading viruses. PMID:21984266

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

    PubMed

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

    2007-05-01

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

  15. A second form of infectious bursal disease virus-associated tubule contains VP4.

    PubMed Central

    Granzow, H; Birghan, C; Mettenleiter, T C; Beyer, J; Köllner, B; Mundt, E

    1997-01-01

    Preparations of density gradient-purified infectious bursal disease virus (IBDV) were found to contain full and empty icosahedral virions, type I tubules with a diameter of about 60 nm, and type II tubules 24 to 26 nm in diameter. By immunoelectron microscopy we demonstrate that virions and both types of tubular structures specifically react with anti-IBDV serum. In infected cells intracytoplasmic and intranuclear type II tubules reacted exclusively with an anti-VP4 monoclonal antibody, as did type II tubules in virion preparations. The immunofluorescence pattern with the anti-VP4 antibody correlated with electron microscopical findings. Neither purified extracellular nor intracellular virions were labeled with the anti-VP4 MAb. Our data show that the type II tubules contain VP4 and suggest that VP4 is not part of the virus particle. PMID:9343252

  16. Lettuce infectious yellows virus-encoded P26 induces plasmalemma deposit cytopathology

    SciTech Connect

    Stewart, Lucy R.; Medina, Vicente; Sudarshana, Mysore R.; Falk, Bryce W.

    2009-05-25

    Lettuce infectious yellows virus (LIYV) encodes a 26 kDa protein (P26) previously shown to associate with plasmalemma deposits (PLDs), unique LIYV-induced cytopathologies located at the plasmalemma over plasmodesmata pit fields in companion cells and phloem parenchyma. To further characterize the relationship of P26 and PLDs, we assessed localization and cytopathology induction of P26 expressed from either LIYV or a heterologous Tobacco mosaic virus (TMV) vector using green fluorescent protein (GFP) fusions, immunofluorescence microscopy, biochemical fractionation, and transmission electron microscopy (TEM). TEM analyses demonstrated that P26 not only associated with, but induced formation of PLDs in the absence of other LIYV proteins. Interestingly, PLDs induced by P26-expressing TMV were no longer confined to phloem cells. Putative P26 orthologs from two other members of the genus Crinivirus which do not induce conspicuous PLDs exhibited fractionation properties similar to LIYV P26 but were not associated with any PLD-like cytopathology.

  17. Absence of infectious human immunodeficiency virus type 1 in "natural" eccrine sweat.

    PubMed

    Wormser, G P; Bittker, S; Forseter, G; Hewlett, I K; Argani, I; Joshi, B; Epstein, J S; Bucher, D

    1992-01-01

    Although human immunodeficiency virus type 1 (HIV-1) has been found in numerous body fluids, there are no reports of attempts to demonstrate this virus in eccrine sweat, a fluid frequently encountered during person-to-person interactions. "Natural" eccrine sweat samples and blood from 50 HIV-1-seropositive patients and 2 HIV-1-seronegative controls were cultured for HIV-1 by a cocultivation method. Polymerase chain reaction for HIV-1 RNA and proviral DNA was done on 40 sweat samples (39 patients, 1 control). HIV-1 was isolated from peripheral blood mononuclear cells of 39 (78%) of 50 patients but from none of 52 sweat samples. No HIV-1 viral DNA or RNA was detected in the 40 sweat samples tested. With present methodology, infectious HIV-1 cannot be demonstrated in "natural" eccrine sweat samples from HIV-infected patients. PMID:1345794

  18. Diagnosis of infectious canine hepatitis virus (CAV-1) infection in puppies with encephalopathy.

    PubMed

    Caudell, D; Confer, A W; Fulton, R W; Berry, A; Saliki, J T; Fent, G M; Ritchey, J W

    2005-01-01

    Nine weaned Labrador Retriever puppies from a litter of 11 were presented with signs of acute central nervous system (CNS) disease that included ataxia and blindness. All puppies died. Gross examination of tissues from 2 puppies revealed regionally diffuse hemorrhages in the brain stem and swollen hemorrhagic lymph nodes. Light microscopic examination of hematoxylin and eosin-stained tissues showed numerous large, basophilic intranuclear inclusion bodies within CNS vascular endothelium and occasionally in individual hepatocytes. Immunohistochemical staining of the tissue was positive using an antibody against canine adenovirus-1. Virus isolation for infectious canine hepatitis virus was achieved using inoculated cell cultures. Polymerase chain reaction amplification of DNA from cell culture material revealed shared homology with other mammalian adenoviruses. PMID:15690952

  19. Infectious laryngotracheitis virus (ILTV) vaccine intake evaluation by detection of virus amplification in feather pulps of vaccinated chickens.

    PubMed

    Davidson, I; Raibshtein, I; Altori, A; Elkin, N

    2016-03-18

    Infectious laryngotracheitis (ILT) is a respiratory disease of poultry caused by an alphaherpesvirus, ILTV. The live vaccine is applied worldwide by drinking water or by the respiratory route, and by the vent application in Israel. No system of direct evaluation of the efficacy of vaccination exists today, except of antibody elicitation, which is an indirect indication of vaccination intake and might happen due to environment exposure. We suggest for the first time an assay for evaluating the accuracy of the vaccination process by spotting the spread of the live vaccine systemically, namely by virus detection in the feather shafts of the vaccinated birds. The feathers are particularly beneficial as they are easy to collect, non-lethal for the bird, therefore advantageous for monitoring purposes. Moreover, the continuous survey of the vaccine virus unveiled the different kinetics of viremia by the different vaccination routes; while after the vent vaccination the systemic viremia peaks during the first week afterwards, after two consecutive vaccine administration by drinking water with 6 day interval, the vireamia peaks only after the second administration. A robust amplification was needed because the vaccine ILTV was present in the bird in minute quantities compared to the wild-type virus. For the vaccine virus identification in feather shafts a nested real-time PCR for the TK ILTV gene was developed. The sensitivity of detection of the nested rtPCR was greater by 1000 compared to conventional nested PCR and 10 times that real-time PCR. PMID:26784685

  20. Viral interference between infectious hypodermal and hematopoietic necrosis virus and white spot syndrome virus in Litopenaeus vannamei.

    PubMed

    Bonnichon, Valérie; Lightner, Donald V; Bonami, Jean-Robert

    2006-10-17

    White spot syndrome virus (WSSV) is highly virulent and has caused significant production losses to the shrimp culture industry over the last decade. Infectious hypodermal and hematopoietic necrosis virus (IHHNV) also infects penaeid shrimp and, while being less important than WSSV, remains a major cause of significant production losses in Litopenaeus vannamei (also called Penaeus vannamei) and L. stylirostris (also called Penaeus stylirostris). These 2 viruses and their interactions were previously investigated in L. stylirostris. We report here laboratory challenge studies carried out to determine if viral interference between IHHNV and WSSV also occurs in L. vannamei, and it was found that experimental infection with IHHNV induced a significant delay in mortality following WSSV challenge. L. vannamei infected per os with IHHNV were challenged with WSSV at 0, 10, 20, 30, 40 and 50 d post-infection. Groups of naïve shrimp infected with WSSV alone died in 3 d whereas shrimp pre-infected with IHHNV for 30, 40 or 50 d died in 5 d. Real-time PCR analysis showed that the delay correlated to the IHHNV load and that WSSV challenge induced a decrease in IHHNV load, indicating some form of competition between the 2 viruses. PMID:17140141

  1. Differential virulence mechanisms of infectious hematopoietic necrosis virus in rainbow trout (Oncorhynchus mykiss) include host entry and virus replication kinetics

    USGS Publications Warehouse

    Penaranda, M.M.D.; Purcell, M.K.; Kurath, G.

    2009-01-01

    Host specificity is a phenomenon exhibited by all viruses. For the fish rhabdovirus infectious hematopoietic necrosis virus (IHNV), differential specificity of virus strains from the U and M genogroups has been established both in the field and in experimental challenges. In rainbow trout (Oncorhynchus mykiss), M IHNV strains are consistently more prevalent and more virulent than U IHNV. The basis of the differential ability of these two IHNV genogroups to cause disease in rainbow trout was investigated in live infection challenges with representative U and M IHNV strains. When IHNV was delivered by intraperitoneal injection, the mortality caused by U IHNV increased, indicating that the low virulence of U IHNV is partly due to inefficiency in entering the trout host. Analyses of in vivo replication showed that U IHNV consistently had lower prevalence and lower viral load than M IHNV during the course of infection. In analyses of the host immune response, M IHNV-infected fish consistently had higher and longer expression of innate immune-related genes such as Mx-1. This suggests that the higher virulence of M IHNV is not due to suppression of the immune response in rainbow trout. Taken together, the results support a kinetics hypothesis wherein faster replication enables M IHNV to rapidly achieve a threshold level of virus necessary to override the strong host innate immune response. ?? 2009 SGM.

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

    PubMed

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

    2010-04-01

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

  3. Enhanced detection of infectious hematopoietic necrosis virus by pretreatment of cell monolayers with polyethylene glycol

    USGS Publications Warehouse

    Batts, W.N.; Winton, J.R.

    1989-01-01

    To improve quantification of very low levels of infectious hematopoietic necrosis virus (IHNV) in samples of tissue, ovarian fluid, or natural water supplies, we tested the ability of polyethylene glycol (PEG) to enhance the sensitivity and speed of the plaque assay system. We compared 4, 7, and 10% solutions of PEG of molecular weight 6,000, 8,000, or 20,000 applied at selected volumes and for various durations. When cell monolayers of epithelioma papulosum cyprini (EPC), fathead minnow (FHM), chinook salmon embryo (CHSE-214), and bluegill fry (BF2) were pretreated with 7% PEG-20,000, they produced 4-17-fold increases in plaque assay titers of IHNV. The plaque assay titers of viral hemorrhagic septicemia virus, chum salmon reovirus, and chinook salmon paramyxovirus were also enhanced by exposure of CHSE-214 cells to PEG, but the titers of infectious pancreatic necrosis virus and Oncorhynchus masou virus were not substantially changed. Plaques formed by IHNV on PEG-treated EPC cells incubated at 15°C had a larger mean diameter at 6 d than those on control cells at 8 d; this suggests the assay could be shortened by use of PEG. Pretreatment of EPC cell monolayers with PEG enabled detection of IHNV in some samples that appeared negative with untreated cells. For example, when ovarian fluid samples from chinook salmon Oncorhynchus tshawytscha were inoculated onto untreated monolayers of EPC cells, IHNV was detected in only 11 of 51 samples; 17 of the samples were positive when PEG-treated EPC cells were used.PDF

  4. Construction of an infectious clone of a plant RNA virus in a binary vector using one-step Gibson Assembly.

    PubMed

    Blawid, Rosana; Nagata, Tatsuya

    2015-09-15

    The construction of full-length infectious clones of RNA viruses is often laborious due to the many cloning steps required and the DNA exclusion within the plasmid during Escherichia coli transformation. We demonstrate single-step cloning procedure of an infectious cDNA of the tomato blistering mosaic virus (ToBMV) using Gibson Assembly (GA), which drastically reduces the number of cloning steps. By agro-inoculation with the construct obtained by this procedure, ToBMV was recovered six days post-inoculation in Nicotiana benthamiana plants. The symptoms induced by the recovered virus were indistinguishable from those caused by the wild-type virus. We conclude that the GA is very useful method particularly to construct a full-length cDNA clone of a plant RNA virus in a binary vector. PMID:25986144

  5. Reverse transcription loop-mediated isothermal amplification for the rapid detection of infectious bronchitis virus in infected chicken tissues.

    PubMed

    Chen, Hao-tai; Zhang, Jie; Ma, Yan-ping; Ma, Li-Na; Ding, Yao-zhong; Liu, Xiang-tao; Cai, Xue-peng; Ma, Li-qing; Zhang, Yong-guang; Liu, Yong-sheng

    2010-04-01

    A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay targeting the nucleocapsid phosphoprotein gene of infectious bronchitis virus (IBV) was developed. The detection limits for the IBV RT-LAMP assay were 10(1) 50% egg infection dose (EID(50)) per 50 microl of titrated viruses and no cross-reaction of IBV RT-LAMP was found when tested with other viruses including Newcastle disease virus (NDV), avian reovirus (ARV), and infectious laryngotrachietis virus (ILTV) due to their mismatch with IBV RT-LAMP primers. A total of 187 clinical tissues samples (88 blood, 62 kidney and 37 lung) were evaluated and compared to conventional RT-PCR. The sensitivity of RT-LAMP and RT-PCR assays for detecting IBV RNA in clinical specimens was 99.5% and 98.4%, respectively. These findings showed that the RT-LAMP assay has potential usefulness for rapid and sensitive diagnosis in outbreak of IBV. PMID:19835950

  6. Comparison of the Levels of Infectious Virus in Respirable Aerosols Exhaled by Ferrets Infected with Influenza Viruses Exhibiting Diverse Transmissibility Phenotypes

    PubMed Central

    Gustin, Kortney M.; Katz, Jacqueline M.; Tumpey, Terrence M.

    2013-01-01

    Influenza viruses pose a major public health burden to communities around the world by causing respiratory infections that can be highly contagious and spread rapidly through the population. Despite extensive research on influenza viruses, the modes of transmission occurring most often among humans are not entirely clear. Contributing to this knowledge gap is the lack of an understanding of the levels of infectious virus present in respirable aerosols exhaled from infected hosts. Here, we used the ferret model to evaluate aerosol shedding patterns and measure the amount of infectious virus present in exhaled respirable aerosols. By comparing these parameters among a panel of human and avian influenza viruses exhibiting diverse respiratory droplet transmission efficiencies, we are able to report that ferrets infected by highly transmissible influenza viruses exhale a greater number of aerosol particles and more infectious virus within respirable aerosols than ferrets infected by influenza viruses that do not readily transmit. Our findings improve our understanding of the ferret transmission model and provide support for the potential for influenza virus aerosol transmission. PMID:23658443

  7. Immunogenicity of a modified-live virus vaccine against bovine viral diarrhea virus types 1 and 2, infectious bovine rhinotracheitis virus, bovine parainfluenza-3 virus, and bovine respiratory syncytial virus when administered intranasally in young calves.

    PubMed

    Xue, Wenzhi; Ellis, John; Mattick, Debra; Smith, Linda; Brady, Ryan; Trigo, Emilio

    2010-05-14

    The immunogenicity of an intranasally-administered modified-live virus (MLV) vaccine in 3-8 day old calves was evaluated against bovine viral diarrhea virus (BVDV) types 1 and 2, infectious bovine rhinotracheitis (IBR) virus, parainfluenza-3 (PI-3) virus and bovine respiratory syncytial virus (BRSV). Calves were intranasally vaccinated with a single dose of a multivalent MLV vaccine and were challenged with one of the respective viruses three to four weeks post-vaccination in five separate studies. There was significant sparing of diseases in calves intranasally vaccinated with the MLV vaccine, as indicated by significantly fewer clinical signs, lower rectal temperatures, reduced viral shedding, greater white blood cell and platelet counts, and less severe pulmonary lesions than control animals. This was the first MLV combination vaccine to demonstrate efficacy against BVDV types 1 and 2, IBR, PI-3 and BRSV in calves 3-8 days of age. PMID:20381643

  8. Immunochromatographic lateral flow test for detection of antibodies to Equine infectious anemia virus.

    PubMed

    Alvarez, I; Gutierrez, G; Barrandeguy, M; Trono, K

    2010-08-01

    The purpose of this study was to develop and evaluate a simple immunochromatographic lateral flow (ICLF) test for specific detection of Equine infectious anemia virus (EIAV) antibodies in equine sera. Viral recombinant p26 capsid protein (rp26) was used as the capture protein in the test line and as the detector reagent conjugated to colloidal gold. The performance of rp26-ICLF was evaluated, and the results obtained were compared with a commercially available agar gel immunodiffusion (AGID) test used as a standard of comparison according to international guidelines. The values obtained for comparative diagnostic sensitivity (98.3%), diagnostic specificity (87.4%) and concordance (92.4%) were similar to those reported for other ICLF tests for animal infectious diseases. Very good repeatability and reproducibility, as well as a total agreement with blind previous results from three proficiency test panels, were obtained, thus indicating that rp26-ICLF is a precise test. The end point of the twofold serial dilution of serum samples was the same as, and even better than, the AGID test, thus demonstrating the same analytical sensitivity as that of the reference method for EIA diagnosis. No cross-reactivity was observed when serum samples from horses with other infectious diseases were analyzed. rp26-ICLF proved to be a precise and rapid test suitable for field screening in veterinary practice, since minimal equipment and operator expertise are required. However, further research should be carried out to increase the level of sensitivity. PMID:20362005

  9. Infectious hematopoietic necrosis virus: Monophyletic origin of European isolates from North American Genogroup M

    USGS Publications Warehouse

    Enzmann, P.-J.; Kurath, G.; Fichtner, D.; Bergmann, S.M.

    2005-01-01

    Infectious hematopoietic necrosis virus (IHNV) was first detected in Europe in 1987 in France and Italy, and later, in 1992, in Germany. The source of the virus and the route of introduction are unknown. The present study investigates the molecular epidemiology of IHNV outbreaks in Germany since its first introduction. The complete nucleotide sequences of the glycoprotein (G) and non-virion (NV) genes from 9 IHNV isolates from Germany have been determined, and this has allowed the identification of characteristic differences between these isolates. Phylogenetic analysis of partial G gene sequences (mid-G, 303 nucleotides) from North American IHNV isolates (Kurath et al. 2003) has revealed 3 major genogroups, designated U, M and L. Using this gene region with 2 different North American IHNV data sets, it was possible to group the European IHNV strains within the M genogroup, but not in any previously defined subgroup. Analysis of the full length G gene sequences indicated that an independent evolution of IHN viruses had occurred in Europe. IHN viruses in Europe seem to be of a monophyletic origin, again most closely related to North American isolates in the M genogroup. Analysis of the NV gene sequences also showed the European isolates to be monophyletic, but resolution of the 3 genogroups was poor with this gene region. As a result of comparative sequence analyses, several different genotypes have been identified circulating in Europe. ?? Inter-Research 2005.

  10. Cross-protective immune responses elicited by a Korean variant of infectious bronchitis virus.

    PubMed

    Kim, Byoung-Yoon; Lee, Dong-Hun; Jang, Jun-Hyuk; Lim, Tae-Hyun; Choi, Soo-Won; Youn, Ha-Na; Park, Jae-Keun; Lee, Joong-Bok; Park, Seung-Yong; Choi, In-Soo; Song, Chang-Seon

    2013-09-01

    Infectious bronchitis virus (IBV) infections cause great economic losses to the poultry industry worldwide. IBVs continuously evolve by developing mutations in antigenic sites; therefore, an IBV vaccine that provides broad cross-protection can be a highly relevant and practical method in IBV control strategies. Although some IBV vaccine strains are known to provide protection against multiple IBV serotypes, in general commercially available IBV vaccine strains provide protection against antigenically related viruses but not distinct heterologous viruses. In the present study we characterized the Korean variant IBV K40/09 strain with regard to its immunogenicity and protective efficacy against seven currently circulating IBV serotypes. Three-week-old specific-pathogen-free chickens were intraocularly immunized with the IBV K40/09 strain at 10(3.5) 50% egg infective dose (EID50). Three weeks after immunization all the birds were challenged with seven different strains at 10(4.5) EID50. Chickens immunized with the IBV K40/09 strain showed significantly high levels of protection against all challenge viruses at the trachea and kidney levels. Our results suggest that IBV K40/09 could be useful to ensure IBV vaccine effectiveness owing to its cross-protective ability. Therefore, the IBV K40/09 strain merits consideration as a vaccine candidate to prevent infection as well as the spread of new IBV strains and many IBV variants that have been reported worldwide. PMID:24283135

  11. Genetic analyses reveal unusually high diversity of infectious haematopoietic necrosis virus in rainbow trout aquaculture

    USGS Publications Warehouse

    Troyer, Ryan M.; LaPatra, Scott E.; Kurath, Gael

    2000-01-01

    Infectious haematopoietic necrosis virus (IHNV) is the most significant virus pathogen of salmon and trout in North America. Previous studies have shown relatively low genetic diversity of IHNV within large geographical regions. In this study, the genetic heterogeneity of 84 IHNV isolates sampled from rainbow trout (Oncorhynchus mykiss) over a 20 year period at four aquaculture facilities within a 12 mile stretch of the Snake River in Idaho, USA was investigated. The virus isolates were characterized using an RNase protection assay (RPA) and nucleotide sequence analyses. Among the 84 isolates analysed, 46 RPA haplotypes were found and analyses revealed a high level of genetic heterogeneity relative to that detected in other regions. Sequence analyses revealed up to 7·6% nucleotide divergence, which is the highest level of diversity reported for IHNV to date. Phylogenetic analyses identified four distinct monophyletic clades representing four virus lineages. These lineages were distributed across facilities, and individual facilities contained multiple lineages. These results suggest that co-circulating IHNV lineages of relatively high genetic diversity are present in the IHNV populations in this rainbow trout culture study site. Three of the four lineages exhibited temporal trends consistent with rapid evolution.

  12. Universal reverse-transcriptase real-time PCR for infectious hematopoietic necrosis virus (IHNV)

    USGS Publications Warehouse

    Purcell, Maureen K.; Thompson, Rachel L.; Garver, Kyle A.; Hawley, Laura M.; Batts, William N.; Sprague, Laura; Sampson, Corie; Winton, James R.

    2013-01-01

    Infectious hematopoietic necrosis virus (IHNV) is an acute pathogen of salmonid fishes in North America, Europe and Asia and is reportable to the World Organization for Animal Health (OIE). Phylogenetic analysis has identified 5 major virus genogroups of IHNV worldwide, designated U, M, L, E and J; multiple subtypes also exist within those genogroups. Here, we report the development and validation of a universal IHNV reverse-transcriptase real-time PCR (RT-rPCR) assay targeting the IHNV nucleocapsid (N) gene. Properties of diagnostic sensitivity (DSe) and specificity (DSp) were defined using laboratory-challenged steelhead trout Oncorhynchus mykiss, and the new assay was compared to the OIE-accepted conventional PCR test and virus isolation in cell culture. The IHNV N gene RT-rPCR had 100% DSp and DSe and a higher estimated diagnostic odds ratio (DOR) than virus culture or conventional PCR. The RT-rPCR assay was highly repeatable within a laboratory and highly reproducible between laboratories. Field testing of the assay was conducted on a random sample of juvenile steelhead collected from a hatchery raceway experiencing an IHN epizootic. The RT-rPCR detected a greater number of positive samples than cell culture and there was 40% agreement between the 2 tests. Overall, the RT-rPCR assay was highly sensitive, specific, repeatable and reproducible and is suitable for use in a diagnostic setting.

  13. Isolation of Infectious Bursal Disease Virus Using Indigenous Chicken Embryos in Kenya

    PubMed Central

    Mutinda, W. U.; Njagi, L. W.; Nyaga, P. N.; Bebora, L. C.; Mbuthia, P. G.; Kemboi, D.; Githinji, J. W. K.; Muriuki, A.

    2015-01-01

    Infectious bursal disease virus (IBDV) isolates were recovered from outbreaks to initiate activities towards developing a local vaccine strain. Use of indigenous chicken embryos was exploited to determine their potential, promote utilization of local resources for research, and enhance household economic activities. Bursa of Fabricius (BFs) samples from outbreaks shown to be IBDV positive was homogenized and inoculated in 4-week-old specific pathogen-free (SPF) IBDV seronegative white leghorn chicks. The harvested virus was inoculated into 11-day-old indigenous chicken embryos that were IBDV seronegative and passaged serially three times after which they were inoculated into 4-week-old indigenous chicks to test for presence and virulence of propagated virus. Out of 153 BFs collected from outbreaks, 43.8% (67/153) were positive for IBDV antigen and 65.7% (44/67) caused disease in SPF chicks. The embryo mean mortalities were 88% on primary inoculation, 94% in 1st passage, 91% in 2nd passage, and 67% in 3rd passage. After the third passage in embryos all the 44 isolates were virulent in 4-week-old indigenous chicks. The results show that indigenous chicken embryos support growth of IBDV and can be used to propagate the virus as an alternative viral propagating tool for respective vaccine preparation. PMID:27347520

  14. Avian diversity and West Nile virus: Testing associations between biodiversity and infectious disease risk

    USGS Publications Warehouse

    Ezenwa, V.O.; Godsey, M.S.; King, R.J.; Guptill, S.C.

    2006-01-01

    The emergence of several high profile infectious diseases in recent years has focused attention on our need to understand the ecological factors contributing to the spread of infectious diseases. West Nile virus (WNV) is a mosquito-borne zoonotic disease that was first detected in the United States in 1999. The factors accounting for variation in the prevalence of WNV are poorly understood, but recent ideas suggesting links between high biodiversity and reduced vector-borne disease risk may help account for distribution patterns of this disease. Since wild birds are the primary reservoir hosts for WNV, we tested associations between passerine (Passeriform) bird diversity, non-passerine (all other orders) bird diversity and virus infection rates in mosquitoes and humans to examine the extent to which bird diversity is associated with WNV infection risk. We found that non-passerine species richness (number of non-passerine species) was significantly negatively correlated with both mosquito and human infection rates, whereas there was no significant association between passerine species richness and any measure of infection risk. Our findings suggest that non-passerine diversity may play a role in dampening WNV amplification rates in mosquitoes, minimizing human disease risk. ?? 2005 The Royal Society.

  15. Avian diversity and West Nile virus: testing associations between biodiversity and infectious disease risk.

    USGS Publications Warehouse

    Ezenwa, V.O.; Godsey, M.S.; King, R.J.; Guptill, S.C.

    2006-01-01

    The emergence of several high profile infectious diseases in recent years has focused attention on our need to understand the ecological factors contributing to the spread of infectious diseases. West Nile virus (WNV) is a mosquito-borne zoonotic disease that was first detected in the United States in 1999. The factors accounting for variation in the prevalence of WNV are poorly understood, but recentideas suggesting links between high biodiversity and reduced vector-borne disease risk may help account for distribution patterns of this disease. Since wild birds are the primary reservoir hosts for WNV, we tested associations between passerine (Passeriform) bird diversity, non-passerine (all other orders) bird diversity and virus infection rates in mosquitoes and humans to examine the extent to which bird diversity is associated with WNV infection risk. We found t h at non-passerine species richness (number of non-passerine species) was significantly negatively correlated with both mosquito and human infection rates, whereas there was no significant association between passerine species richness and any measure of infection risk. Our findings suggest that non-passerine diversity may play a role in dampening WNV amplification rates in mosquitoes, minimizing human disease risk.

  16. Determination of the optimal time of vaccination against infectious bursal disease virus (Gumboro) in Algeria.

    PubMed

    Besseboua, Omar; Ayad, Abdelhanine; Benbarek, Hama

    2015-01-01

    This study was conducted to determine the effect of maternally derived antibody (MDA) on live vaccine against infectious bursal disease. A total of 140 chicks selected from vaccinated parent stock were used in this investigation. In a preset vaccination schedule, blood samples were collected to check for the actual effect. It was noticed that on day 1 the chicks contained a high level (6400.54 ± 2993.67) of maternally derived antibody that gradually decreased below a positive level within 21 days (365.86 ± 634.46). It was found that a high level of MDA interferes with the vaccine virus, resulting in no immune response. For better immune response, it is suggested that the chickens should be vaccinated at day 21, as the uniformity of MDA is poor (coefficient of the variation [CV] > 30%), and boosted at day 28. Indeed, two vaccinations are necessary to achieve good protection against infectious bursal disease virus of the entire flock. PMID:26018110

  17. Evolution of infectious bronchitis virus in China over the past two decades.

    PubMed

    Zhao, Ye; Zhang, Hui; Zhao, Jing; Zhong, Qi; Jin, Ji-Hui; Zhang, Guo-Zhong

    2016-07-01

    Avian infectious bronchitis is a highly contagious disease caused by infectious bronchitis virus (IBV) that affects poultry production worldwide. The absence of vaccine cross-protection and the frequent emergence of new variant strains complicate control of IBV. Here we designed a study to measure the evolution dynamics of IBV strains in China. One hundered and seven complete sequences and 1022 S1-region sequences of Chinese IBVs isolated between 1994 and 2014 were analysed by using MEGA 5.0 software and the Bayesian analysis sampling trees (BEAST) method, and selection pressure on different proteins was assessed. The phylogenetic dissimilarity of different gene trees in the data set indicated possible recombination. Fourteen isolates were identified as recombinants, possibly generated from vaccines of the Massachusetts serotype in recombination with circulating viruses. The earliest IBV in China was found to have existed in the early 1900s, and continues to evolve at a rate of approximately 10-5 substitutions per site per year. We found that purifying selection was the main evolutionary pressure in the protein-coding regions, while the S1 gene bears the greatest positive selection pressure. The proportion of QX-like genotype strains increased over time. These results indicate that the genotypes of Chinese IBVs have undergone a remarkable transition during the past 20 years. PMID:27008625

  18. Infectious hypodermal and hematopoietic necrosis virus from Brazil: Sequencing, comparative analysis and PCR detection.

    PubMed

    Silva, Douglas C D; Nunes, Allan R D; Teixeira, Dárlio I A; Lima, João Paulo M S; Lanza, Daniel C F

    2014-08-30

    A 3739 nucleotide fragment of Infectious hypodermal and hematopoietic necrosis virus (IHHNV) from Brazil was amplified and sequenced. This fragment contains the entire coding sequences of viral proteins, the full 3' untranslated region (3'UTR) and a partial sequence of 5' untranslated region (5'UTR). The genome organization of IHHNV revealed the three typical major coding domains: a left ORF1 of 2001 bp that codes NS1, a left ORF2 (NS2) of 1091 bp that codes NS2 and a right ORF3 of 990 bp that codes VP. Nucleotide and amino acid sequences of the three viral proteins were compared with putative amino acid sequences of viruses reported from different regions. Comparisons among genomes from different geographic locations reveal 31 nucleotide regions that are 100% similar, distributed throughout the genome. An analysis of secondary structure of UTR regions, revealed regions with high probability to form hairpins, that may be involved in mechanisms of viral replication. Additionally, a maximum likelihood analysis indicates that Brazilian IHHNV belongs to lineage III, in the infectious IHHNV group, and is clustered with IHHNV isolates from Hawaii, China, Taiwan, Vietnam and South Korea. A new nested PCR targeting conserved nucleotide regions is proposed to detect IHHNV. PMID:24867614

  19. The Polyomaviridae: Contributions of virus structure to our understanding of virus receptors and infectious entry

    SciTech Connect

    Neu, Ursula; Stehle, Thilo Atwood, Walter J.

    2009-02-20

    This review summarizes the field's major findings related to the characterization of polyomavirus structures and to the characterization of virus receptors and mechanisms of host cell invasion. The four members of the family that have received the most attention in this regard are the mouse polyomavirus (mPyV), the monkey polyomavirus SV40, and the two human polyomaviruses, JCV and BKV. The structures of both the mPyV and SV40 alone and in complex with receptor fragments have been solved to high resolution. The majority of polyomaviruses recognize terminal sialic acid in either an {alpha}2,3 linkage or an {alpha}2,6 linkage to the underlying galactose. Studies on virus structure, receptor utilization and mechanisms of entry have led to new insights into how these viruses interact in an active way with cells to ensure the nuclear delivery and expression of their genomes. Critical work on virus entry has led to the discovery of a pH neutral endocytic compartment that accepts cargo from caveolae and to novel roles for endoplasmic reticulum (ER) associated factors in virus uncoating and penetration of ER membranes. This review will summarize the major findings and compare and contrast the mechanisms used by these viruses to infect cells.

  20. Budding of Equine Infectious Anemia Virus Is Insensitive to Proteasome Inhibitors

    PubMed Central

    Patnaik, Akash; Chau, Vincent; Li, Feng; Montelaro, Ronald C.; Wills, John W.

    2002-01-01

    The only retrovirus protein required for the budding of virus-like particles is the Gag protein; however, recent studies of Rous sarcoma virus (RSV) and human immunodeficiency virus have suggested that modification of Gag with ubiquitin (Ub) is also required. As a consequence, the release of these viruses is reduced in the presence of proteasome inhibitors, which indirectly reduce the levels of free Ub within the cell. Here we show that the budding of equine infectious anemia virus (EIAV) from infected equine cells is largely unaffected by these drugs, although use of one inhibitor (MG-132) resulted in a dramatic block to proteolytic processing of Gag. This lack of sensitivity was also observed in transiently transfected avian cells under conditions that greatly reduce RSV budding. Moreover, insensitivity was observed when the EIAV Gag protein was expressed in the absence of all the other virus products, indicating that they are not required for this phenotype. An activity that enables EIAV to tolerate exposure to proteasome inhibitors was mapped to the C-terminal p9 sequence, as demonstrated by the ability of an RSV Gag-p9 chimera to bud in the presence of the drugs. Intriguingly, the p9 sequence contains a short sequence motif that is similar to a surface-exposed helix of Ub, suggesting that EIAV Gag may have captured a function that allows it to bypass the need for ubiquitination. Thus, the mechanism of EIAV budding may not be substantially different from that of other retroviruses, even though it behaves differently in the presence of proteasome inhibitors. PMID:11861830

  1. Emergence of MD type infectious hematopoietic necrosis virus in Washington State coastal steelhead trout

    USGS Publications Warehouse

    Breyta, Rachel; Jones, Amelia; Stewart, Bruce; Brunson, Ray; Thomas, Joan; Kerwin, John; Bertolini, Jim; Mumford, Sonia; Patterson, Chris; Kurath, Gael

    2013-01-01

    Infectious hematopoietic necrosis virus (IHNV) occurs in North America as 3 major phylogenetic groups designated U, M, and L. In coastal Washington State, IHNV has historically consisted of U genogroup viruses found predominantly in sockeye salmon Oncorhynchus nerka. M genogroup IHNV, which has host-specific virulence for rainbow and steelhead trout O. mykiss, was detected only once in coastal Washington prior to 2007, in an epidemic among juvenile steelhead trout in 1997. Beginning in 2007 and continuing through 2011, there were 8 IHNV epidemics in juvenile steelhead trout, involving 7 different fish culture facilities in 4 separate watersheds. During the same time period, IHNV was also detected in asymptomatic adult steelhead trout from 6 coastal watersheds. Genetic typing of 283 recent virus isolates from coastal Washington revealed that the great majority were in the M genogroup of IHNV and that there were 2 distinct waves of viral emergence between the years 2007 and 2011. IHNV type mG110M was dominant in coastal steelhead trout during 2007 to 2009, and type mG139M was dominant between 2010 and 2011. Phylogenetic analysis of viral isolates indicated that all coastal M genogroup viruses detected in 1997 and 2007 to 2011 were part of the MD subgroup and that several novel genetic variants related to the dominant types arose in the coastal sites. Comparison of spatial and temporal incidence of coastal MD viruses with that of the rest of the Pacific Northwest indicated that the likely source of the emergent viruses was Columbia River Basin steelhead trout. 

  2. Emergence of MD type infectious hematopoietic necrosis virus in Washington State coastal steelhead trout.

    PubMed

    Breyta, Rachel; Jones, Amelia; Stewart, Bruce; Brunson, Ray; Thomas, Joan; Kerwin, John; Bertolini, Jim; Mumford, Sonia; Patterson, Chris; Kurath, Gael

    2013-06-13

    Infectious hematopoietic necrosis virus (IHNV) occurs in North America as 3 major phylogenetic groups designated U, M, and L. In coastal Washington State, IHNV has historically consisted of U genogroup viruses found predominantly in sockeye salmon Oncorhynchus nerka. M genogroup IHNV, which has host-specific virulence for rainbow and steelhead trout O. mykiss, was detected only once in coastal Washington prior to 2007, in an epidemic among juvenile steelhead trout in 1997. Beginning in 2007 and continuing through 2011, there were 8 IHNV epidemics in juvenile steelhead trout, involving 7 different fish culture facilities in 4 separate watersheds. During the same time period, IHNV was also detected in asymptomatic adult steelhead trout from 6 coastal watersheds. Genetic typing of 283 recent virus isolates from coastal Washington revealed that the great majority were in the M genogroup of IHNV and that there were 2 distinct waves of viral emergence between the years 2007 and 2011. IHNV type mG110M was dominant in coastal steelhead trout during 2007 to 2009, and type mG139M was dominant between 2010 and 2011. Phylogenetic analysis of viral isolates indicated that all coastal M genogroup viruses detected in 1997 and 2007 to 2011 were part of the MD subgroup and that several novel genetic variants related to the dominant types arose in the coastal sites. Comparison of spatial and temporal incidence of coastal MD viruses with that of the rest of the Pacific Northwest indicated that the likely source of the emergent viruses was Columbia River Basin steelhead trout. PMID:23759556

  3. A Defective Interfering Influenza RNA Inhibits Infectious Influenza Virus Replication in Human Respiratory Tract Cells: A Potential New Human Antiviral.

    PubMed

    Smith, Claire M; Scott, Paul D; O'Callaghan, Christopher; Easton, Andrew J; Dimmock, Nigel J

    2016-01-01

    Defective interfering (DI) viruses arise during the replication of influenza A virus and contain a non-infective version of the genome that is able to interfere with the production of infectious virus. In this study we hypothesise that a cloned DI influenza A virus RNA may prevent infection of human respiratory epithelial cells with infection by influenza A. The DI RNA (244/PR8) was derived by a natural deletion process from segment 1 of influenza A/PR/8/34 (H1N1); it comprises 395 nucleotides and is packaged in the DI virion in place of a full-length genome segment 1. Given intranasally, 244/PR8 DI virus protects mice and ferrets from clinical influenza caused by a number of different influenza A subtypes and interferes with production of infectious influenza A virus in cells in culture. However, evidence that DI influenza viruses are active in cells of the human respiratory tract is lacking. Here we show that 244/PR8 DI RNA is replicated by an influenza A challenge virus in human lung diploid fibroblasts, bronchial epithelial cells, and primary nasal basal cells, and that the yield of challenge virus is significantly reduced in a dose-dependent manner indicating that DI influenza virus has potential as a human antiviral. PMID:27556481

  4. A Defective Interfering Influenza RNA Inhibits Infectious Influenza Virus Replication in Human Respiratory Tract Cells: A Potential New Human Antiviral

    PubMed Central

    Smith, Claire M.; Scott, Paul D.; O’Callaghan, Christopher; Easton, Andrew J.; Dimmock, Nigel J.

    2016-01-01

    Defective interfering (DI) viruses arise during the replication of influenza A virus and contain a non-infective version of the genome that is able to interfere with the production of infectious virus. In this study we hypothesise that a cloned DI influenza A virus RNA may prevent infection of human respiratory epithelial cells with infection by influenza A. The DI RNA (244/PR8) was derived by a natural deletion process from segment 1 of influenza A/PR/8/34 (H1N1); it comprises 395 nucleotides and is packaged in the DI virion in place of a full-length genome segment 1. Given intranasally, 244/PR8 DI virus protects mice and ferrets from clinical influenza caused by a number of different influenza A subtypes and interferes with production of infectious influenza A virus in cells in culture. However, evidence that DI influenza viruses are active in cells of the human respiratory tract is lacking. Here we show that 244/PR8 DI RNA is replicated by an influenza A challenge virus in human lung diploid fibroblasts, bronchial epithelial cells, and primary nasal basal cells, and that the yield of challenge virus is significantly reduced in a dose-dependent manner indicating that DI influenza virus has potential as a human antiviral. PMID:27556481

  5. Discrimination of infectious hepatitis A virus and rotavirus by combining dyes and surfactants with RT-qPCR

    PubMed Central

    2013-01-01

    Background Human enteric viruses are major agents of foodborne diseases. Because of the absence of a reliable cell culture method for most of the enteric viruses involved in outbreaks, real-time reverse transcriptase PCR is now widely used for the detection of RNA viruses in food samples. However this approach detects viral nucleic acids of both infectious and non infectious viruses, which limits the impact of conclusions with regard to public health concern. The aim of the study was to develop a method to discriminate between infectious and non-infectious particles of hepatitis A virus (HAV) and two strains of rotavirus (RV) following thermal inactivation by using intercalating dyes combined with RT-qPCR. Results Once the binding of propidium monoazide (PMA) or ethidium monoazide (EMA) was shown to be effective on the viral ssRNA of HAV and dsRNA of two strains of RV (SA11 and Wa), their use in conjunction with three surfactants (IGEPAL CA-630, Tween 20, Triton X-100) prior to RT-qPCR assays was evaluated to quantify the infectious particles remaining following heat treatment. The most promising conditions were EMA (20 μM) and IGEPAL CA-630 (0.5%) for HAV, EMA (20 μM) for RV (WA) and PMA (50 μM) for RV (SA11). The effectiveness of the pre-treatment RT-qPCR developed for each virus was evaluated with three RT-qPCR assays (A, B, C) during thermal inactivation kinetics (at 37°C, 68 C, 72°C, 80°C) through comparison with data obtained by RT-qPCR and by infectious titration in cell culture. At 37°C, the quantity of virus (RV, HAV) remained constant regardless of the method used. The genomic titers following heat treatment at 68°C to 80°C became similar to the infectious titers only when a pre-treatment RT-qPCR was used. Moreover, the most effective decrease was obtained by RT-qPCR assay A or B for HAV and RT-qPCR assay B or C for RV. Conclusions We concluded that effectiveness of the pre-treatment RT-qPCR is influenced by the viral target and by the choice

  6. Molecular epidemiology of infectious hematopoietic necrosis virus reveals complex virus traffic and evolution within southern Idaho aquaculture

    USGS Publications Warehouse

    Troyer, R.M.; Kurath, G.

    2003-01-01

    Infectious hematopoietic necrosis virus (IHNV) is a rhabdovirus which infects salmon and trout and may cause disease with up to 90% mortality. In the Hagerman Valley of Idaho, IHNV is endemic or epidemic among numerous fish farms and resource mitigation hatcheries. A previous study characterizing the genetic diversity among 84 IHNV isolates at 4 virus-endemic rainbow trout farms indicated that multiple lineages of relatively high diversity co-circulated at these facilities (Troyer et al. 2000 J Gen Virol. 81:2823-2832). We tested the hypothesis that high IHNV genetic diversity and co-circulating lineages are present in aquaculture facilities throughout this region. In this study, 73 virus isolates from 14 rainbow trout farms and 3 state hatcheries in the Hagerman Valley, isolated between 1978 and 1999, were genetically characterized by sequence analysis of a 303 nucleotide region of the glycoprotein gene. Phylogenetic and epidemiological analyses showed that multiple IHNV lineages co-circulate in a complex pattern throughout private trout farms and state hatcheries in the valley. IHNV maintained within the valley appears to have evolved significantly over the 22 yr study period.

  7. Host Defense Mechanisms Against Infectious Bovine Rhinotracheitis Virus: In Vitro Stimulation of Sensitized Lymphocytes by Virus Antigen

    PubMed Central

    Rouse, Barry T.; Babiuk, Lorne A.

    1974-01-01

    Isolated peripheral blood lymphocytes (PBL) from cattle immunized or infected with infectious bovine rhinotracheitis (IBR) virus were cultured in vitro with ultraviolet light-inactivated IBR virus, and the degree of lymphocyte blastogenesis was quantitated by measurement of the uptake of [3H]thymidine into acid-insoluble material. Lymphocyte blastogenesis only occurred with PBL from immunized or infected animals. The optimal conditions for lymphocyte blastogenesis were defined. Blastogenesis was specific since cells from animals immunized against IBR failed to react with two other herpesvirus antigens tested, herpes simplex and equine rhinopneumonitis viruses. Blastogenesis could be prevented by reacting IBR antigen with IBR-specific antibody before adding to cultures, but incorporating IBR-specific antibody in the culture medium after adding free antigen failed to inhibit blastogenesis. With intranasally infected animals, lymphocyte blastogenesis was detectable after 5 days, reached peak levels between days 7 and 10, and then declined to low levels by day 19. In contrast, levels of neutralizing antibody were barely detectable on day 7 and reached maximal concentrations on day 19. The lymphocyte blastogenesis assay was emphasized as a convenient and useful in vitro correlate of cell-mediated immunity that should help define the role of cell-mediated immunity in immunity to herpesviruses. PMID:4426702

  8. Differential susceptibility in steelhead trout populations to an emergent MD strain of infectious hematopoietic necrosis virus

    USGS Publications Warehouse

    Breyta, R.; Jones, Amelia; Kurath, Gael

    2014-01-01

    A significant emergence of trout-adapted MD subgroup infectious hematopoietic necrosis virus (IHNV) began in the coastal region of Washington State, USA, in 2007. This emergence event lasted until 2011 and caused both asymptomatic adult fish infection and symptomatic epidemic disease and mortality in juvenile fish. Incidence of virus during this emergence demonstrated a heterogeneous distribution among rivers of the coastal region, leaving fish populations of some rivers apparently untouched while others suffered significant and recurrent infection and mortality (Breyta et. al. 2013; Dis Aquat Org 104:179-195). In this study, we examined the possible contribution of variations in susceptibility of fish populations, age-related resistance, and virus virulence to the observed landscape heterogeneity. We found that the most significant variable was host susceptibility: by controlled experimental challenge studies steelhead trout populations with no history of IHNV infection were 1 to 3 orders of magnitude more sensitive than a fish population with a long history of IHNV infection. In addition, 2 fish populations from the same river, which descended relatively recently from a common ancestral population, demonstrated 1 to 2 orders of magnitude difference in susceptibility. Fish age-related development of resistance was most evident in the more susceptible of 2 related fish populations. Finally, the strain of virus involved in the 2007 coastal Washington emergence had high virulence but was within the range of other known M group viruses tested. These results suggest that one major driver of landscape heterogeneity in the 2007 coastal Washington IHNV emergence was variation in fish population susceptibility and that this trait may have a heritable component.

  9. Discerning an Effective Balance between Equine Infectious Anemia Virus Attenuation and Vaccine Efficacy

    PubMed Central

    Craigo, Jodi K.; Li, Feng; Steckbeck, Jonathan D.; Durkin, Shannon; Howe, Laryssa; Cook, Sheila J.; Issel, Charles; Montelaro, Ronald C.

    2005-01-01

    Among the diverse experimental vaccines evaluated in various animal lentivirus models, live attenuated vaccines have proven to be the most effective, thus providing an important model for examining critical immune correlates of protective vaccine immunity. We previously reported that an experimental live attenuated vaccine for equine infectious anemia virus (EIAV), based on mutation of the viral S2 accessory gene, elicited protection from detectable infection by virulent virus challenge (F. Li et al., J. Virol. 77:7244-7253, 2003). To better understand the critical components of EIAV vaccine efficacy, we examine here the relationship between the extent of virus attenuation, the maturation of host immune responses, and vaccine efficacy in a comparative study of three related attenuated EIAV proviral vaccine strains: the previously described EIAVUKΔS2 derived from a virulent proviral clone, EIAVUKΔS2/DU containing a second gene mutation in the virulent proviral clone, and EIAVPRΔS2 derived from a reference avirulent proviral clone. Inoculations of parallel groups of eight horses resulted in relatively low levels of viral replication (average of 102 to 103 RNA copies/ml) and a similar maturation of EIAV envelope-specific antibody responses as determined in quantitative and qualitative serological assays. However, experimental challenge of the experimentally immunized horses by our standard virulent EIAVPV strain by using a low-dose multiple exposure protocol (three inoculations with 10 median horse infective doses, administered intravenously) revealed a marked difference in the protective efficacy of the various attenuated proviral vaccine strains that was evidently associated with the extent of vaccine virus attenuation, time of viral challenge, and the apparent maturation of virus-specific immunity. PMID:15708986

  10. Differential susceptibility in steelhead trout populations to an emergent MD strain of infectious hematopoietic necrosis virus.

    PubMed

    Breyta, Rachel; Jones, Amelia; Kurath, Gael

    2014-11-13

    A significant emergence of trout-adapted MD subgroup infectious hematopoietic necrosis virus (IHNV) began in the coastal region of Washington State, USA, in 2007. This emergence event lasted until 2011 and caused both asymptomatic adult fish infection and symptomatic epidemic disease and mortality in juvenile fish. Incidence of virus during this emergence demonstrated a heterogeneous distribution among rivers of the coastal region, leaving fish populations of some rivers apparently untouched while others suffered significant and recurrent infection and mortality (Breyta et. al. 2013; Dis Aquat Org 104:179-195). In this study, we examined the possible contribution of variations in susceptibility of fish populations, age-related resistance, and virus virulence to the observed landscape heterogeneity. We found that the most significant variable was host susceptibility: by controlled experimental challenge studies steelhead trout populations with no history of IHNV infection were 1 to 3 orders of magnitude more sensitive than a fish population with a long history of IHNV infection. In addition, 2 fish populations from the same river, which descended relatively recently from a common ancestral population, demonstrated 1 to 2 orders of magnitude difference in susceptibility. Fish age-related development of resistance was most evident in the more susceptible of 2 related fish populations. Finally, the strain of virus involved in the 2007 coastal Washington emergence had high virulence but was within the range of other known M group viruses tested. These results suggest that one major driver of landscape heterogeneity in the 2007 coastal Washington IHNV emergence was variation in fish population susceptibility and that this trait may have a heritable component. PMID:25392039

  11. Sequence comparisons of the variable VP2 region of eight infectious bursal disease virus isolates.

    PubMed

    Dormitorio, T V; Giambrone, J J; Duck, L W

    1997-01-01

    The VP2 gene is part of the genomic segment A of infectious bursal disease virus (IBDV). It has been identified as the major host-protective antigen of IBDV and is known to contain conformationally dependent protective epitopes. A 643-base pair segment covering the hypervariable region of this gene from three recent serologic variant IBDV isolates from the southeastern United States, two variants from the Delmarva Peninsula, and three serologic standard viruses were amplified and sequenced using the reverse transcription polymerase chain reaction and cycle sequencing techniques. This was done to determine the molecular similarity among isolates that differ antigenically and pathologically. Sequence analysis suggested that the Arkansas (Ark) and Mississippi (Miss) isolates evolved closely and separately from the Delmarva variants (GLS and DELE), in contrast to the other southeastern variant Georgia (Ga), which is more closely related (98.32%) to Delaware E (DELE). All variants, except for Miss, underwent a shift in amino acid number 222 from proline to threonine. The sequence of Univax BD virus, a commercially available intermediate vaccine, was markedly different, evolving from a separate lineage than the others. Restriction enzyme sites could differentiate most isolates. Except for Miss, variants do not have EcoRII site at the larger hydrophilic domain. All variants lost their HaeIII, StuI, and StyI cutting sites with a change in base number 856. The TaqI site is in DELE, whereas the SpeI site is absent in the standard vaccine viruses. The SWASASGS heptapeptide is conserved in all virulent viruses, including APHIS, but not in the attenuated (Univax BD and Bursa Vac 3) and published (D78 and PBG98) vaccines. PMID:9087318

  12. Different Domains of the RNA Polymerase of Infectious Bursal Disease Virus Contribute to Virulence

    PubMed Central

    Le Nouën, Cyril; Toquin, Didier; Müller, Hermann; Raue, Rüdiger; Kean, Katherine M.; Langlois, Patrick; Cherbonnel, Martine; Eterradossi, Nicolas

    2012-01-01

    Background Infectious bursal disease virus (IBDV) is a pathogen of worldwide significance to the poultry industry. IBDV has a bi-segmented double-stranded RNA genome. Segments A and B encode the capsid, ribonucleoprotein and non-structural proteins, or the virus polymerase (RdRp), respectively. Since the late eighties, very virulent (vv) IBDV strains have emerged in Europe inducing up to 60% mortality. Although some progress has been made in understanding the molecular biology of IBDV, the molecular basis for the pathogenicity of vvIBDV is still not fully understood. Methodology, Principal Findings Strain 88180 belongs to a lineage of pathogenic IBDV phylogenetically related to vvIBDV. By reverse genetics, we rescued a molecular clone (mc88180), as pathogenic as its parent strain. To study the molecular basis for 88180 pathogenicity, we constructed and characterized in vivo reassortant or mosaic recombinant viruses derived from the 88180 and the attenuated Cu-1 IBDV strains. The reassortant virus rescued from segments A of 88180 (A88) and B of Cu-1 (BCU1) was milder than mc88180 showing that segment B is involved in 88180 pathogenicity. Next, the exchange of different regions of BCU1 with their counterparts in B88 in association with A88 did not fully restore a virulence equivalent to mc88180. This demonstrated that several regions if not the whole B88 are essential for the in vivo pathogenicity of 88180. Conclusion, Significance The present results show that different domains of the RdRp, are essential for the in vivo pathogenicity of IBDV, independently of the replication efficiency of the mosaic viruses. PMID:22253687

  13. Evaluation of infectious bronchitis virus Arkansas-type vaccine failure in commercial broilers.

    PubMed

    Roh, Ha-Jung; Hilt, Deborah A; Williams, Susan M; Jackwooda, Mark W

    2013-06-01

    Infectious bronchitis virus (IBV) causes an upper respiratory tract disease in chickens and is highly contagious. Many different types of the virus exist, but only a few types are used as attenuated live vaccines in the commercial poultry industry. Of the vaccine types used, the Arkansas (Ark)-type virus is most frequently reisolated from vaccinated broilers. Previous research has suggested that incomplete clearance of Ark-type vaccine virus plays a role in the inadequate protection observed when vaccinated broilers are challenged with pathogenic Ark virus. In this study, we examine routes of vaccine administration using multiple IBV types including Ark in an effort to understand why Ark vaccines do not provide good protection and persist in commercial broilers. We found that interference between different types of IBV vaccines was not occurring when combined and administered using a commercial hatchery spray cabinet. Also, Ark vaccine virus was not efficacious in 1-day-old broilers when sprayed using a hatchery spray cabinet, but it gave good protection when administrated by eyedrop inoculation. We also found that the amount of Ark vaccine virus was low or undetectable in choanal swabs out to 35 days postvaccination when vaccine was administered by eyedrop or drinking water. Alternatively, a subpopulation of the Ark vaccine isolated from a vaccinated bird, Ark-RI-EP1, showed a peak titer at 7-10 days of age when given by the same routes, suggesting that the Ark-RI-EP1 was more fit with regard to infection, replication in the birds, or both. Moreover, we found that detection of IBV vaccine virus early after administration, regardless of strain or route, correlated with protection against homologous challenge and may thus be a good indicator of vaccine efficacy in the field because humoral antibody titers are typically low or undetectable after vaccination. These experiments provided key findings that can be used to direct efforts for improving the efficacy of IBV

  14. Studies on Dulcamara mottle virus Infectious Clone and Chimeric Genomes with Turnip yellow mosaic virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The genus Tymovirus consists of viruses with positive sense, single-stranded capped RNA genomes that encode three open reading frames and terminate in a tRNA-like structure. An unpublished report of DuMV 3’ UTR indicated that DuMV seemed to terminate in an A-tail, the first such report for a tymovir...

  15. A replication competent lentivirus (RCL) assay for equine infectious anaemia virus (EIAV)-based lentiviral vectors.

    PubMed

    Miskin, J; Chipchase, D; Rohll, J; Beard, G; Wardell, T; Angell, D; Roehl, H; Jolly, D; Kingsman, S; Mitrophanous, K

    2006-02-01

    Lentiviral vectors are being developed to satisfy a wide range of currently unmet medical needs. Vectors destined for clinical evaluation have been rendered multiply defective by deletion of all viral coding sequences and nonessential cis-acting sequences from the transfer genome. The viral envelope and accessory proteins are excluded from the production system. The vectors are produced from separate expression plasmids that are designed to minimize the potential for homologous recombination. These features ensure that the regeneration of the starting virus is impossible. It is a regulatory requirement to confirm the absence of any replication competent virus, so we describe here the development and validation of a replication competent lentivirus (RCL) assay for equine infectious anaemia virus (EIAV)-based vectors. The assay is based on the guidelines developed for testing retroviral vectors, and uses the F-PERT (fluorescent-product enhanced reverse transcriptase) assay to test for the presence of a transmissible reverse transcriptase. We have empirically modelled the replication kinetics of an EIAV-like entity in human cells and devised an amplification protocol by comparison with a replication competent MLV. The RCL assay has been validated at the 20 litre manufacturing scale, during which no RCL was detected. The assay is theoretically applicable to any lentiviral vector and pseudotype combination. PMID:16208418

  16. PKR Activation Favors Infectious Pancreatic Necrosis Virus Replication in Infected Cells

    PubMed Central

    Gamil, Amr A.A.; Xu, Cheng; Mutoloki, Stephen; Evensen, Øystein

    2016-01-01

    The double-stranded RNA-activated protein kinase R (PKR) is a Type I interferon (IFN) stimulated gene that has important biological and immunological functions. In viral infections, in general, PKR inhibits or promotes viral replication, but PKR-IPNV interaction has not been previously studied. We investigated the involvement of PKR during infectious pancreatic necrosis virus (IPNV) infection using a custom-made rabbit antiserum and the PKR inhibitor C16. Reactivity of the antiserum to PKR in CHSE-214 cells was confirmed after IFNα treatment giving an increased protein level. IPNV infection alone did not give increased PKR levels by Western blot, while pre-treatment with PKR inhibitor before IPNV infection gave decreased eukaryotic initiation factor 2-alpha (eIF2α) phosphorylation. This suggests that PKR, despite not being upregulated, is involved in eIF2α phosphorylation during IPNV infection. PKR inhibitor pre-treatment resulted in decreased virus titers, extra- and intracellularly, concomitant with reduction of cells with compromised membranes in IPNV-permissive cell lines. These findings suggest that IPNV uses PKR activation to promote virus replication in infected cells. PMID:27338445

  17. An infectious recombinant foot-and-mouth disease virus expressing a fluorescent marker protein

    PubMed Central

    Juleff, Nicholas; Moffat, Katy; Berryman, Stephen; Christie, John M.; Charleston, Bryan; Jackson, Terry

    2013-01-01

    Foot-and-mouth disease virus (FMDV) is one of the most extensively studied animal pathogens because it remains a major threat to livestock economies worldwide. However, the dynamics of FMDV infection are still poorly understood. The application of reverse genetics provides the opportunity to generate molecular tools to further dissect the FMDV life cycle. Here, we have used reverse genetics to determine the capsid packaging limitations for a selected insertion site in the FMDV genome. We show that exogenous RNA up to a defined length can be stably introduced into the FMDV genome, whereas larger insertions are excised by recombination events. This led us to construct a recombinant FMDV expressing the fluorescent marker protein, termed iLOV. Characterization of infectious iLOV-FMDV showed the virus has a plaque morphology and rate of growth similar to the parental virus. In addition, we show that cells infected with iLOV-FMDV are easily differentiated by flow cytometry using the inherent fluorescence of iLOV and that cells infected with iLOV-FMDV can be monitored in real-time with fluorescence microscopy. iLOV-FMDV therefore offers a unique tool to characterize FMDV infection in vitro, and its applications for in vivo studies are discussed. PMID:23559477

  18. Evolutionary mechanisms involved in the virulence of infectious salmon anaemia virus (ISAV), a piscine orthomyxovirus

    SciTech Connect

    Markussen, Turhan Jonassen, Christine Monceyron Numanovic, Sanela Braaen, Stine Hjortaas, Monika Nilsen, Hanne Mjaaland, Siri

    2008-05-10

    Infectious salmon anaemia virus (ISAV) is an orthomyxovirus causing a multisystemic, emerging disease in Atlantic salmon. Here we present, for the first time, detailed sequence analyses of the full-genome sequence of a presumed avirulent isolate displaying a full-length hemagglutinin-esterase (HE) gene (HPR0), and compare this with full-genome sequences of 11 Norwegian ISAV isolates from clinically diseased fish. These analyses revealed the presence of a virulence marker right upstream of the putative cleavage site R{sub 267} in the fusion (F) protein, suggesting a Q{sub 266} {yields} L{sub 266} substitution to be a prerequisite for virulence. To gain virulence in isolates lacking this substitution, a sequence insertion near the cleavage site seems to be required. This strongly suggests the involvement of a protease recognition pattern at the cleavage site of the fusion protein as a determinant of virulence, as seen in highly pathogenic influenza A virus H5 or H7 and the paramyxovirus Newcastle disease virus.

  19. Detection and isolation of infectious laryngotracheitis virus on a broiler farm after a disease outbreak.

    PubMed

    Dormitorio, Teresa V; Giambrone, Joseph J; Macklin, Kenneth S

    2013-12-01

    A broiler farm in North Alabama suffered a mild infectious laryngotracheitis (ILT) outbreak, as determined by clinical disease and PCR. The poultry integrator sought help to control further outbreaks in subsequent flocks. Samples were collected from various areas of the poultry houses on the farm over an 8-wk period. The first sampling was conducted 8 days after the infected farm was depopulated; the second was conducted 2 days prior to subsequent flock placement; and the third was conducted when the new flock was 5 wk of age. Samples were examined for ILT virus (ILTV) DNA by real-time PCR and virus isolation in embryos. The infected houses were cleaned, disinfected, heated, litter composted, and curtains replaced after the first sampling and prior to placement of the next flock. Samples from all periods were positive for ILTV DNA. However, the number of positive samples and crossing point values indicated a decrease in the amount of viral DNA, while virus isolation in embryos was successful only on the first sampling. The subsequent flock was vaccinated against ILTV by in ovo route using a commercial recombinant vaccine. Cleaning and sanitation after the disease outbreak reduced the amount of ILTV on the farm and together with in ovo vaccination of the new flock may have prevented a recurrence of another ILT outbreak. PMID:24597126

  20. Phylogenetic and molecular epidemiological studies reveal evidence of multiple past recombination events between infectious laryngotracheitis viruses.

    PubMed

    Lee, Sang-Won; Devlin, Joanne M; Markham, John F; Noormohammadi, Amir H; Browning, Glenn F; Ficorilli, Nino P; Hartley, Carol A; Markham, Philip F

    2013-01-01

    In contrast to the RNA viruses, the genome of large DNA viruses such as herpesviruses have been considered to be relatively stable. Intra-specific recombination has been proposed as an important, but underestimated, driving force in herpesvirus evolution. Recently, two distinct field strains of infectious laryngotracheitis virus (ILTV) have been shown to have arisen from independent recombination events between different commercial ILTV vaccines. In this study we sequenced the genomes of additional ILTV strains and also utilized other recently updated complete genome sequences of ILTV to confirm the existence of a number of ILTV recombinants in nature. Multiple recombination events were detected in the unique long and repeat regions of the genome, but not in the unique short region. Most recombinants contained a pair of crossover points between two distinct lineages of ILTV, corresponding to the European origin and the Australian origin vaccine strains of ILTV. These results suggest that there are two distinct genotypic lineages of ILTV and that these commonly recombine in the field. PMID:23383306

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

    PubMed

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

    2013-01-01

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

  2. Phylogenetic and Molecular Epidemiological Studies Reveal Evidence of Multiple Past Recombination Events between Infectious Laryngotracheitis Viruses

    PubMed Central

    Lee, Sang-Won; Devlin, Joanne M.; Markham, John F.; Noormohammadi, Amir H.; Browning, Glenn F.; Ficorilli, Nino P.; Hartley, Carol A.; Markham, Philip F.

    2013-01-01

    In contrast to the RNA viruses, the genome of large DNA viruses such as herpesviruses have been considered to be relatively stable. Intra-specific recombination has been proposed as an important, but underestimated, driving force in herpesvirus evolution. Recently, two distinct field strains of infectious laryngotracheitis virus (ILTV) have been shown to have arisen from independent recombination events between different commercial ILTV vaccines. In this study we sequenced the genomes of additional ILTV strains and also utilized other recently updated complete genome sequences of ILTV to confirm the existence of a number of ILTV recombinants in nature. Multiple recombination events were detected in the unique long and repeat regions of the genome, but not in the unique short region. Most recombinants contained a pair of crossover points between two distinct lineages of ILTV, corresponding to the European origin and the Australian origin vaccine strains of ILTV. These results suggest that there are two distinct genotypic lineages of ILTV and that these commonly recombine in the field. PMID:23383306

  3. Infectious bursal disease virus changes the potassium current properties of chicken embryo fibroblasts.

    PubMed

    Repp, H; Nieper, H; Draheim, H J; Koschinski, A; Müller, H; Dreyer, F

    1998-07-01

    Infectious bursal disease virus (IBDV) is the causative agent of an economically significant poultry disease. IBDV infection leads to apoptosis in chicken embryos and cell cultures. Since changes in cellular ion fluxes during apoptosis have been reported, we investigated the membrane ion currents of chicken embryo fibroblasts (CEFs) inoculated with the Cu-1 strain of IBDV using the patch-clamp recording technique. Incubation of CEFs with IBDV led to marked changes in their K+ outward current properties, with respect to both the kinetics of activation and inactivation and the Ca2+ dependence of the activation. The changes occurred in a time-dependent manner and were complete after 8 h. UV-treated noninfectious virions induced the same K+ current changes as live IBDV. When CEFs were inoculated with IBDV after pretreatment with a neutralizing antibody, about 30% of the cells showed a normal K+ current, whereas the rest exhibited K+ current properties identical to or closely resembling those of IBDV-infected cells. Incubation of CEFs with culture supernatant from IBDV-infected cells from which the virus particles were removed had no influence on the K+ current. Our data strongly suggest that the K+ current changes induced by IBDV are not due to virus replication, but are the result of attachment and/or membrane penetration. Possibly, the altered K+ current may delay the apoptotic process in CEFs after IBDV infection. PMID:9657954

  4. Production of infectious duck hepatitis B virus in a human hepatoma cell line.

    PubMed Central

    Galle, P R; Schlicht, H J; Fischer, M; Schaller, H

    1988-01-01

    The differentiated human hepatoma cell line Hep-G2 was transfected with cloned duck hepatitis B virus (DHBV) DNA. Introduction of closed circular DNA into the human liver cells resulted in the production of viral proteins: core antigen was detected in the cytoplasm, and e antigen, a related product, was secreted into the medium. Moreover, viral particles were released into the tissue culture medium which were indistinguishable from authentic DHBV by density, antigenicity, DNA polymerase activity, and morphology. Intravenous injection of tissue culture-derived DHBV particles into Pekin ducks established DHBV infection. In conclusion, transfection of human hepatoma cells with cloned DHBV DNA results in the production of infectious virus, as occurs with cloned human hepatitis B virus DNA. Human liver cells are therefore competent to support production of the avian and mammalian hepadnaviruses, indicating that liver-specific viral gene expression is controlled by evolutionarily conserved mechanisms. This new DHBV transfection system offers the opportunity to rapidly produce mutated DHBV which then can be further investigated in Pekin ducks. Images PMID:2833623

  5. Psittacid Herpesvirus 1 and Infectious Laryngotracheitis Virus: Comparative Genome Sequence Analysis of Two Avian Alphaherpesviruses

    PubMed Central

    Thureen, Dean R.; Keeler, Calvin L.

    2006-01-01

    Psittacid herpesvirus 1 (PsHV-1) is the causative agent of Pacheco's disease, an acute, highly contagious, and potentially lethal respiratory herpesvirus infection in psittacine birds, while infectious laryngotracheitis virus (ILTV) is a highly contagious and economically significant avian herpesvirus which is responsible for an acute respiratory disease limited to galliform birds. The complete genome sequence of PsHV-1 has been determined and compared to the ILTV sequence, assembled from published data. The PsHV-1 and ILTV genomes exhibit similar structural characteristics and are 163,025 bp and 148,665 bp in length, respectively. The PsHV-1 genome contains 73 predicted open reading frames (ORFs), while the ILTV genome contains 77 predicted ORFs. Both genomes contain an inversion in the unique long region similar to that observed in pseudorabies virus. PsHV-1 is closely related to ILTV, and it is proposed that it be assigned to the Iltovirus genus. These two avian herpesviruses represent a phylogenetically unique clade of alphaherpesviruses that are distinct from the Marek's disease-like viruses (Mardivirus). The determination of the complete genomic nucleotide sequences of PsHV-1 and ILTV provides a tool for further comparative and functional analysis of this unique class of avian alphaherpesviruses. PMID:16873243

  6. Viral genome RNA serves as messenger early in the infectious cycle of murine leukemia virus.

    PubMed Central

    Shurtz, R; Dolev, S; Aboud, M; Salzberg, S

    1979-01-01

    When NIH/3T3 mouse fibroblasts were infected with the Moloney strain of murine leukemia virus, part of the viral genome RNA molecules were detected in polyribosomes of the infected cells early in the infectious cycle. The binding appears to be specific, since we could demonstrate the release of viral RNA from polyribosomes with EDTA. Moreover, when infection occurred in the presence of cycloheximide, most viral RNA molecules were detected in the free cytoplasm. Size analysis on polyribosomal viral RNA molecules indicated that two size class molecules, 38S and 23S, are present in polyribosomes at 3 h after infection. Analysis of the polyriboadenylate [poly(rA)] content of viral RNA extracted from infected polyribosomes demonstrated that such molecules bind with greatest abundance at 3 h after infection, as has been detected with total viral RNA. No molecules lacking poly(rA) stretches could be detected in polyribosomes. Furthermore, when a similar analysis was performed on unbound molecules present in the free cytoplasm, identical results were obtained. We conclude that no selection towards poly(rA)-containing viral molecules is evident on binding to polyribosomes. These findings suggest that the incoming viral genome of the Moloney strain of murine leukemia virus may serve as a messenger for the synthesis of one or more virus-specific proteins early after infection of mouse fibroblasts. PMID:117118

  7. Host tropism of infectious salmon anaemia virus in marine and freshwater fish species.

    PubMed

    Aamelfot, M; Dale, O B; McBeath, A; Falk, K

    2015-08-01

    The aquatic orthomyxovirus infectious salmon anaemia virus (ISAV) causes a severe disease in farmed Atlantic salmon, Salmo salar L. Although some ISA outbreaks are caused by horizontal transmission of virus between farms, the source and reservoir of the virus is largely unknown and a wild host has been hypothesized. Atlantic salmon are farmed in open net-pens, allowing transmission of pathogens from wild fish and the surrounding environment to the farmed fish. In this study, a large number of fish species were investigated for ISAV host potential. For orthomyxoviruses, a specific receptor binding is the first requirement for infection; thus, the fish species were investigated for the presence of the ISAV receptor. The receptor was found to be widely distributed across the fish species. All salmonids expressed the receptor. However, only some of the cod-like and perch-like fish did, and all flat fish were negative. In the majority of the positive species, the receptor was found on endothelial cells and/or on red blood cells. The study forms a basis for further investigations and opens up the possibility for screening species to determine whether a wild host of ISAV exists. PMID:25048819

  8. Molecular characteristics and evolutionary analysis of a very virulent infectious bursal disease virus.

    PubMed

    Li, Zan; Qi, XiaoLe; Ren, XianGang; Cui, Lei; Wang, XiaoMei; Zhu, Ping

    2015-08-01

    Infectious bursal disease virus (IBDV) poses a significant threat to the poultry industry. Viral protein 2 (VP2), the major structural protein of IBDV, has been subjected to frequent mutations that have imparted tremendous genetic diversity to the virus. To determine how amino acid mutations may affect the virulence of IBDV, we built a structural model of VP2 of a very virulent strain of IBDV identified in China, vvIBDV Gx, and performed a molecular dynamics simulation of the interaction between virulence sites. The study showed that the amino acid substitutions that distinguish vvIBDV from attenuated IBDV (H253Q and T284A) favor a hydrophobic and flexible conformation of β-barrel loops in VP2, which could promote interactions between the virus and potential IBDV-specific receptors. Population sequence analysis revealed that the IBDV strains prevalent in East Asia show a significant signal of positive selection at virulence sites 253 and 284. In addition, a signal of co-evolution between sites 253 and 284 was identified. These results suggest that changes in the virulence of IBDV may result from both the interaction and the co-evolution of multiple amino acid substitutions at virulence sites. PMID:26245145

  9. Recovery of Viral RNA and Infectious Foot-and-Mouth Disease Virus from Positive Lateral-Flow Devices

    PubMed Central

    Fowler, Veronica L.; Bankowski, Bartlomiej M.; Armson, Bryony; Di Nardo, Antonello; Valdazo-Gonzalez, Begoña; Reid, Scott M.; Barnett, Paul V.; Wadsworth, Jemma; Ferris, Nigel P.; Mioulet, Valérie; King, Donald P.

    2014-01-01

    Foot-and-mouth disease Virus (FMDV) is an economically important, highly contagious picornavirus that affects both wild and domesticated cloven hooved animals. In developing countries, the effective laboratory diagnosis of foot-and-mouth disease (FMD) is often hindered by inadequate sample preservation due to difficulties in the transportation and storage of clinical material. These factors can compromise the ability to detect and characterise FMD virus in countries where the disease is endemic. Furthermore, the high cost of sending infectious virus material and the biosecurity risk it presents emphasises the need for a thermo-stable, non-infectious mode of transporting diagnostic samples. This paper investigates the potential of using FMDV lateral-flow devices (LFDs) for dry transportation of clinical samples for subsequent nucleic acid amplification, sequencing and recovery of infectious virus by electroporation. FMDV positive samples (epithelial suspensions and cell culture isolates) representing four FMDV serotypes were applied to antigen LFDs: after which it was possible to recover viral RNA that could be detected using real-time RT-PCR. Using this nucleic acid, it was also possible to recover VP1 sequences and also successfully utilise protocols for amplification of complete FMD virus genomes. It was not possible to recover infectious FMDV directly from the LFDs, however following electroporation into BHK-21 cells and subsequent cell passage, infectious virus could be recovered. Therefore, these results support the use of the antigen LFD for the dry, non-hazardous transportation of samples from FMD endemic countries to international reference laboratories. PMID:25313787

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

    PubMed Central

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

    2014-01-01

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

  11. Infectious foot-and-mouth disease virus derived from a cloned full-length cDNA.

    PubMed Central

    Zibert, A; Maass, G; Strebel, K; Falk, M M; Beck, E

    1990-01-01

    A full-length cDNA plasmid of foot-and-mouth disease virus has been constructed. RNA synthesized in vitro by means of a bacteriophage SP6 promoter inserted in front of the cDNA led to the production of infectious particles upon transfection of BHK-21 cells. These particles were also found to be highly infectious for primary bovine kidney cells as well as for baby mice. The difficulty in cloning the foot-and-mouth disease virus cytidyl tract in Escherichia coli was circumvented by joining two separate cloned parts, representing the S and L fragments of the genome, and, in a second step, inserting a dC-dG homopolymer. Homopolymeric sequences of up to 25 cytidyl residues did not lead to the production of virus. Replicons containing poly(C) tracts long enough to permit virus replication were first established in yeast cells. One of these constructs could also be maintained in E. coli and was used to produce infectious RNA in vitro. The length of the poly(C) sequence in this cDNA plasmid was 32 nucleotides. However, the poly(C) tracts of two recombinant viruses found in transfected BHK-21 cells were 60 and 80 nucleotides long, respectively. Possible mechanisms leading to the enlargement of the poly(C) tract during virus replication are discussed. Images PMID:2159523

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

    PubMed

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

    2011-08-01

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

  13. Recovery of NV Knockout Infectious Hematopoietic Necrosis Virus Expressing Foreign Genes

    PubMed Central

    Biacchesi, Stéphane; Thoulouze, Maria-Isabel; Béarzotti, Monique; Yu, Yan-Xing; Brémont, Michel

    2000-01-01

    Infectious hematopoietic necrosis virus (IHNV) is a Novirhabdovirus and is the causative agent of a devastating acute, lethal disease in wild and farmed rainbow trout. The virus is enzootic throughout western North America and has spread to Asia and Europe. A full-length cDNA of the IHNV antigenome (pIHNV-Pst) was assembled from subgenomic overlapping cDNA fragments and cloned in a transcription plasmid between the T7 RNA polymerase promoter and the autocatalytic hepatitis delta virus ribozyme. Recombinant IHNV (rIHNV) was recovered from fish cells at 14°C, following infection with a recombinant vaccinia virus expressing the T7 RNA polymerase (vTF7-3) and cotransfection of pIHNV-Pst together with plasmids encoding the nucleoprotein N (pT7-N), the phosphoprotein P (pT7-P), the RNA polymerase L (pT7-L), and the nonvirion protein NV (pT7-NV). When pT7-N and pT7-NV were omitted, rIHNV was also recovered, although less efficiently. Incidental mutations introduced in pIHNV-Pst were all present in the rIHNV genome; however, a targeted mutation located in the L gene was eliminated from the recombinant genome by homologous recombination with the added pT7-L expression plasmid. To investigate the role of NV protein in virus replication, the pIHNV-Pst construct was engineered such that the entire NV open reading frame was deleted and replaced by the genes encoding green fluorescent protein or chloramphenicol acetyltransferase. The successful recovery of recombinant virus expressing foreign genes instead of the NV gene demonstrated that the NV protein was not absolutely required for viral replication in cell cultures, although its presence greatly improves virus growth. The ability to generate rIHNV from cDNA provides the basis to manipulate the genome in order to engineer new live viral vaccine strains. PMID:11070023

  14. Infectious Pancreatic Necrosis Virus Causing Clinical and Subclinical Infections in Atlantic Salmon Have Different Genetic Fingerprints

    PubMed Central

    Mutoloki, Stephen; Jøssund, Trude B.; Ritchie, Gordon; Munang'andu, Hetron M.; Evensen, Øystein

    2016-01-01

    Infectious pancreatic necrosis virus (IPNV) is the causative agent of IPN, an important disease of salmonids. IPNV infections result in either sub-clinical or overt disease and the basis of this difference is not well-understood. The objective of the present study was to determine the VP2 gene of the virus associated with the different forms of clinical manifestation. Groups of Atlantic salmon (Salmo salar L.) reared in farms located in different IPN disease pressures were monitored from brood stock until grow-out over a 3 year period. Hatcheries A1 and B1 as well as cooperating seawater farms were located in a low disease risk area while hatcheries A2 and B2 as well as their cooperating seawater farms were in high IPN risk areas. Samples including eggs, milt, whole fry, kidney depending on the stage of production were collected during outbreaks or in apparently healthy populations where no outbreaks occurred. The virus was re-isolated in CHSE cells and the VP2 gene amplified by RT-PCR followed by sequencing. During the freshwater stage, there were no disease outbreaks at hatcheries A1, A2, and B1 (except in one fish group that originated from hatchery B2), although IPNV was isolated from some of the fish groups at all 3 hatcheries. By contrast, all fish groups at hatchery B2 suffered IPN outbreaks. In seawater, only groups of fish originating from hatchery A1 had no IPN outbreaks albeit virus being isolated from the fish. On the other hand, fish originating from hatcheries A2, B1, and B2 experienced outbreaks in seawater. The VP2 amino acid fingerprint of the virus associated with subclinical infections from A1 and co-operating seawater sites was V64A137P217T221A247N252S281D282E319. By contrast, all virus isolates associated with clinical infections had the motif I64T137T217A221T247V252T281N282A319, where underlined amino acids represent the avirulent and highly virulent motif, respectively. Phylogenetic analysis of amino acid sequences showed 2 clades, one of

  15. Detection of shrimp infectious myonecrosis virus by reverse transcription loop-mediated isothermal amplification combined with a lateral flow dipstick.

    PubMed

    Puthawibool, Teeranart; Senapin, Saengchan; Kiatpathomchai, Wansika; Flegel, Timothy W

    2009-03-01

    Infectious myonecrosis virus (IMNV) has caused a slowly progressive disease with cumulative mortalities of up to 70% or more in cultured Penaeus (Litopenaeus) vannamei in Northeast Brazil and Indonesia. Rapid detection of viruses by loop-mediated isothermal amplification (LAMP) of genomic material with high specificity and sensitivity can be applied for diagnosis, monitoring and control of diseases in shrimp aquaculture. Using an IMNV template, successful detection was achieved after a 60-min RT-LAMP reaction using biotin-labeled primers followed by 5min hybridization with an FITC-labeled DNA probe and 5min assay using a chromatographic lateral flow dipstick (LFD). Thus, the combined system of RT-LAMP and LFD required a total assay interval of less than 75min, excluding the RNA extraction time. The sensitivity of detection was comparable to that of other commonly used methods for nested RT-PCR detection of IMNV. In addition to reducing amplicon detection time when compared to electrophoresis, LFD confirmed amplicon identity by hybridization and eliminated the need to handle carcinogenic ethidium bromide. The RT-LAMP-LFD method gave negative test results with nucleic acid extracts from normal shrimp and from shrimp infected with other viruses including infectious hypodermal hematopoietic necrosis virus (IHHNV), monodon baculovirus (MBV), a hepatopancreatic parvovirus from P. monodon (PmDNV), white spot syndrome virus (WSSV), yellow head virus (YHV), Taura syndrome virus (TSV), Macrobrachium rosenbergii nodavirus (MrNV) and gill associated virus (GAV). PMID:19022295

  16. Estimation of Parameters Influencing Waterborne Transmission of Infectious Hematopoietic Necrosis Virus (IHNV) in Atlantic Salmon (Salmo salar)

    PubMed Central

    Garver, Kyle A; Mahony, Amelia A. M.; Stucchi, Dario; Richard, Jon; Van Woensel, Cecile; Foreman, Mike

    2013-01-01

    Understanding how pathogenic organisms spread in the environment is crucial for the management of disease, yet knowledge of propagule dispersal and transmission in aquatic environments is limited. We conducted empirical studies using the aquatic virus, infectious hematopoietic necrosis virus (IHNV), to quantify infectious dose, shedding capacity, and virus destruction rates in order to better understand the transmission of IHN virus among Atlantic salmon marine net-pen aquaculture. Transmission of virus and subsequent mortality in Atlantic salmon post-smolts was initiated with as low as 10 plaque forming units (pfu) ml−1. Virus shedding from IHNV infected Atlantic salmon was detected before the onset of visible signs of disease with peak shed rates averaging 3.2×107 pfu fish−1 hour−1 one to two days prior to mortality. Once shed into the marine environment, the abundance of free IHNV is modulated by sunlight (UV A and B) and the growth of natural biota present in the seawater. Virus decayed very slowly in sterilized seawater while rates as high as k =  4.37 d−1 were observed in natural seawater. Decay rates were further accelerated when exposed to sunlight with virus infectivity reduced by six orders of magnitude within 3 hours of full sunlight exposure. Coupling the IHNV transmission parameter estimates determined here with physical water circulation models, will increase the understanding of IHNV dispersal and provide accurate geospatial predictions of risk for IHNV transmission from marine salmon sites. PMID:24340016

  17. Comparison of the replication and transmissibility of two infectious laryngotracheitis virus chicken embryo origin vaccines delivered via drinking water.

    PubMed

    Coppo, Mauricio J C; Devlin, Joanne M; Noormohammadi, Amir H

    2012-01-01

    Infectious laryngotracheitis (ILT) is an acute infectious viral disease that affects chickens, causing respiratory disease, loss of production and mortality in severe cases. Biosecurity measures and administration of attenuated viral vaccine strains are commonly used to prevent ILT. It is notable that most recent ILT outbreaks affecting the intensive poultry industry have been caused by vaccine-related virus strains. The purpose of this study was to characterize and compare viral replication and transmission patterns of two attenuated chicken embryo origin ILT vaccines delivered via the drinking water. Two groups of specific pathogen free chickens were each inoculated with SA-2 ILT or Serva ILT vaccine strains. Unvaccinated birds were then placed in contact with vaccinated birds at regular intervals. Tracheal swabs were collected every 4 days over a period of 60 days and examined for the presence and amount of virus using a quantitative polymerase chain reaction. A rapid increase in viral genome copy numbers was observed shortly after inoculation with SA-2 ILT virus. In contrast, a comparatively delayed virus replication was observed after vaccination with Serva ILT virus. Transmission to in-contact birds occurred soon after exposure to Serva ILT virus but only several days after exposure to SA-2 ILT virus. Results from this study demonstrate in vivo differences between ILT vaccine strains in virus replication and transmission patterns. PMID:22515537

  18. Continuous Exposure to Infectious Pancreatic Necrosis Virus (IPNV) During Early Life Stages of Rainbow Trout, Oncorhynchus mykiss, Walbaum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Rainbow trout (Oncorhynchus mykiss, Walbaum) were exposed continuously to infectious pancreatic necrosis virus (IPNV) at 0, 10, 1,000, or 10,000 pfu/L of water to estimate the effects of chronic IPNV exposure on early life stages. Fish density averaged 35 fish/L (low) or 140 fish/L (high), and wate...

  19. Continuous exposure to infectious pancreatic necrosis virus during early life stages of rainbow trout, Oncorhynchus mykiss(Walbaum)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Rainbow trout (Oncorhynchus mykiss, Walbaum) were exposed continuously to infectious pancreatic necrosis virus (IPNV) at 0, 10, 1,000, or 100,000 pfu/L of water to estimate the effects of chronic IPNV exposure on early life stages. Fish density averaged 35 fish/L or 140 fish/L, with a tank flow rat...

  20. Retrovirus transduction: Segregation of the viral transforming function and the Herpes Simplex virus tk gene in infectious friend spleen focus-forming virus thymidine kinase vectors

    SciTech Connect

    Joyner, A.L.; Bernstein, A.

    1983-12-01

    A series of deletions and insertions utilizing the herpesvirus thymidine kinase gene (tk) were constructed in the murine retrovirus Friend spleen focus-forming virus (SFFV). In all cases, the coding region for the SFFV-specific glycoprotein (gp55), which is implicated in erythroleukemic transformation, was left intact. These SFFV-TK and SFFV deletion vectors were analyzed for expression of tk and gp55 after DNA-mediated gene transfer. In addition, virus rescued by cotranfection of these vectors with Moloney murine leukemia virus was analyzed for infectious TK-transducing virus, gp55 expression, and erythroleukemia-inducing ability. The experiments demonstrated that deletions or insertions within the intron for the gp55 env gene can interfere with expression of gp55 after both DNA-mediated gene transfer and virus infection. In contrast, the gene transfer efficiency of the tk gene was unaffected in the SFFV-TK vectors, and high-titer infectious TK virus could be recovered. Revertant viruses capable of inducing erythroleukemia and expressing gp55 were generated after cotranfection of the SFFV-TK vectors with murine leukemia virus. The revertant viruses lost both tk sequences and the ability to transduce TK/sup -/ fibroblasts to a TK/sup +/ phenotype. These experiments demonstrate that segregation of the TK and erythroleukemia functions can occur in retrovirus vectors which initially carry both markers.

  1. Protection of chickens from Newcastle disease and infectious laryngotracheitis with a recombinant fowlpox virus co-expressing the F, HN genes of Newcastle disease virus and gB gene of infectious laryngotracheitis virus.

    PubMed

    Sun, Hui-Ling; Wang, Yun-Feng; Tong, Guang-Zhi; Zhang, Pei-Jun; Miao, De-Yuan; Zhi, Hai-Dong; Wang, Ming; Wang, Mei

    2008-03-01

    A recombinant fowlpox virus (rFPV) coexpressing the Newcastle disease virus (NDV) fusion and hemagglutinin-neuraminidase genes and infectious laryngothracheitis virus (ILTV) glycoprotein B gene was constructed. This virus was then evaluated for its ability to protect specific-pathogen-free (SPF) chickens against clinical symptoms and death after challenge by virulent NDV and ILTV. SPF chickens were grouped and vaccinated with the rFPV and commercial NDV (La Sota) and ILTV attenuated live vaccine (Nobilis ILT), respectively. After challenge with NDV 10 days postvaccination, 70% of chickens vaccinated with rFPV were protected from death, whereas 100% of the commercial NDV-vaccinated chickens were protected from death. In contrast, 100% of the unvaccinated chickens died after challenge. After challenge with ILTV, both the rFPV and commercial ILTV-vaccinated chickens were completely protected from death and 70% of chickens were protected from respiratory signs. In comparison, 100% of the unvaccinated chickens developed severe respiratory disease and 10% of chickens died. The protective efficacy was also measured by the antibody responses and isolation of challenge viruses. Results showed that this rFPV could be a potential vaccine for preventing NDV and ILTV by a single immunization. PMID:18459306

  2. U.S. response to a report of infectious salmon anemia virus in Western North America

    USGS Publications Warehouse

    Amos, Kevin H; Gustafson, Lori; Warg, Janet; Whaley, Janet; Purcell, Maureen K.; Rolland, Jill B.; Winton, James R.; Snekvik, Kevin; Meyers, Theodore; Stewart, Bruce; Kerwin, John; Blair, Marilyn; Bader, Joel; Evered, Joy

    2014-01-01

    Federal, state, and tribal fishery managers, as well as the general public and their elected representatives in the United States, were concerned when infectious salmon anemia virus (ISAV) was suspected for the first time in free-ranging Pacific Salmon collected from the coastal areas of British Columbia, Canada. This article documents how national and regional fishery managers and fish health specialists of the U.S. worked together and planned and implemented actions in response to the reported finding of ISAV in British Columbia. To date, the reports by Simon Fraser University remain unconfirmed and preliminary results from collaborative U.S. surveillance indicate that there is no evidence of ISAV in U.S. populations of free-ranging or marine-farmed salmonids on the west coast of North America.

  3. Tetraodon nigroviridis as a nonlethal model of infectious spleen and kidney necrosis virus (ISKNV) infection

    SciTech Connect

    Xu Xiaopeng; Huang Lichao; Weng Shaoping; Wang Jing; Lin Ting; Tang Junliang; Li Zhongsheng; Lu Qingxia; Xia Qiong; Yu Xiaoqiang; He Jianguo

    2010-10-25

    Infectious spleen and kidney necrosis virus (ISKNV) is the type species of the genus Megalocytivirus, family Iridoviridae. We have previously established a high mortality ISKNV infection model of zebrafish (Danio rerio). In this study, a nonlethal Tetraodon nigroviridis model of ISKNV infection was established. ISKNV infection did not cause lethal disease in Tetraodon but could infect almost all the organs of this species. Electron microscopy showed ISKNV particles were present in infected tissues. Immunofluorescence and quantitative real-time PCR analysis showed that nearly all the virions and infected cells were cleared at 14 d postinfection. The expression profiles of interferon-{gamma} and tumor necrosis factor-{alpha} gene in response to ISKNV infection were significantly different in Tetraodon and zebrafish. The establishment of the nonlethal Tetraodon model of ISKNV infection can offer a valuable tool complementary to the zebrafish infection model for studying megalocytivirus disease, fish immune systems, and viral tropism.

  4. Expression of infectious woodchuck hepatitis virus in murine and avian fibroblasts.

    PubMed Central

    Seeger, C; Baldwin, B; Tennant, B C

    1989-01-01

    The liver is the primary site for replication of the hepadnavirus genome. We asked whether the posttranscriptional phase of the viral replication cycle would depend on hepatocyte-specific functions. For this purpose, we assayed a previously constructed chimera between sequences of the cytomegalovirus immediate-early promoter-enhancer region and woodchuck hepatitis virus (WHV) (C. Seeger and J. Maragos, J. Virol. 63:1907-1915, 1989) for its ability to direct the synthesis of infectious WHV in hepatoma cells and in murine and avian fibroblast cells. Viruslike particles containing WHV DNA were produced transiently in transfected hepatoma cells and in fibroblasts. Inoculation of woodchucks with culture medium from hepatoma cells or fibroblasts transfected with viral DNA led to productive WHV infection, as observed following infection of woodchucks with serum from WHV-infected animals. These results demonstrate that posttranscriptional events of the hepadnavirus replication cycle are not dependent on hepatocyte-specific functions. Images PMID:2795716

  5. Differentiation of field isolates and vaccine strains of infectious laryngotracheitis virus by DNA sequencing.

    PubMed

    Chacón, Jorge Luis; Ferreira, Antonio J Piantino

    2009-11-12

    Two different regions of the infected cell protein 4 (ICP4) gene of infectious laryngotracheitis virus (ILTV) were amplified and sequenced for characterization of field isolates and tissue culture-origin (TCO) and chicken embryo-origin (CEO) vaccine strains. Phylogenetic analysis of the two regions showed differences in nucleotide and amino acid sequences between field isolates and attenuated vaccines. The PCR-RFLP results were identical to those obtained by DNA sequencing and validated their use to differentiate ILTV strains. The approach using the sequencing of the two fragments of the ICP4 gene showed to be an efficient and practical procedure to differentiate between field isolates and vaccine strains of ILTV. PMID:19747995

  6. Infectious complications of human T cell leukemia/lymphoma virus type I infection.

    PubMed

    Marsh, B J

    1996-07-01

    Infection with human T cell leukemia/lymphoma virus type I (HTLV-I) has been etiologically associated with two diseases: adult T cell leukemia and HTLV-I-associated myelopathy/tropical spastic paraparesis. Increasing evidence suggests that HTLV-I infection may be associated with immunosuppression and, as a consequence, affect the risk and expression of several other infectious diseases, of which the best studied are strongyloidiasis, tuberculosis, and leprosy. In strongyloidiasis, coinfection with HTLV-I appears to result in a higher rate of chronic carriage, an increased parasite load, and a risk of more severe infection. In tuberculosis, a decrease in delayed-type hypersensitivity to Mycobacterium tuberculosis has been established, but whether this decrease is clinically significant has yet to be determined. In leprosy, an increased risk of disease is suggested, but the published studies are all too poorly controlled to draw definite conclusions. PMID:8816143

  7. Detection of variant infectious bronchitis viruses in Sri Lanka (2012-2015).

    PubMed

    Ball, Christopher; Forrester, Anne; Ganapathy, Kannan

    2016-06-01

    Poultry production is an important sector of agriculture in Sri Lanka; however, there is a lack of information regarding circulation of infectious bronchitis virus (IBV). RNA was extracted from chicken tissues, subjected to IBV S1 RT-PCR, and sequenced. Overall, 19 out of 34 (55.88 %) samples were IBV positive and contained the genotype 793B (n = 13; 68.42 %), D274 (n = 4; 21.05 %) or Massachusetts (n = 2; 10.53 %). All three genotypes contained at least one strain with less than 99 % nucleotide sequence identity to the corresponding vaccine strains. This report identified co-circulation of IBV strains 793B, Massachusetts and D274, in Sri Lanka that are divergent from the respective vaccine strains. PMID:27020570

  8. A neutralizing scFv antibody against infectious bursal disease virus screened by flow cytometry.

    PubMed

    Zhou, Yao; Xie, Zhi-Gang

    2015-09-01

    Infectious bursal disease (IBD) is considered a vital viral disease that threatens the poultry industry worldwide. In this study, a recombinant single chain variable fragment (scFv) antibody library derived from chickens immunized with VP2 protein of infectious bursal disease virus (IBDV) was constructed. The library was subjected to three rounds of screening by flow cytometry (FCM) against VP2/IBDV through a bacteria display technology, resulting in the enrichment of scFvs. Three scFv clones with different fluorescence intensity were obtained by colony pick up at random. The obtained scFv antibodies were expressed and purified. Relative affinity assay showed the three clones had different sensitivity to VP2, in accordance with fluorescence activity cell sorting analysis (FACS). The potential use of the isolated IBDV-specific scFv antibodies was demonstrated by the successful application of these antibodies in Western blotting and ELISA assay. What's more, in vitro neutralization measurement showed that one of the three isolated antibodies possessed the neutralization function against IBDV. This study provides new strategies for screening of antibody library, and scFv antibodies isolated in this study may be utilized as lead candidates for further development of diagnostic or therapeutic antibodies for detection and treatment of IBDV infection. PMID:26003676

  9. Infectious Disease Physician Assessment of Hospital Preparedness for Ebola Virus Disease

    PubMed Central

    Polgreen, Philip M.; Santibanez, Scott; Koonin, Lisa M.; Rupp, Mark E.; Beekmann, Susan E.; del Rio, Carlos

    2015-01-01

    Background. The first case of Ebola diagnosed in the United States and subsequent cases among 2 healthcare workers caring for that patient highlighted the importance of hospital preparedness in caring for Ebola patients. Methods. From October 21, 2014 to November 11, 2014, infectious disease physicians who are part of the Emerging Infections Network (EIN) were surveyed about current Ebola preparedness at their institutions. Results. Of 1566 EIN physician members, 869 (55.5%) responded to this survey. Almost all institutions represented in this survey showed a substantial degree of preparation for the management of patients with suspected and confirmed Ebola virus disease. Despite concerns regarding shortages of personal protective equipment, approximately two thirds of all respondents reported that their facilities had sufficient and ready availability of hoods, full body coveralls, and fluid-resistant or impermeable aprons. The majority of respondents indicated preference for transfer of Ebola patients to specialized treatment centers rather than caring for them locally. In general, we found that larger hospitals and teaching hospitals reported higher levels of preparedness. Conclusions. Prior to the Centers for Disease Control and Prevention's plan for a tiered approach that identified specific roles for frontline, assessment, and designated treatment facilities, our query of infectious disease physicians suggested that healthcare facilities across the United States were making preparations for screening, diagnosis, and treatment of Ebola patients. Nevertheless, respondents from some hospitals indicated that they were relatively unprepared. PMID:26180836

  10. Evaluation of infectious bursal disease virus stability at different conditions of temperature and pH.

    PubMed

    Rani, Surabhi; Kumar, Sachin

    2015-11-01

    Infectious bursal disease (IBD) is one of the highly pathogenic viral diseases of poultry. The disease poses a serious threat to the economy of many developing countries where agriculture serves as the primary source of national income. Infectious bursal disease virus (IBDV) belongs to the family Birnaviridae. The IBDV is well characterized to cause immunosuppression in poultry. The live attenuated vaccine is the only way to protect the chickens from IBDV infection. The ineffectiveness of vaccine is one of the major causes of IBDV outbreaks in field condition. In the present study, we discuss briefly about the biology of IBDV genome and its proteins under different conditions of temperature and pH in order to evaluate its infectivity under adverse physical conditions. Our results indicate that the IBDV is non-infective above 42 °C and unstable above 72 °C. However, the change in pH does not significantly contribute to the IBDV stability. The study will be useful in estimating an optimum storage condition for IBDV vaccines without causing any deterioration in its viability and effectiveness. PMID:26265229

  11. Major histocompatibility complex loci are associated with susceptibility of Atlantic salmon to infectious hematopoietic necrosis virus

    USGS Publications Warehouse

    Miller, Kristina M.; Winton, James R.; Schulze, Angela D.; Purcell, Maureen K.; Ming, Tobi J.

    2004-01-01

    Infectious hematopoietic necrosis virus (IHNV) is one of the most significant viral pathogens of salmonids and is a leading cause of death among cultured juvenile fish. Although several vaccine strategies have been developed, some of which are highly protective, the delivery systems are still too costly for general use by the aquaculture industry. More cost effective methods could come from the identification of genes associated with IHNV resistance for use in selective breeding. Further, identification of susceptibility genes may lead to an improved understanding of viral pathogenesis and may therefore aid in the development of preventive and therapeutic measures. Genes of the major histocompatibility complex (MHC), involved in the primary recognition of foreign pathogens in the acquired immune response, are associated with resistance to a variety of diseases in vertebrate organisms. We conducted a preliminary analysis of MHC disease association in which an aquaculture strain of Atlantic salmon was challenged with IHNV at three different doses and individual fish were genotyped at three MHC loci using denaturing gradient gel electrophoresis (PCR-DGGE), followed by sequencing of all differentiated alleles. Nine to fourteen alleles per exon-locus were resolved, and alleles potentially associated with resistance or susceptibility were identified. One allele (Sasa-B-04) from a potentially non-classical class I locus was highly associated with resistance to infectious hematopoietic necrosis (p < 0.01). This information can be used to design crosses of specific haplotypes for family analysis of disease associations.

  12. Interstitial lung disease associated with Equine Infectious Anemia Virus infection in horses.

    PubMed

    Bolfa, Pompei; Nolf, Marie; Cadoré, Jean-Luc; Catoi, Cornel; Archer, Fabienne; Dolmazon, Christine; Mornex, Jean-François; Leroux, Caroline

    2013-01-01

    EIA (Equine Infectious Anemia) is a blood-borne disease primarily transmitted by haematophagous insects or needle punctures. Other routes of transmission have been poorly explored. We evaluated the potential of EIAV (Equine Infectious Anemia Virus) to induce pulmonary lesions in naturally infected equids. Lungs from 77 EIAV seropositive horses have been collected in Romania and France. Three types of lesions have been scored on paraffin-embedded lungs: lymphocyte infiltration, bronchiolar inflammation, and thickness of the alveolar septa. Expression of the p26 EIAV capsid (CA) protein has been evaluated by immunostaining. Compared to EIAV-negative horses, 52% of the EIAV-positive horses displayed a mild inflammation around the bronchioles, 22% had a moderate inflammation with inflammatory cells inside the wall and epithelial bronchiolar hyperplasia and 6.5% had a moderate to severe inflammation, with destruction of the bronchiolar epithelium and accumulation of smooth muscle cells within the pulmonary parenchyma. Changes in the thickness of the alveolar septa were also present. Expression of EIAV capsid has been evidenced in macrophages, endothelial as well as in alveolar and bronchiolar epithelial cells, as determined by their morphology and localization. To summarize, we found lesions of interstitial lung disease similar to that observed during other lentiviral infections such as FIV in cats, SRLV in sheep and goats or HIV in children. The presence of EIAV capsid in lung epithelial cells suggests that EIAV might be responsible for the broncho-interstitial damages observed. PMID:24289102

  13. Immune Responses to Virulent and Vaccine Strains of Infectious Bronchitis Viruses in Chickens.

    PubMed

    Chhabra, Rajesh; Chantrey, Julian; Ganapathy, Kannan

    2015-11-01

    Infectious bronchitis (IB) is an acute and highly contagious chicken viral disease, causing severe economic losses to poultry producers worldwide. In the last few decades, infectious bronchitis virus (IBV) has been extensively studied, but knowledge of immune responses to virulent or vaccine strains of IBVs remains limited. This review focuses on fundamental aspects of immune responses against IBV, including the role of pattern recognition receptors (PRRs) in identification of conserved viral structures and the role of different components of innate immunity (e.g., heterophils, macrophages, dendritic cells, acute phase protein, and cytokines). Studies on adaptive immune activation and the role of humoral and cellular immunity in IBV clearance are also reviewed. Multiple interlinking immune responses are essential for protection against virulent IBVs, including passive, innate, adaptive, and effector T cells active at mucosal surfaces. Although the development of approaches for chicken transcriptome and proteome analyses have greatly helped the understanding of the underlying genetic mechanisms for immunity, there are still major knowledge gaps, such as the role of mucosal and cellular responses to IBVs. In view of recent reports of emergent IBV variants in many countries, there is renewed interest in a more complete understanding of poultry immune responses to both virulent and vaccine strains of IBVs. This will be critical for developing new vaccine or vaccination strategies and other intervention programs. PMID:26301315

  14. Genotypes and phylogeographical relationships of infectious hematopoietic necrosis virus in California, USA

    USGS Publications Warehouse

    Kelley, G.O.; Bendorf, C.M.; Yun, S.C.; Kurath, G.; Hedrick, R.P.

    2007-01-01

    Infectious hematopoietic necrosis virus (IHNV) contains 3 major genogroups in North America with discreet geographic ranges designated as upper (U), middle (M), and lower (L). A comprehensive genotyping of 237 IHNV isolates from hatchery and wild salmonids in California revealed 25 different sequence types (a to y) all in the L genogroup; specifically, the genogroup contained 14 sequence types that were unique to individual isolates as well as 11 sequence types representing 2 or more identical isolates. The most evident trend was the phylogenetic and geographical division of the L genogroup into 2 distinct subgroups designated as LI and LII. Isolates within Subgroup LI were primarily found within waterways linked to southern Oregon and northern California coastal rivers. Isolates in Subgroup LII were concentrated within inland valley watersheds that included the Sacramento River, San Joaquin River, and their tributaries. The temporal and spatial patterns of virus occurrence suggested that infections among adult Chinook salmon in the hatchery or that spawn in the river are a major source of virus potentially infecting other migrating or resident salmonids in California. Serum neutralization results of the California isolates of IHNV corroborated a temporal trend of sequence divergence; specifically, 2 progressive shifts in which more recent virus isolates represent new serotypes. A comparison of the estimates of divergence rates for Subgroup LI (1 ?? ICT5 mutations per nucleotide site per year) indicated stasis similar to that observed in the U genogroup, while the Subgroup LII rate (1 ?? 10 3 mutations per nucleotide site per year) suggested a more active evolution similar to that of the M genogroup. ?? Inter-Research 2007.

  15. Replication characteristics of infectious laryngotracheitis virus in the respiratory and conjunctival mucosa.

    PubMed

    Reddy, Vishwanatha R A P; Steukers, Lennert; Li, Yewei; Fuchs, Walter; Vanderplasschen, Alain; Nauwynck, Hans J

    2014-01-01

    Avian infectious laryngotracheitis virus (ILTV) is an alphaherpesvirus of poultry that is spread worldwide. ILTV enters its host via the respiratory tract and the eyes. Although ILTV has been known for a long time, the replication characteristics of the virus in the respiratory and conjunctival mucosa are still poorly studied. To study these characteristics, two in vitro explant models were developed. Light microscopy and fluorescent terminal deoxynucleotidyl transferase dUTP nick end-labelling staining were used to evaluate the viability of mucosal explants, which were found to be viable up to the end of the experiment at 96 h of cultivation. The tracheal and conjunctival mucosal explants were inoculated with ILTV and collected at 0, 24, 48 and 72 h post inoculation (p.i.). ILTV spread in a plaque-wise manner in both mucosae. A reproducible quantitative analysis of this mucosal spread was evaluated by measuring plaque numbers, plaque latitude and invasion depth underneath the basement membrane. No major differences in plaque numbers were observed over time. Plaque latitude progressively increased to 70.4 ± 12.9 μm in the trachea and 97.8 ± 9.5 μm in the conjunctiva at 72 h p.i. The virus had difficulty crossing the basement membrane and was first observed only at 48 h p.i. The virus was observed at 72 h p.i. in 56% (trachea) and 74% (conjunctiva) of the plaques. Viability analysis of infected explants indicated that ILTV blocks apoptosis in infected cells of both mucosae but activates apoptosis in bystander cells. PMID:25144137

  16. Inhibition of infectious bursal disease virus by vector delivered SiRNA in cell culture.

    PubMed

    Sahare, Amol Ashok; Bedekar, Megha Kadam; Jain, Sudhir Kumar; Singh, Azad; Singh, Sanjeev; Sarkhel, Bikas Chandra

    2015-01-01

    Infectious Bursal Disease (IBD) is major threat to poultry industry. It causes severe immunosuppression and mortality in chicken generally at 3 to 6 weeks of age. RNA intereference (RNAi) emerges as a potent gene regulatory tool in last few years. The present study was conducted to evaluate the efficiency of RNAi to inhibit the IBD virus (IDBV) replication in-vitro. VP2 gene of virus encodes protein involved in capsid formation, cell entry and induction of protective immune responses against it. Thus, VP2 gene of IBDV is the candidate target for the molecular techniques applied for IBDV detection and inhibition assay. In this study, IBDV was isolated from field cases and confirmed by RT-PCR. The virus was then adapted on chicken embryo fibroblast cells (CEF) in which it showed severe cytopathic effects (CPE). The short hairpin RNA (shRNAs) constructs homologous to the VP2 gene were designed and one, having maximum score and fulfilling maximum Reynolds criteria, was selected for evaluation of effective inhibition. Selected shRNA construct (i.e., VP2-shRNA) was observed to be the most effective for inhibiting VP2 gene expression. Real time PCR analysis was performed to measure the relative expression of VP2 gene in different experimental groups. The VP2 gene was less expressed in virus infected cells co-transfected with VP2-shRNA as compared to mock transfected cells and IBDV+ cells (control) at dose 1.6 µ g. The result showed ∼95% efficient down regulation of VP2 gene mRNA in VP2-shRNA treated cells. These findings suggested that designed shRNA construct achieved high level of inhibition of VP2 gene expression in-vitro. PMID:25153457

  17. Isolation of a novel serotype strain of infectious bronchitis virus ZZ2004 from ducks in China.

    PubMed

    Yao, Sixin; Ou, Changbo; Liu, Xingyou; Wang, Xianwen; Yao, Zonghui; Liu, Jinjing

    2016-10-01

    In chickens, the infectious bronchitis virus (IBV) often causes respiratory distress, a decrease in egg production, poor egg quality, and occasional nephritis. However, ZZ2004, a Chinese isolate of IBV, was obtained from ducks with clinical growth suppression and mild respiratory symptoms that had been reared with chickens in the central region of China. Virus isolation, virus neutralization testing, and RT-PCR were employed to identify the causative pathogen, while sequence alignment was used to analyze gene variations of the S1 subunit and M genes. The results showed that the ducks were infected with IBV due to the emergence of a dwarfing phenotype and the death of embryos between 48 and 144 h post-inoculation. RT-PCR also confirmed the presence of the expected fragment sizes of the S1 subunit and M genes by RT-PCR. Meanwhile, the results of the virus neutralization test indicated that the strains of JX/99/01, GD, SAIBK, LDT3 showed cross-reactivity with the ZZ2004 isolate, and hardly any cross-neutralization of IBV ZZ2004 was observed with the strains of M41, H120, Gray, Holte, or Aust-T. Phylogenetic analysis suggested that there were large differences between ZZ2004 and other IBV reference strains on the S1 subunit. Meanwhile, homologies in the nucleotide and amino acid sequences of the M gene of IBV ZZ2004 were 86.9-92.0 % and 91.1-93.9 %, respectively, compared with 35 other IBV reference strains derived from different regions. This result revealed that there were conspicuous variations among the selected strains. Furthermore, the results showed that the prevalent strains of IBV in ducks had no antigen homology with the vaccine strains widely used in China except the LDT3-strain, making it urgent to explore and develop new IBV vaccines. PMID:27164844

  18. Protective immune responses of recombinant VP2 subunit antigen of infectious bursal disease virus in chickens.

    PubMed

    Pradhan, Satya Narayan; Prince, Prabhu Rajaiah; Madhumathi, Jayaprakasam; Roy, Parimal; Narayanan, Rangarajan Badri; Antony, Usha

    2012-08-15

    Infectious bursal disease virus (IBDV) is the causative agent of Gumboro disease and poses a huge threat to poultry industry. The risks associated with conventional attenuated viral vaccines make it indispensable to probe into the development of novel and rationally designed subunit vaccines which are safer as well as effective. VP2 is the major host-protective antigen found in IBDV capsid. It encompasses different independent epitopes responsible for the induction of neutralizing antibody. Here, we report the efficacy of the immunodominant fragment of VP2 which induces both humoral and cellular immunity against infectious bursal disease. A 366 bp fragment (52-417 bp) of the VP2 gene from an IBDV field isolate was amplified and expressed in Escherichia coli as a 21 kDa recombinant protein. The efficacy of rVP2(52-417) antigen was compared with two commercial IBDV whole virus vaccine strains. The rVP2(52-417) induced significantly high antibody titres in chicken compared to commercial vaccines and the anti-rVP2(52-417) sera showed reactivity with viral antigens from both commercial strains (P<0.0001) and field isolates. Also, the chicken splenocytes from rVP2(52-417) immunized group showed a significantly high proliferation (P<0.01) compared to other groups, which implies that the rVP2(52-417) fragment contains immunogenic epitopes capable of eliciting both B and T cell responses. Further, rVP2(52-417) conferred 100% protection against vIBDV challenge in the immunized chickens which was significantly higher (P<0.001) compared to 55-60% protection by commercial vaccine strains. Hence, the study confirms the efficacy of the immunodominant VP2 fragment that could be used as a potent vaccine against IBDV infection in chicken. PMID:22795186

  19. Mucosal, Cellular, and Humoral Immune Responses Induced by Different Live Infectious Bronchitis Virus Vaccination Regimes and Protection Conferred against Infectious Bronchitis Virus Q1 Strain

    PubMed Central

    Chhabra, Rajesh; Forrester, Anne; Lemiere, Stephane; Awad, Faez; Chantrey, Julian

    2015-01-01

    The objectives of the present study were to assess the mucosal, cellular, and humoral immune responses induced by two different infectious bronchitis virus (IBV) vaccination regimes and their efficacy against challenge by a variant IBV Q1. One-day-old broiler chicks were vaccinated with live H120 alone (group I) or in combination with CR88 (group II). The two groups were again vaccinated with CR88 at 14 days of age (doa). One group was kept as the control (group III). A significant increase in lachrymal IgA levels was observed at 4 doa and then peaked at 14 doa in the vaccinated groups. The IgA levels in group II were significantly higher than those in group I from 14 doa. Using immunohistochemistry to examine changes in the number of CD4+ and CD8+ cells in the trachea, it was found that overall patterns of CD8+ cells were dominant compared to those of CD4+ cells in the two vaccinated groups. CD8+ cells were significantly higher in group II than those in group I at 21 and 28 doa. All groups were challenged oculonasally with a virulent Q1 strain at 28 doa, and their protection was assessed. The two vaccinated groups gave excellent ciliary protection against Q1, although group II's histopathology lesion scores and viral RNA loads in the trachea and kidney showed greater levels of protection than those in group I. These results suggest that greater protection is achieved from the combined vaccination of H120 and CR88 of 1-day-old chicks, followed by CR88 at 14 doa. PMID:26202435

  20. A cell culture adapted HCV JFH1 variant that increases viral titers and permits the production of high titer infectious chimeric reporter viruses.

    PubMed

    Liu, Shuanghu; Xiao, Li; Nelson, Cassie; Hagedorn, Curt H; Hagedorn, Curt

    2012-01-01

    The unique properties of the hepatitis C virus (HCV) JFH1 isolate have made it possible to produce and study HCV in an infectious cell culture system. However, relatively low virus titers restrict some of the uses of this system and preparing infectious chimeric reporter viruses have been difficult. In this study, we report cell culture-adapted mutations in wild-type JFH1 yielding higher titers of infectious particles of both JFH1 and chimeric JFH1 viruses carrying reporter genes. Sequencing analyses determined that ten of the sixteen nonsynonymous mutations were in the NS5A region. Individual viruses harboring specific adaptive mutations were prepared and studied. The mutations in the NS5A region, which included all three domains, were most effective in increasing infectious virus production. Insertion of two reporter genes in JFH1 without the adaptive mutations ablated the production of infectious HCV particles. However, the introduction of specific adaptive mutations in the NS5A region permitted reporter genes, Renilla luciferase (Rluc) and EGFP, to be introduced into JHF1 to produce chimeric HCV-NS5A-EGFP and HCV-NS5A-Rluc reporter viruses at relatively high titers of infectious virus. The quantity of hyperphosphorylated NS5A (p58) was decreased in the adapted JFH1 compared wild type JFH1 and is likely be involved in increased production of infectious virus based on previous studies of p58. The JFH1-derived mutant viruses and chimeric reporter viruses described here provide new tools for studying HCV biology, identifying HCV antivirals, and enable new ways of engineering additional infectious chimeric viruses. PMID:23028707

  1. Nucleolin interacts with the dengue virus capsid protein and plays a role in formation of infectious virus particles.

    PubMed

    Balinsky, Corey A; Schmeisser, Hana; Ganesan, Sundar; Singh, Kavita; Pierson, Theodore C; Zoon, Kathryn C

    2013-12-01

    Dengue virus (DENV) is a mosquito-transmitted flavivirus that can cause severe disease in humans and is considered a reemerging pathogen of significant importance to public health. The DENV capsid (C) protein functions as a structural component of the infectious virion; however, it may have additional functions in the virus replicative cycle. Here, we show that the DENV C protein interacts and colocalizes with the multifunctional host protein nucleolin (NCL). Furthermore, we demonstrate that this interaction can be disrupted by the addition of an NCL binding aptamer (AS1411). Knockdown of NCL with small interfering RNA (siRNA) or treatment of cells with AS1411 results in a significant reduction of viral titers after DENV infection. Western blotting and quantitative RT-PCR (qRT-PCR) analysis revealed no differences in viral RNA or protein levels at early time points postinfection, suggesting a role for NCL in viral morphogenesis. We support this hypothesis by showing that treatment with AS1411 alters the migration characteristics of the viral capsid, as visualized by native electrophoresis. Here, we identify a critical interaction between DENV C protein and NCL that represents a potential new target for the development of antiviral therapeutics. PMID:24027323

  2. Nucleolin Interacts with the Dengue Virus Capsid Protein and Plays a Role in Formation of Infectious Virus Particles

    PubMed Central

    Balinsky, Corey A.; Schmeisser, Hana; Ganesan, Sundar; Singh, Kavita; Pierson, Theodore C.

    2013-01-01

    Dengue virus (DENV) is a mosquito-transmitted flavivirus that can cause severe disease in humans and is considered a reemerging pathogen of significant importance to public health. The DENV capsid (C) protein functions as a structural component of the infectious virion; however, it may have additional functions in the virus replicative cycle. Here, we show that the DENV C protein interacts and colocalizes with the multifunctional host protein nucleolin (NCL). Furthermore, we demonstrate that this interaction can be disrupted by the addition of an NCL binding aptamer (AS1411). Knockdown of NCL with small interfering RNA (siRNA) or treatment of cells with AS1411 results in a significant reduction of viral titers after DENV infection. Western blotting and quantitative RT-PCR (qRT-PCR) analysis revealed no differences in viral RNA or protein levels at early time points postinfection, suggesting a role for NCL in viral morphogenesis. We support this hypothesis by showing that treatment with AS1411 alters the migration characteristics of the viral capsid, as visualized by native electrophoresis. Here, we identify a critical interaction between DENV C protein and NCL that represents a potential new target for the development of antiviral therapeutics. PMID:24027323

  3. An infectious molecular clone of an unusual macrophage-tropic and highly cytopathic strain of human immunodeficiency virus type 1.

    PubMed Central

    Collman, R; Balliet, J W; Gregory, S A; Friedman, H; Kolson, D L; Nathanson, N; Srinivasan, A

    1992-01-01

    We isolated and molecularly cloned a human immunodeficiency virus type 1 (HIV-1) strain (89.6) which is unusual because it is both macrophage-tropic and extremely cytopathic in lymphocytes. Moreover, this is the first well-characterized infectious molecularly cloned macrophage-tropic HIV-1 strain derived from peripheral blood. HIV-1 89.6 differs markedly from other macrophage-tropic isolates within the envelope V3 region, which is important in determining cell tropism and cytopathicity. HIV-1 89.6 may thus represent a transitional isolate between noncytopathic macrophage-tropic viruses and cytopathic lymphocyte-tropic viruses. Images PMID:1433527

  4. Infectious salmon anaemia virus replication and induction of alpha interferon in Atlantic salmon erythrocytes

    PubMed Central

    Workenhe, Samuel T; Kibenge, Molly JT; Wright, Glenda M; Wadowska, Dorota W; Groman, David B; Kibenge, Frederick SB

    2008-01-01

    Background Infectious salmon anaemia (ISA) virus (ISAV), which causes ISA in marine-farmed Atlantic salmon, is an orthomyxovirus belonging to the genus Isavirus, family Orthomyxoviridae. ISAV agglutinates erythrocytes of several fish species and it is generally accepted that the ISAV receptor destroying enzyme dissolves this haemagglutination except for Atlantic salmon erythrocytes. Recent work indicates that ISAV isolates that are able to elute from Atlantic salmon erythrocytes cause low mortality in challenge experiments using Atlantic salmon. Previous work on ISAV-induced haemagglutination using the highly pathogenic ISAV strain NBISA01 and the low pathogenic ISAV strain RPC/NB-04-0851, showed endocytosis of NBISA01 but not RPC/NB-04-0851. Real-time RT-PCR was used to assess the viral RNA levels in the ISAV-induced haemagglutination reaction samples, and we observed a slight increase in viral RNA transcripts by 36 hours in the haemagglutination reaction with NBISA01 virus when the experiment was terminated. However, a longer sampling interval was considered necessary to confirm ISAV replication in fish erythrocytes and to determine if the infected cells mounted any innate immune response. This study examined the possible ISAV replication and Type I interferon (IFN) system gene induction in Atlantic salmon erythrocytes following ISAV haemagglutination. Results Haemagglutination assays were performed using Atlantic salmon erythrocytes and one haemagglutination unit of the two ISAV strains, NBISA01 and RPC/NB-04-0851, of differing genotypes and pathogenicities. Haemagglutination induced by the highly pathogenic NBISA01 but not the low pathogenic RPC/NB-04-0851 resulted in productive infection as evidenced by increased ISAV segment 8 transcripts and increase in the median tissue culture infectious dose (TCID50) by 5 days of incubation. Moreover, reverse transcription (RT) quantitative PCR used to compare mRNA levels of key Type I IFN system genes in erythrocyte

  5. Virulence and serological studies of recombinant infectious hematopoietic necrosis virus (IHNV) in rainbow trout.

    PubMed

    Wang, C; Lian, G H; Zhao, L L; Wu, Y; Li, Y J; Tang, L J; Qiao, X Y; Jiang, Y P; Liu, M

    2016-07-15

    Infectious hematopoietic necrosis virus is a highly contagious disease of juvenile salmonid species. From the IHNV HLJ-09 isolated in China, two recombinant viruses were generated by reverse genetics using the RNA polymerase II transcription system. The recombinant viruses were confirmed by RT-PCR, indirect immunofluorescence assay and electron microscopy. They were referred to as rIHNV HLJ-09 and rIHNV-EGFP. rIHNV HLJ-09 and rIHNV-EGFP could stably replicate in EPC cell lines and had the same cellular tropism as wtIHNV HLJ-09. But the titer of rIHNV-EGFP was significantly lower than rIHNV HLJ-09 and wtIHNV HLJ-09. rIHNV-EGFP strain could express EGFP stably at least in 20 passages, and the fluorescence could be observed clearly. To assess the virulence and pathogenicity of the recombinant viruses in vivo, juvenile rainbow trout were challenged by intraperitoneal injection with 20μl of rIHNV HLJ-09, rIHNV-EGFP or wtIHNV HLJ-09 (1×10(6)pfuml(-1)). Fish challenged with rIHNV HLJ-09 and wtIHNV HLJ-09 exhibited clinical signs typical of IHN disease and both produced 90% cumulative percent mortality, whlie rIHNV-EGFP produced only 5%. Pathological sectioning results showed that the tissues (liver, kidney, heart muscle, back muscle) of the fish infected with rIHNV HLJ-09 exhibited pathological changes, with the exception of cerebral neurons and the cheek. However, no lesions of liver, kidney, heart, muscle, brain in rainbow trout of rIHNV-EGFP or the control group were observed. Indirect ELISA results showed that a high level of serum antibody was detected in the experimental fish challenged with rIHNV HLJ-09, just as the same as wtIHNV HLJ-09, while a lower titer was detecred in the fish infected with rIHNV-EGFP. This indicated that the recombinant viruses could induce humoral immune response in the experimental fish. The recombinant viruses had unique genetic tags and could be used for genetic engineering, laying new ground for further investigation of IHNV

  6. Infectious clones of the crinivirus cucurbit chlorotic yellows virus are competent for plant systemic infection and vector transmission.

    PubMed

    Shi, Yan; Shi, Yajuan; Gu, Qinsheng; Yan, Fengming; Sun, Xinyan; Li, Honglian; Chen, Linlin; Sun, Bingjian; Wang, Zhenyue

    2016-06-01

    Cucurbit chlorotic yellows virus (CCYV), a recently identified bipartite crinivirus, causes economic losses in cucurbit plants. CCYV is naturally transmitted only by whitefly Bemisia tabaci. Here we constructed full-length cDNA clones of CCYV (RNA1 and RNA2) fused to the T7 RNA polymerase promoter and the cauliflower mosaic virus 35S promoter. CCYV replicated and accumulated efficiently in Cucumis sativus protoplasts transfected with in vitro transcripts. Without RNA2, RNA1 replicated efficiently in C. sativus protoplasts. Agroinoculation with the infectious cDNA clones of CCYV resulted in systemic infection in the host plants of C. sativus and Nicotiana benthamiana. Virus derived from the infectious clones could be transmitted between cucumber plants by vector whiteflies. This system will greatly enhance the reverse genetic studies of CCYV gene functions. PMID:26982585

  7. Human parainfluenza virus type 3 (HPIV-3); Construction and rescue of an infectious, recombinant virus expressing the enhanced green fluorescent protein (EGFP).

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The ability to rescue an infectious, recombinant, RNA virus from a cDNA clone, has led to new opportunities for measuring viral replication from a viral expressed reporter gene. In this protocol, the process of inserting enhanced green fluorescent protein (EGFP) gene into the human parainfluenza vi...

  8. The infectious particle of insect-borne totivirus-like Omono River virus has raised ridges and lacks fibre complexes.

    PubMed

    Okamoto, Kenta; Miyazaki, Naoyuki; Larsson, Daniel S D; Kobayashi, Daisuke; Svenda, Martin; Mühlig, Kerstin; Maia, Filipe R N C; Gunn, Laura H; Isawa, Haruhiko; Kobayashi, Mutsuo; Sawabe, Kyoko; Murata, Kazuyoshi; Hajdu, Janos

    2016-01-01

    Omono River virus (OmRV) is a double-stranded RNA virus isolated from Culex mosquitos, and it belongs to a group of unassigned insect viruses that appear to be related to Totiviridae. This paper describes electron cryo-microscopy (cryoEM) structures for the intact OmRV virion to 8.9 Å resolution and the structure of the empty virus-like-particle, that lacks RNA, to 8.3 Å resolution. The icosahedral capsid contains 120-subunits and resembles another closely related arthropod-borne totivirus-like virus, the infectious myonecrosis virus (IMNV) from shrimps. Both viruses have an elevated plateau around their icosahedral 5-fold axes, surrounded by a deep canyon. Sequence and structural analysis suggests that this plateau region is mainly composed of the extended C-terminal region of the capsid proteins. In contrast to IMNV, the infectious form of OmRV lacks extensive fibre complexes at its 5-fold axes as directly confirmed by a contrast-enhancement technique, using Zernike phase-contrast cryo-EM. Instead, these fibre complexes are replaced by a short "plug" structure at the five-fold axes of OmRV. OmRV and IMNV have acquired an extracellular phase, and the structures at the five-fold axes may be significant in adaptation to cell-to-cell transmission in metazoan hosts. PMID:27616740

  9. Single-tube, noninterrupted reverse transcription-PCR for detection of infectious bursal disease virus.

    PubMed Central

    Lee, L H; Ting, L J; Shien, J H; Shieh, H K

    1994-01-01

    An assay protocol based on single-tube, noninterrupted reverse transcription-PCR (RT-PCR) for the detection of infectious bursal disease virus (IBDV) is described. After the conditions for RT-PCR had been optimized, a primer set framing a region within the gene coding for IBDV VP2 protein was used to amplify a 318-bp fragment of the IBDV genome. Amplified product was detected with three strains of IBDV, whereas none was obtained from uninfected bursal tissue or seven unrelated avian viruses. The sensitivity of this RT-PCR was tested with purified viral RNA from three strains of IBDV. The detection limit was 10 fg in an ethidium bromide-stained gel. In addition, this assay system was used to detect IBDV in bursal-tissue specimens from commercially reared chickens. The identity of the amplified products from the tissue specimen preparation was determined by using a rapid, simple procedure in which internally nested, end-labeled probes were used. Images PMID:8051255

  10. Isolation, pathogenicity, and H120 protection efficacy of infectious bronchitis viruses isolated in Taiwan.

    PubMed

    Wang, C H; Hsieh, M C; Chang, P C

    1996-01-01

    Seven isolates of infectious bronchitis (IB) virus (IBV) were isolated from two breeder farms and five broiler farms in Taiwan in 1992. The cardinal signs of disease in breeders were egg production drops and watery albumen, and those in broilers were respiratory distress and renal urate deposition or death. All diseased chickens had been vaccinated with IB vaccines (mostly H120). The viruses were isolated and identified by chicken embryo inoculation and electron microscopy. The genomes of the isolates were extracted and amplified by polymerase chain reaction; the restriction fragment length polymorphism analysis suggested that the genotypes of the present IBV isolates were different from the eight reference strains. One-day-old specific-pathogen-free chicks were inoculated with the field isolates in order to test the virulence of those isolates. Respiratory distress and depression commenced at 24 hours after inoculation. Two of the seven isolates were found to be highly virulent, causing 50% or more mortality in inoculated chicks. Vaccine protection tests showed that H120 could protect chickens against challenges with four of six field isolates. PMID:8883793

  11. Molecular characterization of infectious bursal disease virus (IBDV) isolated in Argentina indicates a regional lineage.

    PubMed

    Vera, F; Craig, M I; Olivera, V; Rojas, F; König, G; Pereda, A; Vagnozzi, A

    2015-08-01

    In Argentina, classical vaccines are used to control infectious bursal disease virus (IBDV); however, outbreaks of IBDV are frequently observed. This could be due to failures in the vaccination programs or to the emergence of new strains, which would be able to break through the protection given by vaccines. Hence, genetic characterization of the viruses responsible for the outbreaks that occurred in recent years is crucial for the evaluation of the control programs and the understanding of the epidemiology and evolution of IBDV. In this study, we characterized 51 field samples collected in Argentina (previously identified as IBDV positive) through the analysis of previously identified apomorphic sequences. Phylogenetic analysis of regVP2 showed that 42 samples formed a unique cluster (Argentinean lineage), seven samples were typical classical strains (one of them was a vaccine strain), and two belonged to the very virulent lineage (vvIBDV). Interestingly, when the analysis was performed on the regVP1 sequences, the field samples segregated similarly to regVP2; thus, we observed no evidence of a reassortment event in the Argentinean samples. Amino acid sequence analysis of regVP2 showed a particular pattern of residues in the Argentinean lineage, particularly the presence of T272, P289 and F296, which had not been reported before as signature sequences for any IBDV phenotype. Notably, the residue S254, characteristic of the antigenic variant, was not present in any of the Argentinean samples. PMID:26026955

  12. Equine infectious anemia virus-infected dendritic cells retain antigen presentation capability

    SciTech Connect

    Rivera, Julie A.; McGuire, Travis C. . E-mail: mcguiret@vetmed.wsu.edu

    2005-05-10

    To determine if equine monocyte-derived dendritic cells (DC) were susceptible to equine infectious anemia virus (EIAV) infection, ex vivo-generated DC were infected with virus in vitro. EIAV antigen was detected by immunofluorescence 3 days post-infection with maximum antigen being detected on day 4, whereas there was no antigen detected in DC incubated with the same amount of heat-inactivated EIAV. No cytolytic activity was observed after EIAV{sub WSU5} infection of DC. These monocyte-derived DC were more effective than macrophages and B cells in stimulating allogenic T lymphocytes. Both infected macrophages and DC stimulated similar levels of memory CTL responses in mixtures of CD8+ and CD4+ cells as detected with {sup 51}Cr-release assays indicating that EIAV infection of DC did not alter antigen presentation. However, EIAV-infected DC were more effective than infected macrophages when used to stimulate memory CTL in isolated CD8+ cells. The maintenance of antigen processing and presenting function by EIAV-infected DC in vitro suggests that this function is maintained during in vivo infection.

  13. Rapid detection of infectious laryngotracheitis virus isolates by loop-mediated isothermal amplification.

    PubMed

    Xie, Qing-mei; Ji, Jun; Pickens, Tristan Tyler; Du, Li-qin; Cao, Yong-chang; Li, Hong-mei; Wang, Lin-guo; Ma, Jing-yun; Bi, Ying-zuo

    2010-04-01

    The objective of this study was to develop and evaluate a loop-mediated isothermal amplification (LAMP) method to detect infectious laryngotracheitis virus (ILTV) from commercial broiler and layer flocks in southern China. A set of six specific primers was designed to recognize six distinct genomic sequences of thymidine kinase (TK) from ILTV. The entire assay duration was recorded at 40 min under isothermal condition at 63.5 degrees C. The amplified products were analyzed by electrophoresis and visual judgment by the SYBR Green I dyeing. LAMP assay was 10-fold more sensitive than the routine PCR assay, with a detection limit of 46 copies per reaction. In detecting ILTV, the LAMP assay detected all 5 strains previously isolated, did not cross-react with other avian pathogens, and obtained a 100% sensitivity in 43 positive clinical samples with reference to virus isolation. Therefore, the LAMP assay may be a good alternative method for specific diagnosis of ILTV infection in primary care facilities, and in less well-equipped laboratories. PMID:20100518

  14. Characterization of Equine Infectious Anemia Virus Integration in the Horse Genome

    PubMed Central

    Liu, Qiang; Wang, Xue-Feng; Ma, Jian; He, Xi-Jun; Wang, Xiao-Jun; Zhou, Jian-Hua

    2015-01-01

    Human immunodeficiency virus (HIV)-1 has a unique integration profile in the human genome relative to murine and avian retroviruses. Equine infectious anemia virus (EIAV) is another well-studied lentivirus that can also be used as a promising retro-transfection vector, but its integration into its native host has not been characterized. In this study, we mapped 477 integration sites of the EIAV strain EIAVFDDV13 in fetal equine dermal (FED) cells during in vitro infection. Published integration sites of EIAV and HIV-1 in the human genome were also analyzed as references. Our results demonstrated that EIAVFDDV13 tended to integrate into genes and AT-rich regions, and it avoided integrating into transcription start sites (TSS), which is consistent with EIAV and HIV-1 integration in the human genome. Notably, the integration of EIAVFDDV13 favored long interspersed elements (LINEs) and DNA transposons in the horse genome, whereas the integration of HIV-1 favored short interspersed elements (SINEs) in the human genome. The chromosomal environment near LINEs or DNA transposons potentially influences viral transcription and may be related to the unique EIAV latency states in equids. The data on EIAV integration in its natural host will facilitate studies on lentiviral infection and lentivirus-based therapeutic vectors. PMID:26102582

  15. Research Studies on the Life Cycle of Infectious Hematopoietic Necrosis Virus.

    SciTech Connect

    Ristow, Sandra S.; Arnzen, Jeanene M.; Leong, JoAnn Ching

    1990-11-01

    Seventeen strains of infectious hematopoietic necrosis virus (IHNV) from different geographical regions and from different fish stocks were typed by polyacrylamide gel electrophoresis, indirect fluorescence with 27 monoclonal antibodies against both the G and N proteins of the virus, and by serum neutralization with six monoclonal anti-glycoprotein antibodies. In addition, many other IHNV isolates have been examined. Studying the isolates with the antibodies has shown that a greater amount of variation exists between isolates than was first predicted by the application of the polyacrylamide technique. Isolates within electrophoretic types I-V may be further classified according to their reactions with the monoclonal antibodies in indirect fluorescence. Serum neutralization with selected anti-glycoprotein antibodies in conjunction with fluorescence analysis confirms one of the original findings of Hsu et al. (1986) that two different species in a single facility can be infected with the same isolate. Variation among isolates as measured by reactivity with the monoclonal library appears to be greater within the G protein than within the N protein sequence. 9 refs., 7 figs., 6 tabs.

  16. Structure-dependent efficacy of infectious bursal disease virus (IBDV) recombinant vaccines.

    PubMed

    Martinez-Torrecuadrada, Jorge L; Saubi, Narciís; Pagès-Manté, Albert; Castón, José R; Espuña, Enric; Casal, J Ignacio

    2003-07-01

    The immunogenicity and protective capability of several baculovirus-expressed infectious bursal disease virus (IBDV)-derived assemblies as VP2 capsids, VPX tubules and polyprotein (PP)-derived mixed structures, were tested. Four-week-old chickens were immunised subcutaneously with one dose of each particulate antigen. VP2 icosahedral capsids induced the highest neutralising response, followed by PP-derived structures and then VPX tubules. All vaccinated animals were protected when challenged with a very virulent IBDV (vvIBDV) isolate, however the degree of protection is directly correlated with the levels of neutralising antibodies. VP2 capsids elicited stronger protective immunity than tubular structures and 3 micrograms of them were sufficient to confer a total protection comparable to that induced by an inactivated vaccine. Therefore, VP2 capsids represent a suitable candidate recombinant vaccine instead of virus-like particles (VLPs) for IBDV infections. Our results also provide clear evidence that the recombinant IBDV-derived antigens are structure-dependent in order to be efficient as vaccine components. PMID:12804866

  17. Structure-dependent efficacy of infectious bursal disease virus (IBDV) recombinant vaccines.

    PubMed

    Martinez-Torrecuadrada, Jorge L; Saubi, Narcis; Pagès-Manté, Albert; Castón, José R; Espuña, Enric; Casal, J Ignacio

    2003-05-16

    The immunogenicity and protective capability of several baculovirus-expressed infectious bursal disease virus (IBDV)-derived assemblies as VP2 capsids, VPX tubules and polyprotein (PP)-derived mixed structures, were tested. Four-week-old chickens were immunised subcutaneously with one dose of each particulate antigen. VP2 icosahedral capsids induced the highest neutralising response, followed by PP-derived structures and then VPX tubules. All vaccinated animals were protected when challenged with a very virulent IBDV (vvIBDV) isolate, however the degree of protection is directly correlated with the levels of neutralising antibodies. VP2 capsids elicited stronger protective immunity than tubular structures and 3& mgr;g of them were sufficient to confer a total protection comparable to that induced by an inactivated vaccine. Therefore, VP2 capsids represent a suitable candidate recombinant vaccine instead of virus-like particles (VLPs) for IBDV infections. Our results also provide clear evidence that the recombinant IBDV-derived antigens are structure-dependent in order to be efficient as vaccine components. PMID:12706682

  18. Localised Infection of Atlantic Salmon Epithelial Cells by HPR0 Infectious Salmon Anaemia Virus

    PubMed Central

    Aamelfot, Maria; Christiansen, Debes H.; Dale, Ole Bendik; McBeath, Alastair; Benestad, Sylvie L.; Falk, Knut

    2016-01-01

    Infectious salmon anaemia (ISA) is an important, systemic viral disease of farmed Atlantic salmon, Salmo salar L. Endothelial cells are the main target cells for highly virulent HPR-deleted ISA virus (ISAV) types. Here we examine the pathogenesis of non-virulent ISAV HPR0 infections, presenting evidence of an epithelial tropism for this virus type, including actual infection and replication in the epithelial cells. Whereas all HPR0 RT-qPCR positive gills prepared for cryosection tested positive by immunohistochemistry (IHC) and immunofluorescent labelling, only 21% of HPR0 RT-qPCR positive formalin-fixed paraffin-embedded gills were IHC positive, suggesting different methodological sensitivities. Only specific epithelial cell staining was observed and no staining was observed in endothelial cells of positive gills. Furthermore, using an ISAV segment 7 RT-PCR assay, we demonstrated splicing of HPR0, suggesting initial activation of the replication machinery in the epithelial gill cells. Immunological responses were investigated by the expression of interferon-related genes (e.g. Mx and γIP) and by ELISA for presence of anti-ISAV antibodies on samples taken sequentially over several months during an episode of transient HPR0 infection. All fish revealed a variable, but increased expression of the immunological markers in comparison to normal healthy fish. Taken together, we conclude that HPR0 causes a localized epithelial infection of Atlantic salmon. PMID:26999815

  19. Fish DNA vaccine against infectious hematopoietic necrosis virus: efficacy of various routes of immunization

    USGS Publications Warehouse

    Corbeil, Serge; Kurath, Gael; LaPatra, Scott E.

    2000-01-01

    The DNA vaccine, pIHNVw-G, contains the gene for the glycoprotein (G) of the rhabdovirus infectious hematopoietic necrosis virus (IHNV), a major pathogen of salmon and trout. The relative efficacy of various routes of immunisation with pIHNVw-G was evaluated using 1.8 g rainbow trout fry vaccinated via intramuscular injection, scarification of the skin, intraperitoneal injection, intrabuccal administration, cutaneous particle bombardment using a gene gun, or immersion in water containing DNA vaccine-coated beads. Twenty-seven days after vaccination neutralising antibody titres were determined, and 2 days later groups of vaccinated and control unvaccinated fish were subjected to an IHNV immersion challenge. Results of the virus challenge showed that the intramuscular injection and the gene gun immunisation induced protective immunity in fry, while intraperitoneal injection provided partial protection. Neutralising antibodies were not detected in sera of vaccinated fish regardless of the route of immunisation used, suggesting that cell mediated immunity may be at least partially responsible for the observed protection.

  20. [Rescue of the recombinant infectious bronchitis virus with the ectodomain region of H120 spike glycoprotein].

    PubMed

    Wei, Yan-quan; Guo, Hui-chen; Wang, Hai-ming; Sun, De-hui; Han, Shi-chong; Sun, Shi-qi

    2014-11-01

    To explore the expression potential of heterogeneous genes using the backbone of infectious bronchitis virus (IBV) Beaudette strain, the ectodomain region of the Spike gene (1,302 bp) of IBV H120 strain was amplified by RT-PCR and replaced into the corresponding location of the IBV Beaudette strain full-length cDNA. This recombinant was designated as BeauR-H120(S1). BeauR-H120(S1) was directly used as the DNA template for the transcription of viral genomic RNA in vitro. Then, the transcription product was transfected into Vero cells by electroporation. At 48 h post-transfection, the transfected Vero cells were harvested, and passaging continued. A syncytium was not observed until the recombinant virus had passed through four passages. The presence of rBeau-H120(S1) was verified by the detection of the replaced ectodomain region of the H120 Spike gene using RT-PCR. Western blot analysis of rBeau-H120 (S1)-infected Vero cell lysates demonstrated that the nucleocapsid (N) protein was expressed, which implied that rBeau-H120(S1) could propagate in Vero cells. The TCIDs0 and EIDs0 data demonstrated that the titer levels of rBeau-H120(S1) reached 10(590+/-0.22)TCID50/mL and 10(6.13+/-0.23)EID50/mL in Vero cells and 9-day-old SPF chicken embryos, respectively. Protection studies showed that the percentage of antibody-positive chickens, which were vaccinated with rBeau-H120(S1) at 7 days after hatching, rose to 90% at 21 days post-inoculation. Inoculation provided an 85% rate of immune protection against a challenge of the virulent IBV M41 strain (103EID50/chicken). This recombinant virus constructed using reverse genetic techniques could be further developed as a novel genetic engineering vaccine against infectious bronchitis. PMID:25868282

  1. Experimental co-infection of SPF chickens with low pathogenicity avian influenza virus (LPAIV) subtypes H9N2, H5N2 and H7N9, and infectious bronchitis virus (IBV)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Avian influenza virus (AIV) and infectious bronchitis virus (IBV) are two of the most important respiratory viruses affecting poultry worldwide, but little is known about the effect of co-infection of these two viruses in poultry. Low pathogenicity (LP) AIV can produce from mild to moderate upper r...

  2. Genotyping and pathotyping of diversified strains of infectious bronchitis viruses circulating in Egypt

    PubMed Central

    Zanaty, Ali; Arafa, Abdel-Satar; Hagag, Naglaa; El-Kady, Magdy

    2016-01-01

    AIM To characterize the circulating infectious bronchitis virus (IBV) strains in Egypt depending on the sequence of the spike-1 (S1) gene [hypervariable region-3 (HVR-3)] and to study the pathotypic features of these strains. METHODS In this work, twenty flocks were sampled for IBV detection using RRT-PCR and isolation of IBV in specific pathogen free (SPF) chicks during the period from 2010 to 2015. Partial sequencing and phylogenetic analysis of 400 bp representing the HVR-3 of the S1 gene was conducted. Pathotypic characterization of one selected virus from each group (Egy/Var-I, Egy/Var-II and classic) was evaluated in one day old SPF chicks. The chicks were divided into 4 groups 10 birds each including the negative control group. Birds were inoculated at one day by intranasal instillation of 105EID50/100 μL of IBV viruses [IBV-EG/1212B-2012 (Egy/Var-II), IBV/EG/IBV1-2011 (Egy/Var-I) and IBV-EG/11539F-2011 (classic)], while the remaining negative control group was kept uninfected. The birds were observed for clinical signs, gross lesions and virus pathogenicity. The real-time rRT-PCR test was performed for virus detection in the tissues. Histopathological examinations were evaluated in both trachea and kidneys. RESULTS The results revealed that these viruses were separated into two distinct groups; variant (GI-23) and classic (GI-1), where 16 viruses belonged to a variant group, including 2 subdivisions [Egy/Var-I (6 isolates) and Egy/Var-II (10 isolates)] and 4 viruses clustered to the classic group (Mass-like). IBV isolates in the variant group were grouped with other IBV strains from the Middle East. The variant subgroup (Egy/Var-I) was likely resembling the original Egyptian variant strain (Egypt/Beni-Suif/01) and the Israeli strain (IS/1494/2006). The second subgroup (Egy/Var-II) included the viruses circulating in the Middle East (Ck/EG/BSU-2 and Ck/EG/BSU-3/2011) and the Israeli strain (IS/885/00). The two variant subgroups (Egy/Var-I and Egy

  3. Genetic diversity and epidemiology of infectious hematopoietic necrosis virus in Alaska

    USGS Publications Warehouse

    Emmenegger, E.G; Meyers, T.R.; Burton, T.O.; Kurath, G.

    2000-01-01

    Forty-two infectious hematopoietic necrosis virus (IHNV) isolates from Alaska were analyzed using the ribonuclease protection assay (RPA) and nucleotide sequencing. RPA analyses, utilizing 4 probes, N5, N3 (N gene), GF (G gene), and NV (NV gene), determined that the haplotypes of all 3 genes demonstrated a consistent spatial pattern. Virus isolates belonging to the most common haplotype groups were distributed throughout Alaska, whereas isolates in small haplotype groups were obtained from only 1 site (hatchery, lake, etc.). The temporal pattern of the GF haplotypes suggested a 'genetic acclimation' of the G gene, possibly due to positive selection on the glycoprotein. A pairwise comparison of the sequence data determined that the maximum nucleotide diversity of the isolates was 2.75% (10 mismatches) for the NV gene, and 1.99% (6 mismatches) for a 301 base pair region of the G gene, indicating that the genetic diversity of IHNV within Alaska is notably lower than in the more southern portions of the IHNV North American range. Phylogenetic analysis of representative Alaskan sequences and sequences of 12 previously characterized IHNV strains from Washington, Oregon, Idaho, California (USA) and British Columbia (Canada) distinguished the isolates into clusters that correlated with geographic origin and indicated that the Alaskan and British Columbia isolates may have a common viral ancestral lineage. Comparisons of multiple isolates from the same site provided epidemiological insights into viral transmission patterns and indicated that viral evolution, viral introduction, and genetic stasis were the mechanisms involved with IHN virus population dynamics in Alaska. The examples of genetic stasis and the overall low sequence heterogeneity of the Alaskan isolates suggested that they are evolutionarily constrained. This study establishes a baseline of genetic fingerprint patterns and sequence groups representing the genetic diversity of Alaskan IHNV isolates. This

  4. Both genome segments contribute to the pathogenicity of very virulent infectious bursal disease virus.

    PubMed

    Escaffre, Olivier; Le Nouën, Cyril; Amelot, Michel; Ambroggio, Xavier; Ogden, Kristen M; Guionie, Olivier; Toquin, Didier; Müller, Hermann; Islam, Mohammed R; Eterradossi, Nicolas

    2013-03-01

    Infectious bursal disease virus (IBDV) causes an economically significant disease of chickens worldwide. Very virulent IBDV (vvIBDV) strains have emerged and induce as much as 60% mortality. The molecular basis for vvIBDV pathogenicity is not understood, and the relative contributions of the two genome segments, A and B, to this phenomenon are not known. Isolate 94432 has been shown previously to be genetically related to vvIBDVs but exhibits atypical antigenicity and does not cause mortality. Here the full-length genome of 94432 was determined, and a reverse genetics system was established. The molecular clone was rescued and exhibited the same antigenicity and reduced pathogenicity as isolate 94432. Genetically modified viruses derived from 94432, whose vvIBDV consensus nucleotide sequence was restored in segment A and/or B, were produced, and their pathogenicity was assessed in specific-pathogen-free chickens. We found that a valine (position 321) that modifies the most exposed part of the capsid protein VP2 critically modified the antigenicity and partially reduced the pathogenicity of 94432. However, a threonine (position 276) located in the finger domain of the virus polymerase (VP1) contributed even more significantly to attenuation. This threonine is partially exposed in a hydrophobic groove on the VP1 surface, suggesting possible interactions between VP1 and another, as yet unidentified molecule at this amino acid position. The restored vvIBDV-like pathogenicity was associated with increased replication and lesions in the thymus and spleen. These results demonstrate that both genome segments influence vvIBDV pathogenicity and may provide new targets for the attenuation of vvIBDVs. PMID:23269788

  5. 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.

  6. Genetic characterization of infectious hematopoietic necrosis virus of coastal salmonid stocks in Washington State

    USGS Publications Warehouse

    Emmenegger, E.J.; Kurath, G.

    2002-01-01

    Infectious hematopoietic necrosis virus (IHNV) is a pathogen that infects many Pacific salmonid stocks from the watersheds of North America. Previous studies have thoroughly characterized the genetic diversity of IHNV isolates from Alaska and the Hagerman Valley in Idaho. To enhance understanding of the evolution and viral transmission patterns of IHNV within the Pacific Northwest geographic range, we analyzed the G gene of IHNV isolates from the coastal watersheds of Washington State by ribonuclease protection assay (RPA) and nucleotide sequencing. The RPA analysis of 23 isolates indicated that the Skagit basin IHNV isolates were relatively homogeneous as a result of the dominance of one G gene haplotype (S). Sequence analysis of 303 bases in the middle of the G gene (midG region) of 61 isolates confirmed the high frequency of a Skagit River basin sequence and identified another sequence commonly found in isolates from the Lake Washington basin. Overall, both the RPA and sequence analysis showed that the Washington coastal IHNV isolates are genetically homogeneous and have little genetic diversity. This is similar to the genetic diversity pattern of IHNV from Alaska and contrasts sharply with the high genetic diversity demonstrated for IHNV isolates from fish farms along the Snake River in Idaho. The high degree of sequence and haplotype similarity between the Washington coastal IHNV isolates and those from Alaska and British Columbia suggests that they have a common viral ancestor. Phylogenetic analyses of the isolates we studied and those from different regions throughout the virus's geographic range confirms a conserved pattern of evolution of the virus in salmonid stocks north of the Columbia River, which forms Washington's southern border.

  7. Replication and transmission of live attenuated infectious laryngotracheitis virus (ILTV) vaccines.

    PubMed

    Rodríguez-Avila, Andrés; Oldoni, Ivomar; Riblet, Sylva; García, Maricarmen

    2007-12-01

    The aim of this study was to evaluate the replication of live attenuated infectious laryngotracheitis virus vaccines in selected tissues and their ability to transmit to contact-exposed birds. Four-week-old specific-pathogen-free chickens were eye drop-inoculated with tissue culture origin (TCO) and chicken embryo origin (CEO) vaccines. Contact-exposed chickens were housed in direct contact with eye drop-inoculated chickens from the first day postinoculation. Virus isolation and real-time polymerase chain reaction were used to detect the presence of live virus and viral DNA, respectively, in the trachea, trigeminal ganglia, eye conjunctiva, cecal tonsils, and cloaca from eye drop-inoculated and contact-exposed birds at days 2, 4, 5 to 10, 14, 18, 21, 24, and 28 postinoculation. No differences were observed in the ability of the TCO and CEO vaccines to replicate in the examined tissues. Both vaccines presented a localized replication in the eye conjunctiva and the trachea. Both vaccines were capable of transmitting to contact-exposed birds, attaining peaks of viral DNA as elevated as those observed in inoculated birds. The CEO vaccine replicated faster and reached higher viral genome copy number than the TCO vaccine in the conjunctiva and trachea of eye drop-inoculated and contact-exposed birds. The viral DNA from both vaccines migrated to the trigeminal ganglia during early stages of infection. Although the CEO and TCO vaccines were not recovered from the cecal tonsils and the cloaca, low levels of viral DNA were detected at these sites during the peak of viral replication in the upper respiratory tract. PMID:18251401

  8. The Maturation Process of pVP2 Requires Assembly of Infectious Bursal Disease Virus Capsids

    PubMed Central

    Chevalier, Christophe; Lepault, Jean; Erk, Inge; Da Costa, Bruno; Delmas, Bernard

    2002-01-01

    Infectious bursal disease virus (IBDV) is a nonenveloped avian virus with a two-segment double-stranded RNA genome. Its T=13 icosahedral capsid is most probably assembled with 780 subunits of VP2 and 600 copies of VP3 and has a diameter of about 60 nm. VP1, the RNA-dependent RNA polymerase, resides inside the viral particle. Using a baculovirus expression system, we first observed that expression of the pVP2-VP4-VP3 polyprotein encoded by the genomic segment IBDA results mainly in the formation of tubules with a diameter of about 50 nm and composed of pVP2, the precursor of VP2. Very few virus-like particles (VLPs) and VP4 tubules with a diameter of about 25 nm were also identified. The inefficiency of VLP assembly was further investigated by expression of additional IBDA-derived constructs. Expression of pVP2 without any other polyprotein components results in the formation of isometric particles with a diameter of about 30 nm. VLPs were observed mainly when a large exogeneous polypeptide sequence (the green fluorescent protein sequence) was fused to the VP3 C-terminal domain. Large numbers of VLPs were visualized by electron microscopy, and single particles were shown to be fluorescent by standard and confocal microscopy analysis. Moreover, the final maturation process converting pVP2 into the VP2 mature form was observed on generated VLPs. We therefore conclude that the correct scaffolding of the VP3 can be artificially induced to promote the formation of VLPs and that the final processing of pVP2 to VP2 is controlled by this particular assembly. To our knowledge, this is the first report of the engineering of a morphogenesis switch to control a particular type of capsid protein assembly. PMID:11836416

  9. Both Genome Segments Contribute to the Pathogenicity of Very Virulent Infectious Bursal Disease Virus

    PubMed Central

    Escaffre, Olivier; Le Nouën, Cyril; Amelot, Michel; Ambroggio, Xavier; Ogden, Kristen M.; Guionie, Olivier; Toquin, Didier; Müller, Hermann; Islam, Mohammed R.

    2013-01-01

    Infectious bursal disease virus (IBDV) causes an economically significant disease of chickens worldwide. Very virulent IBDV (vvIBDV) strains have emerged and induce as much as 60% mortality. The molecular basis for vvIBDV pathogenicity is not understood, and the relative contributions of the two genome segments, A and B, to this phenomenon are not known. Isolate 94432 has been shown previously to be genetically related to vvIBDVs but exhibits atypical antigenicity and does not cause mortality. Here the full-length genome of 94432 was determined, and a reverse genetics system was established. The molecular clone was rescued and exhibited the same antigenicity and reduced pathogenicity as isolate 94432. Genetically modified viruses derived from 94432, whose vvIBDV consensus nucleotide sequence was restored in segment A and/or B, were produced, and their pathogenicity was assessed in specific-pathogen-free chickens. We found that a valine (position 321) that modifies the most exposed part of the capsid protein VP2 critically modified the antigenicity and partially reduced the pathogenicity of 94432. However, a threonine (position 276) located in the finger domain of the virus polymerase (VP1) contributed even more significantly to attenuation. This threonine is partially exposed in a hydrophobic groove on the VP1 surface, suggesting possible interactions between VP1 and another, as yet unidentified molecule at this amino acid position. The restored vvIBDV-like pathogenicity was associated with increased replication and lesions in the thymus and spleen. These results demonstrate that both genome segments influence vvIBDV pathogenicity and may provide new targets for the attenuation of vvIBDVs. PMID:23269788

  10. Genotyping of infectious bronchitis viruses from broiler farms in Iraq during 2014-2015.

    PubMed

    Seger, Waleed; GhalyanchiLangeroudi, Arash; Karimi, Vahid; Madadgar, Omid; Marandi, Mehdi Vasfi; Hashemzadeh, Masoud

    2016-05-01

    Infectious bronchitis virus (IBV) is one of the most critical pathogens in the poultry industry, causing serious economic losses in all countries including Iraq. IBV has many genotypes that do not confer any cross-protection. This virus has been genotyped by sequence analysis of the S1 glycoprotein gene. A total of 100 tracheal and kidney tissue specimens from different commercial broiler flocks in the middle and south of Iraq were collected from September 2013 to September 2014. Thirty-two IBV-positive samples were selected from among the total and were further characterized by nested PCR. Phylogenetic analysis revealed that isolates belong to four groups (group I, variant 2 [IS/1494-like]; group II, 793/B-like; group III, QX-like; group IV, DY12-2-like). Sequence analysis revealed nucleotide sequence identities within groups I, II, and III of 99.68 %-100 %, 99.36 %-100 %, and 96.42 %-100 %, respectively. Group I (variant 2) was the dominant IBV genotype. One Chinese-like recombinant virus (DY12-2-like) that had not been reported in the Middle East was detected. In addition, the presence of QX on broiler chicken farms in the area studied was confirmed. This is the first comprehensive study on the genotyping of IBV in Iraq with useful information regarding the molecular epidemiology of IBV. The phylogenetic relationship of the strains with respect to different time sequences and geographical regions displayed complexity and diversity. Further studies are needed and should include the isolation and full-length molecular characterization of IBV in this region. PMID:26887967

  11. Structural View and Substrate Specificity of Papain-like Protease from Avian Infectious Bronchitis Virus*

    PubMed Central

    Kong, Lingying; Shaw, Neil; Yan, Lingming; Lou, Zhiyong; Rao, Zihe

    2015-01-01

    Papain-like protease (PLpro) of coronaviruses (CoVs) carries out proteolytic maturation of non-structural proteins that play a role in replication of the virus and performs deubiquitination of host cell factors to scuttle antiviral responses. Avian infectious bronchitis virus (IBV), the causative agent of bronchitis in chicken that results in huge economic losses every year in the poultry industry globally, encodes a PLpro. The substrate specificities of this PLpro are not clearly understood. Here, we show that IBV PLpro can degrade Lys48- and Lys63-linked polyubiquitin chains to monoubiquitin but not linear polyubiquitin. To explain the substrate specificities, we have solved the crystal structure of PLpro from IBV at 2.15-Å resolution. The overall structure is reminiscent of the structure of severe acute respiratory syndrome CoV PLpro. However, unlike the severe acute respiratory syndrome CoV PLpro that lacks blocking loop (BL) 1 of deubiquitinating enzymes, the IBV PLpro has a short BL1-like loop. Access to a conserved catalytic triad consisting of Cys101, His264, and Asp275 is regulated by the flexible BL2. A model of ubiquitin-bound IBV CoV PLpro brings out key differences in substrate binding sites of PLpros. In particular, P3 and P4 subsites as well as residues interacting with the β-barrel of ubiquitin are different, suggesting different catalytic efficiencies and substrate specificities. We show that IBV PLpro cleaves peptide substrates KKAG-7-amino-4-methylcoumarin and LRGG-7-amino-4-methylcoumarin with different catalytic efficiencies. These results demonstrate that substrate specificities of IBV PLpro are different from other PLpros and that IBV PLpro might target different ubiquitinated host factors to aid the propagation of the virus. PMID:25609249

  12. Recombinant infectious bronchitis virus (IBV) H120 vaccine strain expressing the hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) protects chickens against IBV and NDV challenge.

    PubMed

    Yang, Xin; Zhou, Yingshun; Li, Jianan; Fu, Li; Ji, Gaosheng; Zeng, Fanya; Zhou, Long; Gao, Wenqian; Wang, Hongning

    2016-05-01

    Infectious bronchitis (IB) and Newcastle disease (ND) are common viral diseases of chickens, which are caused by infectious bronchitis virus (IBV) and Newcastle disease virus (NDV), respectively. Vaccination with live attenuated strains of IBV-H120 and NDV-LaSota are important for the control of IB and ND. However, conventional live attenuated vaccines are expensive and result in the inability to differentiate between infected and vaccinated chickens. Therefore, there is an urgent need to develop new efficacious vaccines. In this study, using a previously established reverse genetics system, we generated a recombinant IBV virus based on the IBV H120 vaccine strain expressing the haemagglutinin-neuraminidase (HN) protein of NDV. The recombinant virus, R-H120-HN/5a, exhibited growth dynamics, pathogenicity and viral titers that were similar to those of the parental IBV H120, but it had acquired hemagglutination activity from NDV. Vaccination of SPF chickens with the R-H120-HN/5a virus induced a humoral response at a level comparable to that of the LaSota/H120 commercial bivalent vaccine and provided significant protection against challenge with virulent IBV and NDV. In summary, the results of this study indicate that the IBV H120 strain could serve as an effective tool for designing vaccines against IB and other infectious diseases, and the generation of IBV R-H120-HN/5a provides a solid foundation for the development of an effective bivalent vaccine against IBV and NDV. PMID:26873815

  13. DEVELOPMENT AND EVALUATION OF A REAL-TIME TAQMAN RT-PCR ASSAY FOR THE DETECTION OF INFECTIOUS BRONCHITIS VIRUS FROM INFECTED CHICKENS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    It is important to rapidly differentiate infectious bronchitis virus (IBV) from disease agents like highly pathogenic avian influenza virus and exotic Newcastle disease virus, because those diseases can be extremely similar in the early stages of their pathogenicity. In this study, we report the dev...

  14. Virus-like particles of hepatitis B virus core protein containing five mimotopes of infectious bursal disease virus (IBDV) protect chickens against IBDV.

    PubMed

    Wang, Yong-shan; Ouyang, Wei; Liu, Xiao-juan; He, Kong-wang; Yu, Sheng-qing; Zhang, Hai-bin; Fan, Hong-jie; Lu, Cheng-ping

    2012-03-01

    Current infectious bursal disease virus (IBDV) vaccines suffer from maternal antibody interference and mimotope vaccines might be an alternative. Previously we demonstrated an IBDV VP2 five-mimotope polypeptide, 5EPIS, elicited protective immunity in chickens. In the current study, the 5epis gene was inserted into a plasmid carrying human hepatitis B virus core protein (HBc) gene at its major immunodominant region site. The recombinant gene was efficiently expressed in Escherichia coli to produce chimeric protein HBc-5EPIS which self-assembles to virus-like particles (VLP). Two-week old specific-pathogen-free chickens were immunized intramuscularly with HBc-5EPIS VLP or 5EPIS polypeptide without adjuvant (50 μg/injection) on day 0, 7, 14 and 21. Anti-5EPIS antibody was first detected on day 7 and day 21 in HBc-5EPIS and 5EPIS groups, respectively; on day 28, anti-5EPIS titers reached 12,800 or 1600 by ELISA, and 3200 or 800 by virus neutralization assay in HBc-5EPIS and 5EPIS groups, respectively. No anti-5EPIS antibody was detected in the buffer control group throughout the experiment. Challenge on day 28 with a virulent IBDV strain (GX8/99) resulted in 100%, 40.0% and 26.7% survival for chickens immunized with HBc-5EPIS, 5EPIS and buffer, respectively. These data suggest epitope presentation on chimeric VLP is a promising approach for improving mimotope vaccines for IBDV. PMID:22285269

  15. Real-time cell analysis--a new method for dynamic, quantitative measurement of infectious viruses and antiserum neutralizing activity.

    PubMed

    Teng, Zheng; Kuang, Xiaozhou; Wang, Jiayu; Zhang, Xi

    2013-11-01

    A newly developed electronic cell sensor array--the xCELLigence real-time cell analysis (RTCA) system is tested currently for dynamic monitoring of cell attachment, proliferation, damage, and death. In this study, human enterovirus (HEV71) infection of human rhabdomyosarcoma (RD) was used as an in vitro model to validate the application of this novel system as a straightforward and efficient assay for quantitative measurement of infectious viruses based on virus-induced cytopathic effect (CPE). Several experimental tests were performed including the determination of optimal seeding density of the RD cells in 96-well E-plates, RTCA real-time monitoring of the virus induced CPE and virus titer calculation, and viral neutralization test to determine HEV71 antibody titer. Traditional 50% tissue culture infective dose (TCID50) assay was also conducted for methodology comparison and validation, which indicated a consistent result between the two assays. These findings indicate that the xCELLigence RTCA system can be a valuable addition to current viral assays for quantitative measurement of infectious viruses and quantitation of neutralization antibody titer in real-time, warranting for future research and exploration of applications to many other animal and human viruses. PMID:23835032

  16. Retention of infectious haematopoietic necrosis virus infectivity in fish tissue homogenates and fluids stored at three temperatures.

    USGS Publications Warehouse

    Burke, J.; Mulcahy, D.

    1983-01-01

    Pools of brain, kidney, spleen, liver and gut tissues from several rainbow trout, Salmo gairdneri Richardson, and whole sockeye salmon, Oncorhynchus nerka (Walbaum), fry were homogenized with a known amount of infectious haematopoietic necrosis virus (IHNV). Virus was also added to ovarian fluids and sera pooled from several rainbow trout. The plaque assay was used to determine the retention of IHNV infectivity after different storage periods at 20°C, 4°C and —20°C. The work was used to evaluate homogenization as a remote field treatment of IHNV samples before shipment to the laboratory. Maintenance of viral infectivity varied widely among different homogenates and fluids. For short-term storage, 4°C was generally the most efficient temperature for preserving infectious virus in ovarian fluids, Sera and homogenates of eggs, spleen, whole fry and brain, while infectivity was most efficiently preserved in kidney and liver homogenates by storage at −20°C. Infectious virus was not detected in any sample stored for one year at −20°C. Variations in retention of viral infectivity make homogenization of samples in the field followed by transfer to the laboratory unacceptable.

  17. Genetic variation underlying resistance to infectious hematopoietic necrosis virus in a steelhead trout (Oncorhynchus mykiss) population

    USGS Publications Warehouse

    Brieuc, Marine S. O.; Purcell, Maureen K.; Palmer, Alexander D.; Naish, Kerry A.

    2015-01-01

    Understanding the mechanisms of host resistance to pathogens will allow insights into the response of wild populations to the emergence of new pathogens. Infectious hematopoietic necrosis virus (IHNV) is endemic to the Pacific Northwest and infectious to Pacific salmon and trout (Oncorhynchus spp.). Emergence of the M genogroup of IHNV in steelhead trout O. mykiss in the coastal streams of Washington State, between 2007 and 2011, was geographically heterogeneous. Differences in host resistance due to genetic change were hypothesized to be a factor influencing the IHNV emergence patterns. For example, juvenile steelhead trout losses at the Quinault National Fish Hatchery (QNFH) were much lower than those at a nearby facility that cultures a stock originally derived from the same source population. Using a classical quantitative genetic approach, we determined the potential for the QNFH steelhead trout population to respond to selection caused by the pathogen, by estimating the heritability for 2 traits indicative of IHNV resistance, mortality (h2 = 0.377 (0.226 - 0.550)) and days to death (h2 = 0.093 (0.018 - 0.203)). These results confirm that there is a genetic basis for resistance and that this population has the potential to adapt to IHNV. Additionally, genetic correlation between days to death and fish length suggests a correlated response in these traits to selection. Reduction of genetic variation, as well as the presence or absence of resistant alleles, could affect the ability of populations to adapt to the pathogen. Identification of the genetic basis for IHNV resistance could allow the assessment of the susceptibility of other steelhead populations.

  18. Genotyping of infectious laryngotracheitis virus using allelic variations from multiple genomic regions.

    PubMed

    Choi, Eun-Jung; La, Tae-Min; Choi, In-Soo; Song, Chang-Seon; Park, Seung-Yong; Lee, Joong-Bok; Lee, Sang-Won

    2016-08-01

    Live attenuated vaccines are extensively used worldwide to control the outbreak of infectious laryngotracheitis. Virulent field strains showing close genetic relationship with the infectious laryngotracheitis virus (ILTV) vaccines of chicken embryo origin have been detected in the poultry industry. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis, a reliable molecular epidemiological method, of multiple genomic regions was performed. The PCR-RFLP is a time-consuming method that requires considerable amount of intact viral genomic DNA to amplify genomic regions greater than 4 kb. In this study, six variable genomic regions were selected and amplified for sequencing. The multi-allelic PCR-sequence genotyping showed better discrimination power than that of previous PCR-sequencing schemes using single or two target regions. The allelic variation patterns yielded 16 strains of ILTV classified into 14 different genotypes. Three Korean field strains, 550/05/Ko, 0010/05/Ko and 40032/08/Ko, were found to have the same genotype as the commercial vaccine strain, Laryngo Vac (Zoetis, Florham Park, NJ, USA). Three other Korean field strains, 40798/10/Ko, 12/07/Ko, and 30678/14/Ko, showed recombined allelic patterns. The multi-allelic PCR-sequencing method was proved to be an efficient and practical procedure to classify the different strains of ILTV. The method could serve as an alternate diagnostic and differentiating tool for the classification of ILTV, and contribute to understanding of the epidemiology of the disease at a global level. PMID:26956802

  19. Complete Genome Sequence of the First Chinese Virulent Infectious Laryngotracheitis Virus

    PubMed Central

    Cui, Xianlan; Zhang, Xiaomin; Cui, Hongyu; Xue, Mei; Wang, Yunfeng

    2013-01-01

    Infectious laryngotracheitis (ILT) is an acute respiratory disease caused by infectious laryngotracheitis virus (ILTV). The complete genome sequences of five attenuated ILTV vaccine strains and six virulent ILTV strains as well as two Australian ILTV field strains have been published in Australia and the USA so far. To provide the complete genome sequence information of ILTVs from different geographic regions, the whole genome of ILTV LJS09 isolated in China was sequenced. The genome of ILTV LJS09 was 153,201 bp in length, and contained 79 ORFs. Most of the ORFs had high sequence identity with homologous ORFs of reference strains. There was a large fragment deletion within the noncoding region of unique long region (UL) of ILTV LJS09 compared with SA2 and A20 strains. Though the origin binding protein of ILTV LJS09 existed, there was no AT-rich region in strain LJS09. Alignments of the amino acid sequences revealed seven mutations at amino acids 71 (Arg → Lys), 116 (Ala → Val), 207 (Thr → Ile) and 644 (Thr → Ile) on glycoprotein B, 155 (Phe → Ser) and 376 (Arg → His) on glycoprotein D and 8 (Gln→Pro) on glycoprotein L of ILTV LJS09 compared to those of virulent strain (USDA) as ILTV LJS09 did not grow on chicken embryo fibroblasts, suggesting the role of the key seven amino acids in determination of the cell tropism of ILTV LJS09. This is the first complete genome sequence of the virulent strain of ILTV in Asia using the conventional PCR method, which will help to facilitate the future molecular biological research of ILTVs. PMID:23922947

  20. Complete genome sequence of the first Chinese virulent infectious laryngotracheitis virus.

    PubMed

    Kong, Congcong; Zhao, Yan; Cui, Xianlan; Zhang, Xiaomin; Cui, Hongyu; Xue, Mei; Wang, Yunfeng

    2013-01-01

    Infectious laryngotracheitis (ILT) is an acute respiratory disease caused by infectious laryngotracheitis virus (ILTV). The complete genome sequences of five attenuated ILTV vaccine strains and six virulent ILTV strains as well as two Australian ILTV field strains have been published in Australia and the USA so far. To provide the complete genome sequence information of ILTVs from different geographic regions, the whole genome of ILTV LJS09 isolated in China was sequenced. The genome of ILTV LJS09 was 153,201 bp in length, and contained 79 ORFs. Most of the ORFs had high sequence identity with homologous ORFs of reference strains. There was a large fragment deletion within the noncoding region of unique long region (UL) of ILTV LJS09 compared with SA2 and A20 strains. Though the origin binding protein of ILTV LJS09 existed, there was no AT-rich region in strain LJS09. Alignments of the amino acid sequences revealed seven mutations at amino acids 71 (Arg → Lys), 116 (Ala → Val), 207 (Thr → Ile) and 644 (Thr → Ile) on glycoprotein B, 155 (Phe → Ser) and 376 (Arg → His) on glycoprotein D and 8 (Gln→Pro) on glycoprotein L of ILTV LJS09 compared to those of virulent strain (USDA) as ILTV LJS09 did not grow on chicken embryo fibroblasts, suggesting the role of the key seven amino acids in determination of the cell tropism of ILTV LJS09. This is the first complete genome sequence of the virulent strain of ILTV in Asia using the conventional PCR method, which will help to facilitate the future molecular biological research of ILTVs. PMID:23922947

  1. Genetic and serological typing of European infectious haematopoietic necrosis virus (IHNV) isolates

    USGS Publications Warehouse

    Johansson, T.; Einer-Jensen, K.; Batts, W.; Ahrens, P.; Bjorkblom, C.; Kurath, G.; Bjorklund, H.; Lorenzen, N.

    2009-01-01

    Infectious haematopoietic necrosis virus (IHNV) causes the lethal disease infectious haematopoietic necrosis (IHN) in juvenile salmon and trout. The nucleocapsid (N) protein gene and partial glycoprotein (G) gene (nucleotides 457 to 1061) of the European isolates IT-217A, FR-32/87, DE-DF 13/98 11621, DE-DF 4/99-8/99, AU-9695338 and RU-FR1 were sequenced and compared with IHNV isolates from the North American genogroups U, M and L. In phylogenetic studies the N gene of the Italian, French, German and Austrian isolates clustered in the M genogroup, though in a different subgroup than the isolates from the USA. Analyses of the partial G gene of these European isolates clustered them in the M genogroup close to the root while the Russian isolate clustered in the U genogroup. The European isolates together with US-WRAC and US-Col-80 were also tested in an enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies (MAbs) against the N protein. MAbs 136-1 and 136-3 reacted equally at all concentrations with the isolates tested, indicating that these antibodies identify a common epitope. MAb 34D3 separated the M and L genogroup isolates from the U genogroup isolate. MAb 1DW14D divided the European isolates into 2 groups. MAb 1DW14D reacted more strongly with DE-DF 13/98 11621 and RU-FR1 than with IT-217A, FR- 32/87, DE-DF 4/99-8/99 and AU-9695338. In the phylogenetic studies, the Italian, French, German and Austrian isolates clustered in the M genogroup, whereas in the serological studies using MAbs, the European M genogroup isolates could not be placed in the same specific group. These results indicate that genotypic and serotypic classification do not correlate. ?? 2009 Inter-Research.

  2. Molecular characterization of chicken infectious anemia virus circulating in Argentina during 2007.

    PubMed

    Craig, M I; Rimondi, A; Delamer, M; Sansalone, P; König, G; Vagnozzi, A; Pereda, A

    2009-09-01

    Chicken infectious anemia virus (CAV) is a worldwide-distributed infectious agent that affects commercial poultry. Although this agent was first detected in Argentina in 1994, no further studies on CAV in this country were reported after that. The recent increased occurrence of clinical cases of immunosuppression that could be caused by CAV has prompted this study. Our results confirmed that CAV is still circulating in commercial flocks in Argentina. Phylogenetic analysis focusing on the VP1 nucleotide sequence showed that all Argentinean isolates grouped together in a cluster, sharing a high similarity (> 97%) with genotype B reference strains. However, Argentinean isolates were distantly related to other strains commonly used for vaccination in this country, such as Del-Ros and Cux-1. Sequence analysis of predicted VP1 peptides showed that most of the Argentinean isolates have a glutamine residue at positions 139 and 144, suggesting that these isolates might have a reduced spread in cell culture compared with Cux-1. In addition, a particular amino acid substitution at position 290 is present in all studied Argentinean isolates, as well as in several VP1 sequences from Malaysia, Australia, and Japan isolates. Our results indicate that it is possible to typify CAV strains by comparison of VPI nucleotide sequences alone because the same tree topology was obtained when using the whole genome sequence. The molecular analysis of native strains sheds light into the epidemiology of CAV in Argentinean flocks. In addition, this analysis could be considered in future control strategies focused not only on breeders but on broilers and layer flocks. PMID:19848068

  3. Emergence and maintenance of infectious salmon anaemia virus (ISAV) in Europe: a new hypothesis.

    PubMed

    Nylund, A; Devold, M; Plarre, H; Isdal, E; Aarseth, M

    2003-08-15

    The present study describes the use of molecular methods in studying infectious salmon anaemia virus (ISAV), an important pathogen of farmed salmon in Norway, Scotland, the Faeroe Islands, Canada, USA and Chile. The nucleotide sequences of the haemagglutinin gene (HA) from 70 ISAV isolates have been analysed for phylogenetic relationship and the average mutation rate of nucleotide substitutions calculated. The isolates constitute 2 major groups, 1 European and 1 North American group. The isolate from Chile is closely related to the North American isolates. The European isolates can be further divided into 3 separate groups reflecting geographical distribution, time of collection, and transmission connected with farming activity. Based on existing information about infectious salmon anaemia (ISA) and new information emerging from the present study, it is hypothesised that: (1) ISAV is maintained in wild populations of trout and salmon in Europe; (2) it is transmitted between wild hosts mainly during their freshwater spawning phase in rivers; (3) wild salmonids, mainly trout, possibly carry benign wild-type ISAV isolates; (4) a change (mutation) in virulence probably results from deletions of amino acid segments from the highly polymorphic region (HPR) of benign wild-type isolates; (5) ISA emerges in farmed Atlantic salmon when mutated isolates are transmitted from wild salmonids or, following mutation of benign isolates, in farmed salmon after transmission from wild salmonids; (6) farming activity is an important factor in transmission of ISAV between farming sites in addition to transmission of ISAV from wild salmonids to farmed salmon; (7) transmission of ISAV from farmed to wild salmonids probably occurs less frequently than transmission from wild to farmed fish due to lower frequency of susceptible wild individuals; (8) the frequency of new outbreaks of ISA in farmed salmon probably reflects natural variation in the prevalence of ISAV in wild populations of

  4. Subclinical Reactivation and Shed of Infectious Varicella Zoster Virus in Saliva of Astronauts

    NASA Technical Reports Server (NTRS)

    Cohrs, Randall J.; Mehta, Satish K.; Schmid, D. Scott; Gilden, Donald H.; Pierson, Duane L.

    2007-01-01

    were found to be the European genotype which also contained a rare MspI restriction enodnuclease site in VZV ORF62 at position 107,252. These findings extend our previous demonstration of VZV DNA in saliva of astronauts by showing that infectious VZV is also present. Thus, like HSV-1 and HSV-2, VZV can reactivate and shed infectious virus in the absence of clinical disease.

  5. Genetic analysis of heptad-repeat regions in the G2 fusion subunit of the Junin arenavirus envelope glycoprotein

    SciTech Connect

    York, Joanne . E-mail: joanne.york@umontana.edu; Agnihothram, Sudhakar S. . E-mail: sudhakar.agnihothram@umontana.edu; Romanowski, Victor . E-mail: victor@biol.unlp.edu.ar; Nunberg, Jack H. . E-mail: jack.nunberg@umontana.edu

    2005-12-20

    The G2 fusion subunit of the Junin virus envelope glycoprotein GP-C contains two hydrophobic heptad-repeat regions that are postulated to form a six-helix bundle structure required for the membrane fusion activity of Class I viral fusion proteins. We have investigated the role of these heptad-repeat regions and, specifically, the importance of the putative interhelical a and d position sidechains by using alanine-scanning mutagenesis. All the mutant glycoproteins were expressed and transported to the cell surface. Proteolytic maturation at the subtilisin kexin isozyme-1/site-1-protease (SKI-1/S1P) cleavage site was observed in all but two of the mutants. Among the adequately cleaved mutant glycoproteins, four positions in the N-terminal region (I333, L336, L347 and L350) and two positions in the C-terminal region (R392 and W395) were shown to be important determinants of cell-cell fusion. Taken together, our results indicate that {alpha}-helical coiled-coil structures are likely critical in promoting arenavirus membrane fusion. These findings support the inclusion of the arenavirus GP-C among the Class I viral fusion proteins and suggest pharmacologic and immunologic strategies for targeting arenavirus infection and hemorrhagic fever.

  6. Restriction of Equine Infectious Anemia Virus by Equine APOBEC3 Cytidine Deaminases ▿ †

    PubMed Central

    Zielonka, Jörg; Bravo, Ignacio G.; Marino, Daniela; Conrad, Elea; Perković, Mario; Battenberg, Marion; Cichutek, Klaus; Münk, Carsten

    2009-01-01

    The mammalian APOBEC3 (A3) proteins comprise a multigene family of cytidine deaminases that act as potent inhibitors of retroviruses and retrotransposons. The A3 locus on the chromosome 28 of the horse genome contains multiple A3 genes: two copies of A3Z1, five copies of A3Z2, and a single copy of A3Z3, indicating a complex evolution of multiple gene duplications. We have cloned and analyzed for expression the different equine A3 genes and examined as well the subcellular distribution of the corresponding proteins. Additionally, we have tested the functional antiretroviral activity of the equine and of several of the human and nonprimate A3 proteins against the Equine infectious anemia virus (EIAV), the Simian immunodeficiency virus (SIV), and the Adeno-associated virus type 2 (AAV-2). Hematopoietic cells of horses express at least five different A3s: A3Z1b, A3Z2a-Z2b, A3Z2c-Z2d, A3Z2e, and A3Z3, whereas circulating macrophages, the natural target of EIAV, express only part of the A3 repertoire. The five A3Z2 tandem copies arose after three consecutive, recent duplication events in the horse lineage, after the split between Equidae and Carnivora. The duplicated genes show different antiviral activities against different viruses: equine A3Z3 and A3Z2c-Z2d are potent inhibitors of EIAV while equine A3Z1b, A3Z2a-Z2b, A3Z2e showed only weak anti-EIAV activity. Equine A3Z1b and A3Z3 restricted AAV and all equine A3s, except A3Z1b, inhibited SIV. We hypothesize that the horse A3 genes are undergoing a process of subfunctionalization in their respective viral specificities, which might provide the evolutionary advantage for keeping five copies of the original gene. PMID:19458006

  7. The Infectious Bursal Disease Virus RNA-Binding VP3 Polypeptide Inhibits PKR-Mediated Apoptosis

    PubMed Central

    Busnadiego, Idoia; Maestre, Ana M.; Rodríguez, Dolores; Rodríguez, José F.

    2012-01-01

    Infectious bursal disease virus (IBDV) is an avian pathogen responsible for an acute immunosuppressive disease that causes major losses to the poultry industry. Despite having a bipartite dsRNA genome, IBDV, as well as other members of the Birnaviridae family, possesses a single capsid layer formed by trimers of the VP2 capsid protein. The capsid encloses a ribonucleoprotein complex formed by the genome associated to the RNA-dependent RNA polymerase and the RNA-binding polypeptide VP3. A previous report evidenced that expression of the mature VP2 IBDV capsid polypeptide triggers a swift programmed cell death response in a wide variety of cell lines. The mechanism(s) underlying this effect remained unknown. Here, we show that VP2 expression in HeLa cells activates the double-stranded RNA (dsRNA)-dependent protein kinase (PKR), which in turn triggers the phosphorylation of the eukaryotic initiation factor 2α (eIF2α). This results in a strong blockade of protein synthesis and the activation of an apoptotic response which is efficiently blocked by coexpression of a dominant negative PKR polypeptide. Our results demonstrate that coexpression of the VP3 polypeptide precludes phosphorylation of both PKR and eIF2α and the onset of programmed cell death induced by VP2 expression. A mutation blocking the capacity of VP3 to bind dsRNA also abolishes its capacity to prevent PKR activation and apoptosis. Further experiments showed that VP3 functionally replaces the host-range vaccinia virus (VACV) E3 protein, thus allowing the E3 deficient VACV deletion mutant WRΔE3L to grow in non-permissive cell lines. According to results presented here, VP3 can be categorized along with other well characterized proteins such us VACV E3, avian reovirus sigmaA, and influenza virus NS1 as a virus-encoded dsRNA-binding polypeptide with antiapoptotic properties. Our results suggest that VP3 plays a central role in ensuring the viability of the IBDV replication cycle by preventing programmed

  8. Differential effects of viroporin inhibitors against feline infectious peritonitis virus serotypes I and II.

    PubMed

    Takano, Tomomi; Nakano, Kenta; Doki, Tomoyoshi; Hohdatsu, Tsutomu

    2015-05-01

    Feline infectious peritonitis virus (FIP virus: FIPV), a feline coronavirus of the family Coronaviridae, causes a fatal disease called FIP in wild and domestic cat species. The genome of coronaviruses encodes a hydrophobic transmembrane protein, the envelope (E) protein. The E protein possesses ion channel activity. Viral proteins with ion channel activity are collectively termed "viroporins". Hexamethylene amiloride (HMA), a viroporin inhibitor, can inhibit the ion channel activity of the E protein and replication of several coronaviruses. However, it is not clear whether HMA and other viroporin inhibitors affect replication of FIPV. We examined the effect of HMA and other viroporin inhibitors (DIDS [4,4'-disothiocyano-2,2'-stilbenedisulphonic acid] and amantadine) on infection by FIPV serotypes I and II. HMA treatment drastically decreased the titers of FIPV serotype I strains Black and KU-2 in a dose-dependent manner, but it only slightly decreased the titer of FIPV serotype II strain 79-1146. In contrast, DIDS treatment decreased the titer of FIPV serotype II strain 79-1146 in dose-dependent manner, but it only slightly decreased the titers of FIPV serotype I strains Black and KU-2. We investigated whether there is a difference in ion channel activity of the E protein between viral serotypes using E. coli cells expressing the E protein of FIPV serotypes I and II. No difference was observed, suggesting that a viroporin other than the E protein influences the differences in the actions of HMA and DIDS on FIPV serotypes I and II. PMID:25701212

  9. Different architectures in the assembly of infectious bursal disease virus capsid proteins expressed in insect cells.

    PubMed

    Martinez-Torrecuadrada, J L; Castón, J R; Castro, M; Carrascosa, J L; Rodriguez, J F; Casal, J I

    2000-12-20

    Infectious bursal disease virus (IBDV) capsid is formed by the processing of a large polyprotein and subsequent assembly of VPX/VP2 and VP3. To learn more about the processing of the polyprotein and factors affecting the correct assembly of the viral capsid in vitro, different constructs were made using two baculovirus transfer vectors, pFastBac and pAcYM1. Surprisingly, the expression of the capsid proteins gave rise to different types of particles in each system, as observed by electron microscopy and immunofluorescence. FastBac expression led to the production of only rigid tubular structures, similar to those described as type I in viral infection. Western blot analysis revealed that these rigid tubules are formed exclusively by VPX. These tubules revealed a hexagonal arrangement of units that are trimer clustered, similar to those observed in IBDV virions. In contrast, pAcYM1 expression led to the assembly of virus-like particles (VLPs), flexible tubules, and intermediate assembly products formed by icosahedral caps elongated in tubes, suggesting an aberrant morphogenesis. Processing of VPX to VP2 seems to be a crucial requirement for the proper morphogenesis and assembly of IBDV particles. After immunoelectron microscopy, VPX/VP2 was detected on the surface of tubules and VLPs. We also demonstrated that VP3 is found only on the inner surfaces of VLPs and caps of the tubular structures. In summary, assembly of VLPs requires the internal scaffolding of VP3, which seems to induce the closing of the tubular architecture into VLPs and, thereafter, the subsequent processing of VPX to VP2. PMID:11118356

  10. Appearance and quantification of infectious hematopoietic necrosis virus in female sockeye salmon (Oncorhynchus nerka) during their spawning migration

    USGS Publications Warehouse

    Mulcahy, D.; Jenes, C.K.; Pascho, R.J.

    1984-01-01

    The incidence and amount of infectious hematopoietic necrosis (IHN) virus was determined in 10 organs and body fluids from each of 100 female sockeye salmon(Oncorhynchus nerka) before, during, and after their spawning migration into freshwater. Virus was found in high concentrations only in fish sampled during and after spawning. Infection rates increased from nil to 100 percent within 2 weeks. In spawning fish, incidences of IHN virus were high in all organs and fluids except brain and serum, and the highest concentrations were in the pyloric caeca and lower gut. Immediately before spawning, IHN virus was found most frequently in the gills, less frequently in the pyloric caeca and spleen, and rarely in other organs.

  11. Combination of Two Marek's Disease Virus Vectors Shows Effective Vaccination Against Marek's Disease, Infectious Bursal Disease, and Newcastle Disease.

    PubMed

    Ishihara, Yukari; Esaki, Motoyuki; Saitoh, Shuji; Yasuda, Atsushi

    2016-06-01

    Herpesvirus of turkeys (HVT) is a widely used vector for poultry vaccines. However, different HVTs expressing different foreign antigens cannot always be used simultaneously because of the risk of recombination and interference. In this study, we inoculated a mixture of an HVT-expressing the antigen of Newcastle disease virus (NDV; HVT/ND) and Marek's disease virus (MDV) serotype 1 Rispens virus expressing the antigen of infectious bursal disease virus (IBD; Ripens/IBD) into chickens. This mixture showed 94%, 100%, or 94% protection against MDV, IBDV, or NDV challenge, respectively. In conclusion, the combination of Rispens/IBD and HVT/ND is effective for vaccination against MDV, IBDV, and NDV without significant interference. PMID:27309290

  12. Determination of the infectious titer and virulence of an original US porcine epidemic diarrhea virus PC22A strain.

    PubMed

    Liu, Xinsheng; Lin, Chun-Ming; Annamalai, Thavamathi; Gao, Xiang; Lu, Zhongyan; Esseili, Malak A; Jung, Kwonil; El-Tholoth, Mohamed; Saif, Linda J; Wang, Qiuhong

    2015-01-01

    The infectious dose of a virus pool of original US PEDV strain PC22A was determined in 4-day-old, cesarean-derived, colostrum-deprived (CDCD) piglets. The median pig diarrhea dose (PDD50) of the virus pool was determined as 7.35 log10 PDD50/mL, similar to the cell culture infectious titer, 7.75 log10 plaque-forming units (PFU)/mL. 100 PDD50 caused watery diarrhea in all conventional suckling piglets (n = 12) derived from a PEDV-naive sow, whereas 1000 and 10 000 PDD50 did not cause diarrhea in piglets derived from two PEDV-field exposed-recovered sows. This information is important for future PEDV challenge studies and validation of PEDV vaccines. PMID:26408019

  13. Testing of male sockeye salmon (Oncorhynchus nerka) and steelhead trout (Salmo gairdneri) for infectious hematopoietic necrosis virus

    USGS Publications Warehouse

    Mulcahy, D.; Pascho, R.J.; Batts, W.N.

    1987-01-01

    Infectious hematopoietic necrosis (IHN) virus has been isolated only rarely from whole milt samples of male sockeye salmon (Oncorhynchus nerka). In 3 yr of testing, virus incidences in males ranged from 0 to 13% when milt was sampled but were 60–100% with spleen or kidney. When IHN virus was isolated from sockeye salmon milt at titers less than 3.00 log10 plaque-forming units (pfu)/mL, the level of virus in the kidney or spleen exceeded 7.00 log10 pfu/g. Higher rates of IHN virus isolation from kidney or spleen than from milt were also generally found in steelhead trout (Salmo gairdneri), although the differences were less pronounced than in sockeye salmon. Furthermore, virus was sometimes isolated from steelhead trout milt when the level of virus in kidney or spleen samples was very low, and was recovered from some milt samples when none was isolated from the corresponding spleen sample. When male salmonids are tested for IHN virus, kidney or spleen samples are superior to whole milt, but milt should be included for critical examinations.

  14. Airborne influenza virus detection with four aerosol samplers using molecular and infectivity assays: considerations for a new infectious virus aerosol sampler

    PubMed Central

    Fabian, P.; McDevitt, J. J.; Houseman, E. A.; Milton, D. K.

    2013-01-01

    As a first step in conducting studies of airborne influenza transmission, we compared the collection performance of an SKC Biosampler, a compact cascade impactor (CCI), Teflon filters, and gelatin filters by collecting aerosolized influenza virus in a one-pass aerosol chamber. Influenza virus infectivity was determined using a fluorescent focus assay and influenza virus nucleic acid (originating from viable and non-viable viruses) was measured using quantitative PCR. The results showed that the SKC Biosampler recovered and preserved influenza virus infectivity much better than the other samplers – the CCI, Teflon, and gelatin filters recovered only 7–22% of infectious viruses compared with the Biosampler. Total virus collection was not significantly different among the SKC Biosampler, the gelatin, and Teflon filters, but was significantly lower in the CCI. Results from this study show that a new sampler is needed for virus aerosol sampling, as commercially available samplers do not efficiently collect and conserve virus infectivity. Applications for a new sampler include studies of airborne disease transmission and bioterrorism monitoring. Design parameters for a new sampler include high collection efficiency for fine particles and liquid sampling media to preserve infectivity. PMID:19689447

  15. The surface envelope protein gene region of equine infectious anemia virus is not an important determinant of tropism in vitro.

    PubMed Central

    Perry, S T; Flaherty, M T; Kelley, M J; Clabough, D L; Tronick, S R; Coggins, L; Whetter, L; Lengel, C R; Fuller, F

    1992-01-01

    Virulent, wild-type equine infectious anemia virus (EIAV) is restricted in one or more early steps in replication in equine skin fibroblast cells compared with cell culture-adapted virus, which is fully competent for replication in this cell type. We compared the sequences of wild-type EIAV and a full-length infectious proviral clone of the cell culture-adapted EIAV and found that the genomes were relatively well conserved with the exception of the envelope gene region, which showed extensive sequence differences. We therefore constructed several wild-type and cell culture-adapted virus chimeras to examine the role of the envelope gene in replication in different cell types in vitro. Unlike wild-type virus, which is restricted by an early event(s) for replication in equine dermis cells, the wild-type outer envelope gene chimeras are replication competent in this cell type. We conclude that even though there are extensive sequence differences between wild-type and cell culture-adapted viruses in the surface envelope gene region, this domain is not a determinant of the differing in vitro cell tropisms. Images PMID:1318398

  16. Mycoplasma pneumoniae preceding Lemierre's syndrome due to Fusobacterium nucleatum complicated by acute Epstein-Barr virus (EBV) infectious mononucleosis in an immunocompetent host.

    PubMed

    Klein, Natalie C; Petelin, Andrew; Cunha, Burke A

    2013-01-01

    We report an unusual case of Lemierre's syndrome due to a rare species of Fusobacterium, that is, Fusobacterium nucleatum preceded by Mycoplasma pneumoniae pharyngitis and followed later by Epstein-Barr virus infectious mononucleosis. PMID:22464641

  17. Synthesis of coronavirus mRNAs: kinetics of inactivation of infectious bronchitis virus RNA synthesis by UV light. [Chickens

    SciTech Connect

    Stern, D.F.; Sefton, B.M.

    1982-05-01

    Infection of cells with the avian coronavirus infectious bronchitis virus results in the synthesis of five major subgenomic RNAs. These RNAs and the viral genome form a 3' coterminal nested set. We found that the rates of inactivation of synthesis of the RNAs by UV light were different and increased with the length of the transcript. These results show that each RNA is transcribed from a unique promoter and that extensive processing of the primary transcripts probably does not occur.

  18. Immunogenicity of synthetic peptides representing neutralizing epitopes on the glycoprotein of infectious hematopoietic necrosis virus

    USGS Publications Warehouse

    Emmenegger, E.; Landolt, M.; LaPatra, S.; Winton, J.

    1997-01-01

    Three peptides, P76, P226, and P268 representing 3 putative antigen~c determinants on the glycoprotein of infectious hematopoietic necrosis virus (IHNV), were synthesized and injected into rainbow trout Oncorhynchus mykiss to assess their immunogen~city. Antisera extracted from the immunized trout were analyzed uslng an enzyme linked imrnunosorbent assay (ELISA) for the presence of antibodies that could bind to the peptides or to intact virions of IHNV. The antisera were also tested for neutralizing activity against IHNV by a complement-mediated neutralization assay. In general, recognition of the peptides and IHNV was low and only a few antibody binding patterns were demonstrated. Antisera from fish injected with P76 constructs recognized the homologous peptide more than the heterologous peptides, whereas antisera from fish inoculated with either P226 or P268 constructs recognized P76 equally, or better, than the homologous peptide; however, there was a high degree of individual variation within each treatment group. Neutralization actlvlty was demonstrated by serum from a single flsh lnlected with one of the pept~des (P268) and from 7 of 10 positive control f~sh Infected with an attenuated strain of IHNV Possible explanations for the dichotomous immune responses are discussed. These results indicate we need to improve our overall understanding of the

  19. Antibody against infectious salmon anaemia virus among feral Atlantic salmon (Salmo salar)

    USGS Publications Warehouse

    Cipriano, R.C.

    2009-01-01

    Archived sera from Atlantic salmon (Salmo salar) that returned to the Penobscot River (Maine), Merrimack River (Massachusetts), and Connecticut River (in Massachusetts) from 1995 to 2002 were analysed for antibodies against infectious salmon anaemia virus (ISAV) using an enzyme-linked immunosorbent assay (ELISA). Up to 60 samples were archived per river system per year. In a given year, the number of fish sampled by ELISA for ISAV antibodies in the Penobscot River ranged from 2.9 to 11.2, and the range of salmon sampled in the Merrimack River and the Connecticut River was 31.3-100 and 20.0-67.5, respectively. Archived sera were not available for the 1995 and 2002 year classes from the Connecticut River. In all, 1141 samples were processed; 14 serum samples tested positive for antibodies to ISAV. In the Penobscot River, serum from one fish tested positive in each of the 1995 and 1999 year-class returns, and sera from two fish tested positive in the 1998 returns. In the Merrimack River, sera from four fish tested positive in each of the 1996 and 1997 returns, and sera from two fish were positive in the 2002 return. None of the archived sera from Atlantic salmon that returned to the Connecticut River tested positive. ?? 2009 United States Government, Department of the Interior.

  20. Epidemiologic investigation of the re-emergence of infectious salmon anemia virus in Chile.

    PubMed

    Mardones, Fernando O; Perez, Andrés M; Carpenter, Tim E

    2009-04-01

    Reports from surveillance activities were analyzed to determine the epidemiology of the re-emergence of infectious salmon anemia virus (ISAV) in Atlantic salmon farms in Chile. The epidemic and spatial and spatiotemporal patterns were described, taking into account commercial compartmentalization of the farms. During the 64 wk study period, 76 ISAV-infected salmon farms, representing 17 companies, were reported in 65% of the management geographic zones of the 10th region in southern Chile. Approximately 20% of the farms at risk became infected, with the incidence rate increasing slightly over time. Results from epidemic analyses and observed spatial and spatiotemporal patterns suggested an initial dispersal and subsequent clustering of cases around the index case (IC) in a propagated epidemic mode. Results suggested that delayed depopulation may have been a key factor in the spread and persistence of ISAV. Clustering of cases supported the assumption that passive transmission in seawater from ISAV-infected farms (proximity) is a critical factor in controlling disease. The re-emergence of ISAV in Chile has resulted in one of the largest ISAV epidemics reported in the world and this study generates new hypotheses and provides useful information for spatial disease control planning in salmon farming areas. PMID:19476280

  1. [Immunogenicity of recombinant Lactobacillus casei expressing VP2 protein of infectious bursal disease virus in chickens].

    PubMed

    Lin, Hongli; Hou, Shenda; Wang, Song; Wang, Yupeng; LuanI, Yunyan; Hou, Xilin

    2014-11-01

    In order to determine immunogenicity and protective effect in chickens, we used the IBDV (Infectious bursal disease virus)-Vp2/Lactobacillus casei as antigen transfer system. First, the immunized and control chickens were challenged by IBDV/DQ at lethal dose to determine the protective ratio. Second, chickens were orallyand intranasally vaccinated twice with 10(9) CFU/mL pLA-VP2/L. casei, pLA/L. casei and PBS as negativecontrol and commercial vaccine as positive control. The bursa injury and the lesion score wererecorded post challenge. The level of specific IgG and sIgA in pLA-VP2/L. casei and positive control groups was significantly higher than that in negativecontrol groups. The protection efficacy in pLA-VP2/L. casei oral group was higher than that inintranasal group. The SI. of pLA-VP2/L. casei oral group was significant higher than other groups. The lesion score indicated the pLA-VP2/L. casei was safer than commercial vaccine for bursa. Collectively, the pLA-VP2/L. casei could be a vaccine candidate for IBDV. PMID:25985519

  2. Suppressor T cell clones from patients with acute Epstein-Barr virus-induced infectious mononucleosis.

    PubMed Central

    Wang, F; Blaese, R M; Zoon, K C; Tosato, G

    1987-01-01

    Suppression and/or cytotoxicity are believed to play an important role in the defense against Epstein-Barr virus (EBV) infection. To analyze the role of suppressor T cells in relation to EBV, we sought to clone and study these T cells. Analysis of 152 T cell clones derived from the peripheral blood of two patients with acute EBV-induced infectious mononucleosis (IM) yielded 11 highly suppressive clones that had no cytotoxic activity for the natural killer sensitive K562 cell line, an autologous EBV-infected cell line, or an allogeneic EBV-infected B cell line. Four of six suppressor T cell clones also profoundly inhibited EBV-induced immunoglobulin production, and five of five clones delayed the outgrowth of immortalized cells. These results indicate that during acute IM, suppressor T cells capable of inhibiting B cell activation in the absence of cytotoxicity can be identified, and may play a key role in the control of EBV infection. Images PMID:3025263

  3. Analysis of synonymous codon usage in spike protein gene of infectious bronchitis virus.

    PubMed

    Makhija, Aditi; Kumar, Sachin

    2015-12-01

    Infectious bronchitis virus (IBV) is responsible for causing respiratory, renal, and urogenital diseases in poultry. IBV infection in poultry leads to high mortality rates in affected flocks and to severe economic losses due to a drop in egg production and a reduced gain in live weight of the broiler birds. IBV-encoded spike protein (S) is the major protective immunogen for the host. Although the functions of the S protein have been well studied, the factors shaping synonymous codon usage bias and nucleotide composition in the S gene have not been reported yet. In the present study, we analyzed the relative synonymous codon usage and effective number of codons (Nc) using the 53 IBV S genes. The major trend in codon usage variation was studied using correspondence analysis. The plot of Nc values against GC3 as well as the correlation between base composition and codon usage bias suggest that mutational pressure rather than natural selection is the main factor that determines the codon usage bias in the S gene. Interestingly, no association of aromaticity, degree of hydrophobicity, and aliphatic index was observed with the codon usage variation in IBV S genes. The study represents a comprehensive analysis of IBV S gene codon usage patterns and provides a basic understanding of the codon usage bias. PMID:26452019

  4. Nodular Scleritis Associated with Herpes Zoster Virus: An Infectious and Immune-Mediated Process.

    PubMed

    Loureiro, Mónica; Rothwell, Renata; Fonseca, Sofia

    2016-01-01

    Purpose. To describe a case of anterior nodular scleritis, preceded by an anterior hypertensive uveitis, which was primarily caused by varicella zoster virus (VZV). Case Report. A 54-year-old woman presented with anterior uveitis of the right eye presumably caused by herpetic viral disease and was successfully treated. Two months later, she developed a nodular scleritis and started oral nonsteroidal anti-inflammatory without effect. A complete laboratory workup revealed positivity for HLA-B27; the infectious workup was negative. Therapy was changed to oral prednisolone and an incomplete improvement occurred. Therefore, a diagnostic anterior paracentesis was performed and the polymerase chain reaction (PCR) analysis revealed VZV. She was treated with valacyclovir and the oral prednisolone began to decrease; however, a marked worsening of the scleritis occurred with the reduction of the daily dose; subsequently, methotrexate was introduced allowing the suspension of the prednisolone and led to clinical resolution of the scleritis. Conclusion. This report of anterior nodular scleritis caused by VZV argues in favor of an underlying immune-mediated component, requiring immunosuppressive therapy for clinical resolution. The PCR analysis of the aqueous humor was revealed to be a valuable technique and should be considered in cases of scleritis with poor response to treatment. PMID:27298747

  5. Nodular Scleritis Associated with Herpes Zoster Virus: An Infectious and Immune-Mediated Process

    PubMed Central

    Loureiro, Mónica; Rothwell, Renata; Fonseca, Sofia

    2016-01-01

    Purpose. To describe a case of anterior nodular scleritis, preceded by an anterior hypertensive uveitis, which was primarily caused by varicella zoster virus (VZV). Case Report. A 54-year-old woman presented with anterior uveitis of the right eye presumably caused by herpetic viral disease and was successfully treated. Two months later, she developed a nodular scleritis and started oral nonsteroidal anti-inflammatory without effect. A complete laboratory workup revealed positivity for HLA-B27; the infectious workup was negative. Therapy was changed to oral prednisolone and an incomplete improvement occurred. Therefore, a diagnostic anterior paracentesis was performed and the polymerase chain reaction (PCR) analysis revealed VZV. She was treated with valacyclovir and the oral prednisolone began to decrease; however, a marked worsening of the scleritis occurred with the reduction of the daily dose; subsequently, methotrexate was introduced allowing the suspension of the prednisolone and led to clinical resolution of the scleritis. Conclusion. This report of anterior nodular scleritis caused by VZV argues in favor of an underlying immune-mediated component, requiring immunosuppressive therapy for clinical resolution. The PCR analysis of the aqueous humor was revealed to be a valuable technique and should be considered in cases of scleritis with poor response to treatment. PMID:27298747

  6. DNA prime-protein boost vaccination enhances protective immunity against infectious bursal disease virus in chickens.

    PubMed

    Gao, Honglei; Li, Kai; Gao, Li; Qi, Xiaole; Gao, Yulong; Qin, Liting; Wang, Yongqiang; Wang, Xiaomei

    2013-05-31

    Infectious bursal disease virus causes an acute contagious immunosuppressive disease in chickens. Using VP2 protein from IBDV (Gx strain) as the immunogen, the goal of the current study was to evaluate the immune responses and protective efficacy elicited by different prime-boost vaccination regimens (DNA only, protein only, and DNA plus protein) in chickens. The results indicated that both pCAGoptiVP2 plasmid and rVP2 protein induced humoral and cellular immune responses. Chickens in the DNA prime-protein boost group developed significantly higher levels of ELISA and neutralizing antibodies to IBDV compared with those immunized with either the DNA vaccine or the protein vaccine alone (P<0.05). Furthermore, the highest levels of lymphocyte proliferation response, IL-4 and IFN-γ production were induced following priming with the DNA vaccine and boosting with the rVP2 protein. Additionally, chickens inoculated with the DNA prime-protein boost vaccine had 100% protection against challenge with vvIBDV, as evidenced by the absence of clinical signs, mortality, and bursal atrophy. In contrast, chickens receiving the DNA vaccine and the rVP2 protein vaccine had 67% and 80% protection, respectively. These findings demonstrated that the DNA prime-protein boost immunization strategy was effective in eliciting both humoral and cellular immune responses in chickens, highlighting the potential value of such an approach in the prevention of vvIBDV infection. PMID:23419823

  7. Cyclophilin A Interacts with Viral VP4 and Inhibits the Replication of Infectious Bursal Disease Virus.

    PubMed

    Wang, Nian; Zhang, Lizhou; Chen, Yuming; Lu, Zhen; Gao, Li; Wang, Yongqiang; Gao, Yulong; Gao, Honglei; Cui, Hongyu; Li, Kai; Liu, Changjun; Zhang, Yanping; Qi, Xiaole; Wang, Xiaomei

    2015-01-01

    Nonstructural protein VP4, a serine protease of infectious bursal disease virus (IBDV) that catalyzes the hydrolysis of polyprotein pVP2-VP4-VP3 to form the viral proteins VP2, VP4, and VP3, is essential to the replication of IBDV. However, the interacting partners of VP4 in host cells and the effects of the interaction on the IBDV lifecycle remain incompletely elucidated. In this study, using the yeast two-hybrid system, the putative VP4-interacting partner cyclophilin A (CypA) was obtained from a chicken embryo fibroblast (CEF) expression library. CypA was further confirmed to interact with VP4 of IBDV using co-immunoprecipitation (CO-IP), GST pull-down, and confocal microscopy assays. Moreover, we found that the overexpression of CypA suppressed IBDV replication, whereas the knock-down of CypA by small interfering RNAs promoted the replication of IBDV. Taken together, our findings indicate that the host cell protein CypA interacts with viral VP4 and inhibits the replication of IBDV. PMID:26090438

  8. A zebrafish (Danio rerio) model of infectious spleen and kidney necrosis virus (ISKNV) infection

    SciTech Connect

    Xu Xiaopeng; Zhang Lichun; Weng Shaoping; Huang Zhijian; Lu Jing; Lan Dongming; Zhong Xuejun; Yu Xiaoqiang; Xu Anlong He Jianguo

    2008-06-20

    Zebrafish is a model animal for studies of genetics, development, toxicology, oncology, and immunology. In this study, infectious spleen and kidney necrosis virus (ISKNV) was used to establish an infection in zebrafish, and the experimental conditions were established and characterized. Mortality of adult zebrafish infected with ISKNV by intraperitoneal (i.p.) injection exceeded 60%. ISKNV can be passed stably in zebrafish for over ten passages. The ailing zebrafish displayed petechial hemorrhaging and scale protrusion. Histological analysis of moribund fish revealed necrosis of tissue and enlarged cells in kidney and spleen. The real-time RT-PCR analysis of mRNA level confirmed that ISKNV was replicated in zebrafish. Immunohistochemistry and immunofluorescence analyses further confirmed the presence of ISKNV-infected cells in almost all organs of the infected fish. Electron microscope analyses showed that the ISKNV particle was present in the infected tissues. The establishment of zebrafish infection model of ISKNV can offer a valuable tool for studying the interactions between ISKNV and its host.

  9. Comprehensive gene expression profiling following DNA vaccination of rainbow trout against infectious hematopoietic necrosis virus

    USGS Publications Warehouse

    Purcell, Maureen K.; Nichols, Krista M.; Winton, James R.; Kurath, Gael; Thorgaard, Gary H.; Wheeler, Paul; Hansen, John D.; Herwig, Russell P.; Park, Linda K.

    2006-01-01

    The DNA vaccine based on the glycoprotein gene of Infectious hematopoietic necrosis virus induces a non-specific anti-viral immune response and long-term specific immunity against IHNV. This study characterized gene expression responses associated with the early anti-viral response. Homozygous rainbow trout were injected intra-muscularly (I.M.) with vector DNA or the IHNV DNA vaccine. Gene expression in muscle tissue (I.M. site) was evaluated using a 16,008 feature salmon cDNA microarray. Eighty different genes were significantly modulated in the vector DNA group while 910 genes were modulated in the IHNV DNA vaccinate group relative to control group. Quantitative reverse-transcriptase PCR was used to examine expression of selected immune genes at the I.M. site and in other secondary tissues. In the localized response (I.M. site), the magnitudes of gene expression changes were much greater in the vaccinate group relative to the vector DNA group for the majority of genes analyzed. At secondary systemic sites (e.g. gill, kidney and spleen), type I IFN-related genes were up-regulated in only the IHNV DNA vaccinated group. The results presented here suggest that the IHNV DNA vaccine induces up-regulation of the type I IFN system across multiple tissues, which is the functional basis of early anti-viral immunity.

  10. Attenuation, transmission, and immunogenicity of an ORF-C gene deleted strain of infectious laryngotracheitis virus (ILTV) in specific pathogen free chickens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Infectious laryngotracheitis (ILT) is a very serious and widespread respiratory disease of chickens caused by infectious laryngotracheitis virus (ILTV). Conventional attenuated ILT vaccines, obtained by continuous passages in chicken embryos and tissue culture, had been the main tools utilized by th...

  11. Model for inactivation and disposal of infectious human immunodeficiency virus and radioactive waste in a BL3 facility.

    PubMed Central

    Stinson, M C; Galanek, M S; Ducatman, A M; Masse, F X; Kuritzkes, D R

    1990-01-01

    A method is described for autoclaving low levels of solid infectious, radioactive waste. The method permits steam penetration to inactivate biologic waste, while any volatile radioactive compounds generated during the autoclave process are absorbed. Inactivation of radiolabeled infectious waste has been problematic because the usual sterilization techniques result in unacceptable radiation handling practices. If autoclaved under the usual conditions, there exists a high probability of volatilization or release of radioisotopes from the waste. This results in the radioactive contamination of the autoclave and the laboratory area where steam is released from the autoclave. Our results provide a practical method to inactivate and dispose of infectious radioactive waste. For our research, Bacillus pumilus spore strips and vaccinia virus were used as more heat-resistant surrogates of the human immunodeficiency virus (HIV). These surrogates were used because HIV is difficult to grow under most conditions and is less heat tolerant than the surrogates. In addition, B. pumilus has defined cell death values, whereas such values have not been established for HIV. Both B. pumilus and vaccinia virus are less hazardous to work with. The autoclave method is time efficient and can be performed by laboratory personnel with minimal handling of the waste. Furthermore, waste site handlers are able to visually inspect the solid waste containers and ascertain that inactivation procedures have been implemented. PMID:2310182

  12. Recombinant Adeno-Associated Virus Utilizes Cell-Specific Infectious Entry Mechanisms

    PubMed Central

    Weinberg, Marc S.; Nicolson, Sarah; Bhatt, Aadra P.; McLendon, Michael; Li, Chengwen

    2014-01-01

    ABSTRACT Understanding the entry and trafficking mechanism(s) of recombinant adeno-associated virus (rAAV) into host cells can lead to evolution in capsid and vector design and delivery methods, resulting in enhanced transduction and therapeutic gene expression. Variability of findings regarding the early entry pathway of rAAV supports the possibility that rAAV, like other viruses, can utilize more than one infectious entry pathway. We tested whether inhibition of macropinocytosis impacted rAAV transduction of HeLa cells compared to hepatocellular carcinoma cell lines. We found that macropinocytosis inhibitor cytochalasin D blocked rAAV transduction of HeLa cells (>2-fold) but enhanced (10-fold) transduction in HepG2 and Huh7 lines. Similar results were obtained with another macropinocytosis inhibitor, 5-(N-ethyl-N-isopropyl) amiloride (EIPA). The augmented transduction was due to neither viral binding nor promoter activity, affected multiple rAAV serotypes (rAAV2, rAAV2-R585E, and rAAV8), and influenced single-stranded and self-complementary virions to comparable extents. Follow-up studies using CDC42 inhibitor ML141 and p21-activated kinase 1 (PAK1) siRNA knockdown also resulted in enhanced HepG2 transduction. Microscopy revealed that macropinocytosis inhibition correlated with expedited nuclear entry of the rAAV virions into HepG2 cells. Enhancement of hepatocellular rAAV transduction extended to the mouse liver in vivo (4-fold enhancement) but inversely blocked heart tissue transduction (13-fold). This evidence of host cell-specific rAAV entry pathways confers a potent means for controlling and enhancing vector delivery and could help unify the divergent accounts of rAAV cellular entry mechanisms. IMPORTANCE There is a recognized need for improved rAAV vector targeting strategies that result in delivery of fewer total particles, averting untoward toxicity and/or an immune response against the vector. A critical step in rAAV transduction is entry and early

  13. Prevalence of infectious salmon anaemia virus (ISAV) in wild salmonids in western Norway.

    PubMed

    Plarre, H; Devold, M; Snow, M; Nylund, A

    2005-08-01

    Studies of infectious salmon anaemia virus (ISAV), an important pathogen of farmed salmon in Norway, Scotland, the Faeroe Islands, Ireland, Canada, the USA and Chile, suggest that natural reservoirs for this virus can be found on both sides of the North Atlantic. Based on existing information about ISAV it is believed to be maintained in wild populations of trout and salmon in Europe. It has further been suggested that ISAV is transmitted between wild hosts, mainly during their freshwater spawning phase in rivers, and that wild salmonids, mainly trout, are possible carriers of benign wild-type variants of ISAV. Change in virulence is probably a result of deletions of amino acid segments from the highly polymorphic region (HPR) of benign wild-type isolates after transmission to farmed salmon. Hence, it has been suggested that the frequency of new outbreaks of ISA in farmed salmon could partly reflect natural variation in the prevalence of ISAV in wild populations of salmonids. The aims of the present study were to screen for ISAV in wild salmonids during spawning in rivers and to determine the pathogenicity of resultant isolates from wild fish. Tissues from wild salmonids were screened by RT-PCR and real-time PCR. The prevalence of ISAV in wild trout Salmo trutta varied from 62 to 100% between tested rivers in 2001. The prevalence dropped in 2002, ranging from 13 to 36% in the same rivers and to only 6% in 2003. All ISAV were nonpathogenic when injected into disease-free Atlantic salmon, but were capable of propagation, as indicated by subsequent viral recovery. However, non-pathogenic ISAV has also been found in farmed salmon, where a prevalence as high as 60% has been registered, but with no mortalities occurring. Based on the results of the present and other studies, it must be concluded that vital information about the importance of wild and man-made reservoirs for the emergence of ISA in salmon farming is still lacking. This information can only be gained by

  14. trans-Packaged West Nile virus-like particles: infectious properties in vitro and in infected mosquito vectors.

    PubMed

    Scholle, Frank; Girard, Yvette A; Zhao, Qizu; Higgs, Stephen; Mason, Peter W

    2004-11-01

    A trans-packaging system for West Nile virus (WNV) subgenomic replicon RNAs (repRNAs), deleted for the structural coding region, was developed. WNV repRNAs were efficiently encapsidated by the WNV C/prM/E structural proteins expressed in trans from replication-competent, noncytopathic Sindbis virus-derived RNAs. Infectious virus-like particles (VLPs) were produced in titers of up to 10(9) infectious units/ml. WNV VLPs established a single round of infection in a variety of different cell lines without production of progeny virions. The infectious properties of WNV and VLPs were indistinguishable when efficiencies of infection of a number of different cell lines and inhibition of infection by neutralizing antibodies were determined. To investigate the usefulness of VLPs to address biological questions in vivo, Culex pipiens quinquefasciatus mosquitoes were orally and parenterally infected with VLPs, and dissected tissues were analyzed for WNV antigen expression. Antigen-positive cells in midguts of orally infected mosquitoes were detected as early as 2 days postinfection and as late as 8 days. Intrathoracic inoculation of VLPs into mosquitoes demonstrated a dose-dependent pattern of infection of secondary tissues and identified fat body, salivary glands, tracheal cells, and midgut muscle as susceptible WNV VLP infection targets. These results demonstrate that VLPs can serve as a valuable tool for the investigation of tissue tropism during the early stages of infection, where virus spread and the need for biosafety level 3 containment complicate the use of wild-type virus. PMID:15479801

  15. Core bead chromatography for preparation of highly pure, infectious respiratory syncytial virus in the negative purification mode.

    PubMed

    Mundle, Sophia T; Kishko, Michael; Groppo, Rachel; DiNapoli, Joshua; Hamberger, John; McNeil, Bryan; Kleanthous, Harry; Parrington, Mark; Zhang, Linong; Anderson, Stephen F

    2016-07-12

    Respiratory syncytial virus (RSV) is an important human pathogen, and is the most frequent viral cause of severe respiratory disease in infants. In addition, it is increasingly being recognized as an important cause of respiratory disease in the elderly and immunocompromised. Although a passive prophylactic treatment does exist for high-risk neonates and children, the overall disease burden warrants the development of a safe and effective prophylactic vaccine for use in otherwise healthy newborns and children. RSV is known to be an extremely labile virus, prone to aggregation and loss of infectious titer during virus handling and preparation procedures. To date infective RSV virions have been prepared by methods which are not readily scalable, such as density gradient ultracentrifugation. In this study we describe a scalable, chromatography-based purification procedure for preparation of highly pure, infectious RSV. The purification scheme is based on core bead technology and hollow fiber tangential flow filtration (TFF) and results in a ∼60% recovery of infectious virus titer. This method can be used to prepare highly purified wild type or live-attenuated vaccine strain viruses with titers as high as 1×10(8) plaque forming units per mL. A live-attenuated RSV vaccine prepared by this method was found to be immunogenic and protective in vivo, and its purity was 50-200-fold greater with respect to host cell dsDNA and Vero host cell proteins, than the raw feed stream. The results presented here can be considered a starting point for downstream process development of a live-attenuated vaccine approach for prevention of disease by RSV. PMID:27238375

  16. Resistance and Protective Immunity in Redfish Lake Sockeye Salmon Exposed to M Type Infectious Hematopoietic Necrosis Virus (IHNV)

    USGS Publications Warehouse

    Kurath, Gael; Garver, Kyle; Purcell, Maureen K.; LaPatra, Scott E.

    2010-01-01

    Differential virulence of infectious hematopoietic necrosis virus (IHNV) isolates from the U and M phylogenetic subgroups is clearly evident in the Redfish Lake (RFL) strain of sockeye salmon Oncorhynchus nerka. In these fish, experimental immersion challenges with U isolates cause extremely high mortality and M isolates cause low or no mortality. When survivors of M virus immersion challenges were exposed to a secondary challenge with virulent U type virus they experienced high mortality, indicating that the primary M challenge did not elicit protective immunity. Delivery of a moderate dose (2 × 104 plaque-forming units [PFU]/fish) of virus by intraperitoneal injection challenge did not overcome RFL sockeye salmon resistance to M type IHNV. Injection challenge with a high dose (5 × 106 PFU/fish) of M type virus caused 10% mortality, and in this case survivors did develop protective immunity against a secondary U type virus challenge. Thus, although it is possible for M type IHNV to elicit cross-protective immunity in this disease model, it does not develop after immersion challenge despite entry, transient replication of M virus to low levels, stimulation of innate immune genes, and development of neutralizing antibodies in some fish.

  17. Molecular confirmation of infectious spleen and kidney necrosis virus (ISKNV) in farmed and imported ornamental fish in Australia.

    PubMed

    Mohr, Peter G; Moody, Nicholas J G; Williams, Lynette M; Hoad, John; Cummins, David M; Davies, Kelly R; StJ Crane, Mark

    2015-10-16

    Viruses of the genus Megalocytivirus have not been detected in wild populations of fish in Australia but circulate in imported ornamental fish. In 2012, detection of a megalocytivirus in healthy platys Xiphophorus maculatus was reported from a farm in Australia during surveillance testing as part of a research project undertaken at the University of Sydney. Confirmatory testing of the original samples at the AAHL Fish Diseases Laboratory verified the presence of an infectious spleen and kidney necrosis virus (ISKNV)-like virus. Additional sampling at the positive farm confirmed the persistence of the virus in the platys, with 39 of 265 (14.7%) samples testing positive. Comparison of 3 separate gene regions of the virus with those of ISKNV confirmed the detection of a virus indistinguishable from ISKNV. Subsequently, ISKNV was also detected in a range of imported ornamental fish from several countries between 2013 and 2014, by screening with real-time PCR and confirmation by conventional PCR and sequence analysis. Accordingly, the current importation of live ornamental fish acts as a potential perpetual source for the establishment of ISKNV viruses within Australia. The testing of the farmed and imported ornamental fish verified the utility of the probe-based real-time PCR assay for screening of ornamental fish for Megalocytivirus. PMID:26480913

  18. Pathophysiology of infectious hematopoietic necrosis virus disease in rainbow trout (Salmo gairdneri): early changes in blood and aspects of the immune Response after Injection of IHN Virus

    USGS Publications Warehouse

    Amend, Donald F.; Smith, Lynnwood

    1974-01-01

    Juvenile rainbow trout (Salmo gairdneri) were injected with infectious hematopoietic necrosis (IHN) virus and various hematological and blood chemical changes were monitored over 9 days. The packed cell volume, hemoglobin, red blood cell count, and plasma bicarbonate were significantly depressed by day 4. Plasma chloride, calcium, phosphorus, total protein, and blood cell types did not change during the 9 days. Furthermore, plasma  LDH isozyme was significantly increased by the fourth day, and fish infected with infectious pancreatic necrosis virus, Vibrio anguillarum, Aeromonas salmonicida, and redmouth bacterium did not show specific LDH isozyme alterations. Acid-base alterations occurred at 10 C but not at 18 C. The acid-base imbalance and elevation of the  LDH isozyme were consistently associated with the early development of the disease.The immune response after injection of IHN virus was determined and protection from disease was tested by passive immunization. Actively immunized fish developed IHN-neutralizing antibodies within 54 days after injection of virus, and the antibodies were protective when juvenile fish were passively immunized and experimentally challenged with IHN virus.

  19. Bursal transcriptome of chickens protected by DNA vaccination versus those challenged with infectious bursal disease virus.

    PubMed

    Lee, Chih-Chun; Kim, Bong-Suk; Wu, Ching Ching; Lin, Tsang Long

    2015-01-01

    Infectious bursal disease virus (IBDV) infection destroys the bursa of Fabricius, causing immunosuppression and rendering chickens susceptible to secondary bacterial or viral infections. IBDV large-segment-protein-expressing DNA has been shown to confer complete protection of chickens from infectious bursal disease (IBD). The purpose of the present study was to compare DNA-vaccinated chickens and unvaccinated chickens upon IBDV challenge by transcriptomic analysis of bursa regarding innate immunity, inflammation, immune cell regulation, apoptosis and glucose transport. One-day-old specific-pathogen-free chickens were vaccinated intramuscularly three times at weekly intervals with IBDV large-segment-protein-expressing DNA. Chickens were challenged orally with 8.2 × 10(2) times the egg infective dose (EID)50 of IBDV strain variant E (VE) one week after the last vaccination. Bursae collected at 0.5, 1, 3, 5, 7, and 10 days post-challenge (dpc) were subjected to real-time RT-PCR quantification of bursal transcripts related to innate immunity, inflammation, immune cell regulation, apoptosis and glucose transport. The expression levels of granzyme K and CD8 in DNA-vaccinated chickens were significantly (p < 0.05) higher than those in unvaccinated chickens upon IBDV challenge at 0.5 or 1 dpc. The expression levels of other genes involved in innate immunity, inflammation, immune cell regulation, apoptosis and glucose transport were not upregulated or downregulated in DNA-vaccinated chickens during IBDV challenge. Bursal transcripts related to innate immunity and inflammation, including TLR3, MDA5, IFN-α, IFN-β, IRF-1, IRF-10, IL-1β, IL-6, IL-8, iNOS, granzyme A, granzyme K and IL-10, were upregulated or significantly (p < 0.05) upregulated at 3 dpc and later in unvaccinated chickens challenged with IBDV. The expression levels of genes related to immune cell regulation, apoptosis and glucose transport, including CD4, CD8, IL-2, IFN-γ, IL-12(p40), IL-18, GM-CSF, GATA-3

  20. The N-terminal Helical Region of the Hepatitis C Virus p7 Ion Channel Protein Is Critical for Infectious Virus Production

    PubMed Central

    Scull, Margaret A.; Schneider, William M.; Fung, Canny; Jones, Christopher T.; van de Belt, Marieke; Penin, François; Rice, Charles M.

    2015-01-01

    The hepatitis C virus (HCV) p7 protein is required for infectious virus production via its role in assembly and ion channel activity. Although NMR structures of p7 have been reported, the location of secondary structural elements and orientation of the p7 transmembrane domains differ among models. Furthermore, the p7 structure-function relationship remains unclear. Here, extensive mutagenesis, coupled with infectious virus production phenotyping and molecular modeling, demonstrates that the N-terminal helical region plays a previously underappreciated yet critical functional role, especially with respect to E2/p7 cleavage efficiency. Interrogation of specific N-terminal helix residues identified as having p7-specific defects and predicted to point toward the channel pore, in a context of independent E2/p7 cleavage, further supports p7 as a structurally plastic, minimalist ion channel. Together, our findings indicate that the p7 N-terminal helical region is critical for E2/p7 processing, protein-protein interactions, ion channel activity, and infectious HCV production. PMID:26588073

  1. Immunomodulatory effect of baculovirus in chickens: How it modifies the immune response against infectious bursal disease virus.

    PubMed

    Chimeno Zoth, Silvina; Carballeda, Juan Manuel; Gravisaco, María José; Lucero, María Soledad; Richetta, Matías; Gómez, Evangelina; Berinstein, Analía

    2016-07-01

    Several reports have shown that baculoviruses (BVs) have strong adjuvant properties on the mammalian immune system. Recent studies of our group demonstrated the ability of BV to stimulate the innate immunity in chickens. In this investigation, we aimed to assess the potential antiviral effect of BV given both, before and after infectious bursal disease virus (IBDV). In the first case, specific pathogen free chickens were intravenously inoculated with 5 × 10(7) pfu of Autographa californica nuclear polyhedrosis virus and 3 h later were orally administered 2.5 × 10(5) egg infectious doses 50 of IBDV. In the second case, chickens received IBDV 3 h before BV inoculation. Five days later, chickens were bled and euthanized. RNA from the bursa was analyzed for cytokine production. Also, bursae were used for virus recovery, and processed for lymphocyte isolation. The results showed that the administration of BV 3 h after the inoculation with IBDV produced important changes in the effect that IBDV causes in the bursa. BV reduced the infiltration of T lymphocytes, decreased the expression pattern of IL-6 and IFN-γ and inhibited IBDV replication. The results herein presented demonstrate that this Lepidopteran virus shows antiviral activity in chickens under experimental conditions. Investigations under field conditions have to be done to probe this strategy as a valuable sanitary tool for the treatment and prevention of chicken diseases. PMID:27063861

  2. RNA 1 and RNA 2 Genomic Segments of Chronic Bee Paralysis Virus Are Infectious and Induce Chronic Bee Paralysis Disease

    PubMed Central

    Youssef, Ibrahim; Schurr, Frank; Goulet, Adeline; Cougoule, Nicolas; Ribière-Chabert, Magali; Darbon, Hervé; Thiéry, Richard; Dubois, Eric

    2015-01-01

    Chronic bee paralysis virus (CBPV) causes an infectious and contagious disease of adult honeybees. Its segmented genome is composed of two major positive single-stranded RNAs, RNA 1 (3,674 nt) and RNA 2 (2,305 nt). Three minor RNAs (about 1,000 nt each) have been described earlier but they were not detected by sequencing of CBPV genome. In this study, the results of in vivo inoculation of the two purified CBPV major RNAs are presented and demonstrate that RNA 1 and RNA 2 are infectious. Honeybees inoculated with 109 RNA copies per bee developed paralysis symptoms within 6 days after inoculation. The number of CBPV RNA copies increased significantly throughout the infection. Moreover, the negative strand of CBPV RNA was detected by RT-PCR, and CBPV particles were visualized by electronic microscopy in inoculated honeybees. Taken together, these results show that CBPV RNA 1 and CBPV RNA 2 segments can induce virus replication and produce CBPV virus particles. Therefore, the three minor RNAs described in early studies are not essential for virus replication. These data are crucial for the development of a reverse genetic system for CBPV. PMID:26583154

  3. Differential diagnosis of fowlpox and infectious laryngotracheitis viruses in chicken diphtheritic manifestations by mono and duplex real-time polymerase chain reaction.

    PubMed

    Davidson, Irit; Raibstein, Israel; Altory, Amira

    2015-01-01

    Infectious laryngotracheitis virus (ILTV) and fowlpox virus (FPV) cause diphtheritic lesions in chicken tracheas and can simultaneously infect the same bird. A differential molecular diagnostic test, the duplex real-time polymerase chain reaction, is now reported using ILTV and FPV vaccine viruses and clinical samples from chickens, either uninfected or naturally infected with ILTV or FPV, or with both viruses. The dual virus amplification by real-time polymerase chain reaction was demonstrated to behave similarly to monoplex amplification, in spite of the fact that the real-time exponential amplification plots of the vaccine viruses were more illustrative than those of the clinical samples. PMID:25317604

  4. Protection of rainbow trout against infectious hematopoietic necrosis virus four days after specific or semi-specific DNA vaccination

    USGS Publications Warehouse

    LaPatra, S.E.; Corbeil, S.; Jones, G.R.; Shewmaker, W.D.; Lorenzen, N.; Anderson, E.D.; Kurath, G.

    2001-01-01

    A DNA vaccine against a fish rhabdovirus, infectious hematopoietic necrosis virus (IHNV), was shown to provide significant protection as soon as 4 d after intramuscular vaccination in 2 g rainbow trout (Oncorhynchus mykiss) held at 15??C. Nearly complete protection was also observed at later time points (7, 14, and 28 d) using a standardized waterborne challenge model. In a test of the specificity of this early protection, immunization of rainbow trout with a DNA vaccine against another fish rhabdovirus, viral hemorrhagic septicemia virus, provided a significant level of cross-protection against IHNV challenge for a transient period of time, whereas a rabies virus DNA vaccine was not protective. This indication of distinct early and late protective mechanisms was not dependent on DNA vaccine doses from 0.1 to 2.5 ??g. ?? 2001 Elsevier Science Ltd.

  5. Protection of rainbow trout against infectious hematopoietic necrosis virus four days after specific or semi-specific DNA vaccination.

    PubMed

    LaPatra, S E; Corbeil, S; Jones, G R; Shewmaker, W D; Lorenzen, N; Anderson, E D; Kurath, G

    2001-07-16

    A DNA vaccine against a fish rhabdovirus, infectious hematopoietic necrosis virus (IHNV), was shown to provide significant protection as soon as 4 d after intramuscular vaccination in 2 g rainbow trout (Oncorhynchus mykiss) held at 15 degrees C. Nearly complete protection was also observed at later time points (7, 14, and 28 d) using a standardized waterborne challenge model. In a test of the specificity of this early protection, immunization of rainbow trout with a DNA vaccine against another fish rhabdovirus, viral hemorrhagic septicemia virus, provided a significant level of cross-protection against IHNV challenge for a transient period of time, whereas a rabies virus DNA vaccine was not protective. This indication of distinct early and late protective mechanisms was not dependent on DNA vaccine doses from 0.1 to 2.5 microg. PMID:11427277

  6. Assembly and release of infectious hepatitis C virus involving unusual organization of the secretory pathway

    PubMed Central

    Triyatni, Miriam; Berger, Edward A; Saunier, Bertrand

    2016-01-01

    AIM: To determine if calnexin (CANX), RAB1 and alpha-tubulin were involved in the production of hepatitis C virus (HCV) particles by baby hamster kidney-West Nile virus (BHK-WNV) cells. METHODS: Using a siRNA-based approach complemented with immuno-fluorescence confocal microscope and Western blot studies, we examined the roles of CANX, RAB1 and alpha-tubulin in the production of HCV particles by permissive BHK-WNV cells expressing HCV structural proteins or the full-length genome of HCV genotype 1a. Immuno-fluorescence studies in producer cells were performed with monoclonal antibodies against HCV structural proteins, as well as immunoglobulin from the serum of a patient recently cured from an HCV infection of same genotype. The cellular compartment stained by the serum immunoglobulin was also observed in thin section transmission electron microscopy. These findings were compared with the JFH-1 strain/Huh-7.5 cell model. RESULTS: We found that CANX was necessary for the production of HCV particles by BHK-WNV cells. This process involved the recruitment of a subset of HCV proteins, detected by immunoglobulin of an HCV-cured patient, in a compartment of rearranged membranes bypassing the endoplasmic reticulum-Golgi intermediary compartment and surrounded by mitochondria. It also involved the maturation of N-linked glycans on HCV envelope proteins, which was required for assembly and/or secretion of HCV particles. The formation of this specialized compartment required RAB1; upon expression of HCV structural genes, this compartment developed large vesicles with viral particles. RAB1 and alpha-tubulin were required for the release of HCV particles. These cellular factors were also involved in the production of HCVcc in the JFH-1 strain/Huh-7.5 cell system, which involves HCV RNA replication. The secretion of HCV particles by BHK-WNV cells presents similarities with a pathway involving caspase-1; a caspase-1 inhibitor was found to suppress the production of HCV

  7. Fulminant Epstein-Barr virus - infectious mononucleosis in an adult with liver failure, splenic rupture, and spontaneous esophageal bleeding with ensuing esophageal necrosis: a case report

    PubMed Central

    2014-01-01

    Introduction Infectious mononucleosis is a clinical syndrome most commonly associated with primary Epstein-Barr virus infection. The majority of patients with infectious mononucleosis recovers without apparent sequelae. However, infectious mononucleosis may be associated with several acute complications. In this report we present a rare case of esophageal rupture that has never been described in the literature before. Case presentation We present the case of an 18-year-old Caucasian man affected by severe infectious mononucleosis complicated by fulminant hepatic failure, splenic rupture and esophageal necrosis. Conclusions Although primary Epstein-Barr virus infection is rarely fatal, fulminant infection may occur - in this case leading to hepatic failure, splenic rupture and esophageal necrosis, subsequently making several surgical interventions necessary. We show here that infectious mononucleosis is not only a strictly medical condition, but can also lead to severe surgical complications. PMID:24499457

  8. DNA vaccination with VP2 gene fragment confers protection against Infectious Bursal Disease Virus in chickens.

    PubMed

    Pradhan, Satya Narayan; Prince, Prabhu Rajaiah; Madhumathi, Jayaprakasam; Arunkumar, Chakkaravarthy; Roy, Parimal; Narayanan, Rangarajan Badri; Antony, Usha

    2014-06-25

    Infectious Bursal Disease Virus (IBDV) causes immunosuppression in young chickens by destruction of antibody producing B cells in the Bursa of Fabricius and poses a potential threat to the poultry industry. We have examined the protective efficacy of a subunit DNA vaccine against IBDV infection in chickens in this study. An immunodominant VP2 gene fragment (VP252-417) was cloned into CMV promoter based DNA vaccine vector pVAX1 and in vitro expression of the DNA encoded antigens was confirmed by transfection of CHO cells with vaccine constructs followed by RT-PCR and western blot analysis using IBDV-antiserum. Two weeks old chickens were immunized intramuscularly with pVAXVP252-417 and the in vivo transcription of the plasmid DNA was confirmed by RT-PCR analysis of DNA injected muscle tissue at different intervals of post immunization. Tissue distribution analysis revealed that the plasmid DNA was extensively distributed in muscle, spleen, kidney, liver, and bursa tissues. Chickens immunized with pVAXVP252-417 developed high titer (1:12,000) of anti-VP252-417 antibodies. Further, chicken splenocytes from pVAXVP252-417 immunized group showed a significantly high proliferation to the whole viral and recombinant antigen (P<0.01) compared to control groups, which implies that pVAXVP252-417 codes for immunogenic fragment which has epitopes capable of eliciting both B and T cell responses. This is evident by the fact that, pVAXVP252-417 immunized chicken conferred 75% protection against virulent IBDV (vIBDV) challenge compared to the control group. Thus, the present study confirms that the immunodominant VP2 fragment can be used as a potential DNA vaccine against IBDV infection in chickens. PMID:24745626

  9. Factors associated with introduction of infectious laryngotracheitis virus on broiler farms during a localized outbreak.

    PubMed

    Volkova, Victoriya; Thornton, Danny; Hubbard, Sue Ann; Magee, Danny; Cummings, Tim; Luna, Lynne; Watson, Jim; Wills, Robert

    2012-09-01

    We analyzed factors involved in the introduction of infectious laryngotracheitis (ILT) virus (ILTV) onto broiler farms during a localized outbreak in an immunologically naive broiler population. The outbreak occurred in the state of Mississippi, United States in 2002-2003. From the responses to a retrospective survey questionnaire administered via personal interviews, 181 farm-level risk factors were defined and analyzed for their association with ILTV introduction using logistic regression. There were 27 case farms (93% of all the infected broiler farms) and two sets of controls: farms matched to the cases by location and those randomly selected among the broiler farms in Mississippi. We found that farm suppliers such as gas company representatives, who are likely to visit other farms, and farm-workers who visit other chicken farms, are likely vehicles of ILTV introduction onto broiler farms. These risks can be greatly reduced by following biosecurity procedures, in particular if farm workers bathe and change footwear prior to entering broiler houses on their own farm. Footbaths for farm visitors can provide a false sense of security during an ILT outbreak when, indeed, other practices such as plastic boots or changing boots are more effective in preventing ILTV transmission. Sharing of equipment used for removal of caked broiler litter between subsequent flocks may also serve as an important vehicle of ILTV transmission. During the 2002-2003 outbreak, shared litter removal equipment was associated with ILTV transmission despite a requirement being put in place for litter decontamination. We also found that tunnel-ventilated broiler houses with inlets toward a neighboring poultry farm are more likely to get infected with ILTV. In addition to this analysis, the data collected provide a good overview of the actual practices and deficiencies of biosecurity undertaken on broiler farms in this part of the United States. PMID:23050469

  10. Genome sequence comparison of two United States live attenuated vaccines of infectious laryngotracheitis virus (ILTV).

    PubMed

    Chandra, Yohanna Gita; Lee, Jeongyoon; Kong, Byung-Whi

    2012-06-01

    This study was conducted to identify unique nucleotide differences in two U.S. chicken embryo origin (CEO) vaccines [LT Blen (GenBank accession: JQ083493) designated as vaccine 1; Laryngo-Vac(®) (GenBank accession: JQ083494) designated as vaccine 2] of infectious laryngotracheitis virus (ILTV) genomes compared to an Australian Serva vaccine reference ILTV genome sequence [Gallid herpesvirus 1 (GaHV-1); GenBank accession number: HQ630064]. Genomes of the two vaccine ILTV strains were sequenced using Illumina Genome Analyzer 2X of 36 cycles of single-end reads. Results revealed that few nucleotide differences (23 in vaccine 1; 31 in vaccine 2) were found and indicate that the US CEO strains are practically identical to the Australian Serva CEO strain, which is a European-origin vaccine. The sequence differences demonstrated the spectrum of variability among vaccine strains. Only eight amino acid differences were found in ILTV proteins including UL54, UL27, UL28, UL20, UL1, ICP4, and US8 in vaccine 1. Similarly, in vaccine 2, eight amino acid differences were found in UL54, UL27, UL28, UL36, UL1, ICP4, US10, and US8. Further comparison of US CEO vaccines to several ILTV genome sequences revealed that US CEO vaccines are genetically close to both the Serva vaccine and 63140/C/08/BR (GenBank accession: HM188407) and are distinct from the two Australian-origin CEO vaccines, SA2 (GenBank accession: JN596962) and A20 (GenBank accession: JN596963), which showed close similarity to each other. These data demonstrate the potential of high-throughput sequencing technology to yield insight into the sequence variation of different ILTV strains. This information can be used to discriminate between vaccine ILTV strains and further, to identify newly emerging mutant strains of field isolates. PMID:22382591

  11. Genome-wide host responses against infectious laryngotracheitis virus vaccine infection in chicken embryo lung cells

    PubMed Central

    2012-01-01

    Background Infectious laryngotracheitis virus (ILTV; gallid herpesvirus 1) infection causes high mortality and huge economic losses in the poultry industry. To protect chickens against ILTV infection, chicken-embryo origin (CEO) and tissue-culture origin (TCO) vaccines have been used. However, the transmission of vaccine ILTV from vaccinated- to unvaccinated chickens can cause severe respiratory disease. Previously, host cell responses against virulent ILTV infections were determined by microarray analysis. In this study, a microarray analysis was performed to understand host-vaccine ILTV interactions at the host gene transcription level. Results The 44 K chicken oligo microarrays were used, and the results were compared to those found in virulent ILTV infection. Total RNAs extracted from vaccine ILTV infected chicken embryo lung cells at 1, 2, 3 and 4 days post infection (dpi), compared to 0 dpi, were subjected to microarray assay using the two color hybridization method. Data analysis using JMP Genomics 5.0 and the Ingenuity Pathway Analysis (IPA) program showed that 213 differentially expressed genes could be grouped into a number of functional categories including tissue development, cellular growth and proliferation, cellular movement, and inflammatory responses. Moreover, 10 possible gene networks were created by the IPA program to show intermolecular connections. Interestingly, of 213 differentially expressed genes, BMP2, C8orf79, F10, and NPY were expressed distinctly in vaccine ILTV infection when compared to virulent ILTV infection. Conclusions Comprehensive knowledge of gene expression and biological functionalities of host factors during vaccine ILTV infection can provide insight into host cellular defense mechanisms compared to those of virulent ILTV. PMID:22530940

  12. Pathogenicity and growth characteristics of selected infectious laryngotracheitis virus strains from the United States.

    PubMed

    Oldoni, Ivomar; Rodríguez-Avila, Andrés; Riblet, Sylva M; Zavala, Guillermo; García, Maricarmen

    2009-02-01

    In a recent study, several US infectious laryngotracheitis virus (ILTV) strains and field isolates were genotyped by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) into nine different genotypes. All of the commercial poultry isolates were identified within genotypes IV, V, and VI. Based on the PCR-RFLP, Group IV isolates were characterized as genetically identical to the chicken embryo origin (CEO) vaccines, Group V as genetically closely related to the CEO vaccines, and Group VI as genetically different to the vaccine strains. The objective of this study was to determine the pathogenicity and growth characteristics of six ILTV commercial poultry isolates as compared with the CEO vaccine. Two isolates representative of PCR-RFLP Groups IV, V, and VI were selected. Differences in disease severity, viral tissue distribution in chickens, and plaque formation ability in cell culture were observed among viral genotypes IV, V, and VI, and between V-A and V-B isolates. Mild respiratory clinical signs were produced by IV-A, IV-B and the CEO vaccine, while VI-A and VI-B isolates produced severe respiratory signs and severe depression, and during the peak of clinical signs both isolates were re-isolated from the conjunctiva, sinus, trachea and thymus. Similarly to Group VI isolates, V-A and V-B produced severe respiratory signs, depression, and were re-isolated from conjunctiva, sinus, and trachea; on cell culture, both isolates produced significant larger plaques than any of the other isolates analysed. Overall, differences in pathogenicity and growth characteristics were observed among genetically closely related US ILTV isolates; however, complete genomes will be necessary to identify molecular determinants linked to the pathogenic viral phenotypes. PMID:19156579

  13. Identification and expression analysis of infectious laryngotracheitis virus encoding microRNAs.

    PubMed

    Rachamadugu, Rakesh; Lee, Jeong Yoon; Wooming, Ann; Kong, Byung-Whi

    2009-12-01

    MicroRNAs (miRNAs) are a class of small non-coding RNA molecules that play a pivotal role in the regulation of gene expression at the post transcriptional level. Infectious laryngotracheitis virus (ILTV) is an alphaherpesvirus belonging to the herpesviridae family. It causes an acute respiratory disorder in chicken resulting in high mortality rates. ILTV encoding miRNAs have been identified in cell cultures infected by ILTV. Seven candidates ILTV encoding miRNAs were identified by the 454 FLX genome sequencing method. Five ILTV miRNAs identified in this study were identical to those previously reported by Waidner et al. (Virology 388:128, 2009). Two unique ILTV miRNAs, iltv-miR-I1-3p and iltv-miR-I7-3p, were identified in this study. The iltv-miR-I1-3p is the passenger strand of I1-5p, which was previously known. The iltv-miR-I7-3p showed a perfect match with the complementary passenger strand in contrast to other miRNA species showing imperfect complementarity with the passenger strand. The I7-3p was mapped in the replication origin (oriL) of the palindrome stem loop sequence of the ILTV genome. Expression of all ILTV miRNAs were confirmed by the end point PCR using small RNA libraries generated from either ILTV infected or uninfected control chicken embryo kidney (CEK) cells. PMID:19728068

  14. Sequence analysis of both genome segments of three Croatian infectious bursal disease field viruses.

    PubMed

    Lojkić, I; Bidin, Z; Pokrić, B

    2008-09-01

    In order to determine the mutations responsible for virulence, three Croatian field infectious bursal disease viruses (IBDV), designated Cro-Ig/02, Cro-Po/00, and Cro-Pa/98 were characterized. Coding regions of both genomic segments were sequenced, and the nucleotide and deduced amino acid sequences were compared with previously reported full-length sequenced IBDV strains. Phylogenetic analysis, based on the nucleotide and deduced amino acid sequences of polyprotein and VP1, was performed. Eight characteristic amino acid residues, that were common to very virulent (vv) IBDV, were detected on polyprotein: 222A, 256I, 294I, 451L, 685N, 715S, 751D, and 1005A. All eight were found in Cro-Ig/02 and Cro-Po/00. C-Pa/98 had all the characteristics of an attenuated strain, except for glutamine on residue 253, which is common for vv, classical virulent, and variant strains. Between less virulent and vvIBDV, three substitutions were found on VP5: 49 G --> R, 79 --> F, and 137 R --> W. In VP1, there were nine characteristic amino acid residues common to vvwIBDV: 146D, 147N, 242E, 390M, 393D, 511S, 562P, 687P, and 695R. All nine residues were found in A-Ig/02, and eight were found in B-Po/00, which had isoleucine on residue 390. Based on our analyses, isolates Cro-Ig/02 and Cro-Po/00 were classified with vv IBDV strains. C-Pa/98 shared all characteristic amino acid residues with attenuated and classical virulence strains, so it was classified with those. PMID:18939645

  15. Detection of infectious bursal disease virus isolates with unknown antigenic properties by reverse genetics.

    PubMed

    Icard, Alan H; Sellers, Holly S; Mundt, Egbert

    2008-12-01

    Infectious bursal disease virus (IBDV) serotype 1 is the causative agent of a highly contagious immunosuppressive disease of young chickens. In the past, a number of antigenic, as well as pathogenic, subtypes have been described. The determination of the antigenic makeup of circulating strains is of vital interest to the poultry industry because changes in the antigenicity of circulating field strains have an impact on the use of vaccines. To obtain a more comprehensive overview of the relationship between the nucleotide and amino acid sequence and the antigenic makeup of field isolates, a system based on reverse genetics of IBDV was established. Using this approach, a database for field isolates from three different states in the United States (Georgia, Alabama, and Louisiana), consisting of nucleotide sequence, amino acid sequence, and a reaction pattern based on a panel of monoclonal antibodies, was established. The obtained results showed that phylogenic analysis, which is based on the similarity of sequences, would lead to false conclusions regarding a possible antigenic makeup of the particular isolate. Sequences of field samples were divided into three groups: 1) those that grouped with variant strain E/Del sequences but were antigenically different, 2) those that did not group with sequences of E/Del but were similar in their antigenic makeup, and 3) those that did not group with E/Del sequences and were antigenically different. In addition, using the reverse-genetics approach, a number of field isolates showed no reactivity with any of the used monoclonal antibodies, indicating that an unknown, antigenic subtype of IBDV serotype 1 is circulating in the field. PMID:19166049

  16. Serologically silent, occult equine infectious anemia virus (EIAV) infections in horses.

    PubMed

    Ricotti, Sonia; Garcia, Maria Inés; Veaute, Carolina; Bailat, Alejandra; Lucca, Eduardo; Cook, R Frank; Cook, Sheila J; Soutullo, Adriana

    2016-05-01

    Molecular and serological techniques for Equine Infectious Anemia Virus (EIAV) diagnosis were compared using samples from 59 clinically normal horses stabled on five farms in the Santa Fe Province of Argentina. Of these 26 (44.1%) were positive in official AGID tests and/or gp45/gp90-based ELISA. Surprisingly 18 of the 33 seronegative horses were positive in a PCR against viral sequences encoding gp45 (PCR-positive/AGID-negative) with all but one remaining EIAV-antibody negative throughout a two year observation period. The gp45 PCR results are supported by fact that 7/18 of these horses were positive in the Office International des Epizooties (OIE) recommended EIAV gag gene specific PCR plus 2 of this 7 also reacted in a PCR directed predominantly against the 5' untranslated region of the viral genome. Furthermore sufficient quantities of serum were available from 8 of these horses to verify their seronegative status in sensitive Western Blot tests and demonstrate by ELISA the absence of EIAV-specific antibodies was not attributable to abnormalities in total IgG concentration. Studies involving 7 of the PCR-positive/AGID-negative horses to measure lymphocyte proliferation in the presence of PHA showed no significant differences between this group and control animals. In addition, lymphocytes from 2 of these 7 horses responded to peptides derived from gp90 and gp45. Together these results demonstrate that apparently clinically normal horses with no gross signs of immunodeficiency in terms of total IgG concentration or T helper-cell function can remain seronegative for at least 24 months while harboring EIAV specific nucleic acid sequences. PMID:27066707

  17. Tracking fluorescence-labeled rabies virus: enhanced green fluorescent protein-tagged phosphoprotein P supports virus gene expression and formation of infectious particles.

    PubMed

    Finke, Stefan; Brzózka, Krzysztof; Conzelmann, Karl-Klaus

    2004-11-01

    Rhabdoviruses such as rabies virus (RV) encode only five multifunctional proteins accomplishing viral gene expression and virus formation. The viral phosphoprotein, P, is a structural component of the viral ribonucleoprotein (RNP) complex and an essential cofactor for the viral RNA-dependent RNA polymerase. We show here that RV P fused to enhanced green fluorescent protein (eGFP) can substitute for P throughout the viral life cycle, allowing fluorescence labeling and tracking of RV RNPs under live cell conditions. To first assess the functions of P fusion constructs, a recombinant RV lacking the P gene, SAD DeltaP, was complemented in cell lines constitutively expressing eGFP-P or P-eGFP fusion proteins. P-eGFP supported the rapid accumulation of viral mRNAs but led to low infectious-virus titers, suggesting impairment of virus formation. In contrast, complementation with eGFP-P resulted in slower accumulation of mRNAs but similar infectious titers, suggesting interference with polymerase activity rather than with virus formation. Fluorescence microscopy allowed the detection of eGFP-P-labeled extracellular virus particles and tracking of cell binding and temperature-dependent internalization into intracellular vesicles. Recombinant RVs expressing eGFP-P or an eGFP-P mutant lacking the binding site for dynein light chain 1 (DLC1) instead of P were used to track interaction with cellular proteins. In cells expressing a DsRed-labeled DLC1, colocalization of DLC1 with eGFP-P but not with the mutant P was observed. Fluorescent labeling of RV RNPs will allow further dissection of virus entry, replication, and egress under live-cell conditions as well as cell interactions. PMID:15507620

  18. Expression of the infectious salmon anemia virus receptor on atlantic salmon endothelial cells correlates with the cell tropism of the virus.

    PubMed

    Aamelfot, Maria; Dale, Ole Bendik; Weli, Simon Chioma; Koppang, Erling Olaf; Falk, Knut

    2012-10-01

    Infectious salmon anemia (ISA) is a World Organization for Animal Health (OIE)-listed disease of farmed Atlantic salmon, characterized by slowly developing anemia and circulatory disturbances. The disease is caused by ISA virus (ISAV) in the Orthomyxoviridae family; hence, it is related to influenza. Here we explore the pathogenesis of ISA by focusing on virus tropism, receptor tissue distribution, and pathological changes in experimentally and naturally infected Atlantic salmon. Using immunohistochemistry on ISAV-infected Atlantic salmon tissues with antibody to viral nucleoprotein, endotheliotropism was demonstrated. Endothelial cells lining the circulatory system were found to be infected, seemingly noncytolytic, and without vasculitis. No virus could be found in necrotic parenchymal cells. From endothelium, the virus budded apically and adsorbed to red blood cells (RBCs). No infection or replication within RBCs was detected, but hemophagocytosis was observed, possibly contributing to the severe anemia in fish with this disease. Similarly to what has been done in studies of influenza, we examined the pattern of virus attachment by using ISAV as a probe. Here we detected the preferred receptor of ISAV, 4-O-acetylated sialic acid (Neu4,5Ac(2)). To our knowledge, this is the first report demonstrating the in situ distribution of this sialic acid derivate. The pattern of virus attachment mirrored closely the distribution of infection, showing that the virus receptor is important for cell tropism, as well as for adsorption to RBCs. PMID:22811536

  19. Studies on uptake and intracellular processing of infectious pancreatic necrosis virus by Atlantic cod scavenger endothelial cells.

    PubMed

    Martin-Armas, M; Sommer, A-I; Smedsrød, B

    2007-11-01

    Previous work in our group has identified the scavenger endothelial cells (SECs) of heart endocardium in cod, Gadus morhua L., as the major site for elimination of both physiological and foreign macromolecular waste from the circulation. The present study was undertaken to establish the role of cod SECs in the clearance of virus. We focused on infectious pancreatic necrosis virus (IPNV) as it is a well-known virus with a broad host range, and causes significant economic losses in the salmon industry. Our results showed that cod SEC cultures infected by the IPNV produce high titres of new virus. Ligand-receptor inhibition experiments suggested that the virus did not enter the cells through any of the major endocytosis receptors of cod SECs. Yet, the infection lowered the capacity of the cells to endocytose ligands via the scavenger receptor. Inhibitors of receptor recycling and vesicle acidification did not affect virus infectivity. The finding that SEC cultures prepared from 25% of the cod produced high titres of IPNV without being infected in the laboratory, suggests that SECs of cod may serve as reservoirs for IPNV in persistently infected cod. PMID:17958614

  20. Development and Evaluation of the Protective Efficacy of Novel Marek's Disease Virus Rispens Vector Vaccines Against Infectious Bursal Disease.

    PubMed

    Ishihara, Yukari; Esaki, Motoyuki; Saitoh, Shuji; Sato, Takanori; Yasuda, Atsushi

    2016-09-01

    Infectious bursal disease (IBD) is a major disease affecting the poultry industry and is caused by infection with IBD virus (IBDV). To develop a novel vaccine to prevent IBD in chickens, recombinant Marek's disease virus Rispens viruses carrying the VP2 gene of IBDV driven by five different promoters (Rispens/IBD) were constructed using homologous recombination and a bacterial artificial chromosome (BAC). Rispens/IBD driven by the chicken beta-actin (Bac) promoter (Rispens/Bac-IBD), Rous sarcoma virus promoter, or simian virus 40 promoter were administered to 1-day-old SPF chicks, and the protective efficacy against IBDV was evaluated by challenging chicks with virulent IBDV. As a result, Rispens/Bac-IBD showed the best protection (87%). Next, we constructed the virus driven by the Bac-derived Coa5 promoter (Rispens/Coa5-IBD) for a secondary in vivo trial using commercial layer chickens since Rispens/Bac-IBD was thought to be genetically unstable. Rispens/Coa5-IBD showed stability in vitro and exhibited better antibody production and protection during challenge against virulent IBDV at both 5 (95%) and 7 wk of age (91%) compared with that of Rispens/Bac-IBD (90% at 5 wk of age and 84% at 7 wk of age). Thus, Rispens/Coa5-IBD may be a novel promising vaccine against IBD and virulent Marek's disease. PMID:27610721

  1. Biochemical and antigenic properties of the first isolates of infectious hematopoietic necrosis virus from salmonid fish in Europe

    USGS Publications Warehouse

    Arkush, K.D.; Bovo, G.; deKinkelin, P.; Winton, J.R.; Wingfield, W.H.; Hedrick, R.P.

    1989-01-01

    The first isolates of infectious hematopoietic necrosis virus (IHNV) recovered from rainbow trout Oncorhynchus mykiss (formerly Salmo gairdneri) in France and Italy were compared to six representative strains from North America by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of virion polypeptides and neutralization by monoclonal antibodies (MAbs). All three IHNV isolates from Europe had similar polypeptide profiles when compared by SDS-PAGE. An analysis of the antigenic relatedness of the European isolates to representative strains from North America showed that they were clearly different from viruses obtained from salmonids in California. The RB/B5 MAb, which was developed against virus isolated from adult steelhead (anadromous rainbow trout) reared in central Oregon, neutralized all isolates examined. The 193–110/B4 MAb, developed against IHNV isolated from infected yearling rainbow trout in southern Idaho, neutralized all isolates tested except those from California. The SRCV/A4 MAb, developed against Sacramento River chinook virus (SRCV) isolated from adult spring chinook salmon O. tshawytscha in central California, was the least reactive, and strong neutralization was observed only with the SRCV strain of IHNV from California. However, partial reactivity of the virus isolates from France with the SRCV/A4 MAb distinguished them from the virus recovered from salmonids in Italy.

  2. Persistent infections with infectious pancreatic necrosis virus (IPNV) of different virulence in Atlantic salmon, Salmo salar L.

    PubMed

    Julin, K; Johansen, L-H; Sommer, A-I; Jørgensen, J B

    2015-11-01

    Infectious pancreatic necrosis virus (IPNV) is a prevalent pathogen in fish worldwide. The virus causes substantial mortality in Atlantic salmon juveniles and smolts when transferred to sea water and persistent infection in surviving fish after disease outbreaks. Here, we have investigated the occurrence of the virus as well as the innate immune marker Mx in the head kidney (HK) of Atlantic salmon throughout an experimental challenge covering both a fresh and a seawater phase. The fish were challenged with a high (HV) and low virulence (LV) IPNV. Both isolates caused mortality due to reactivation of the virus after transfer to sea water. In the freshwater phase, higher levels of virus transcripts were detected in the HK of fish infected with LV IPNV compared to HV, suggesting that the HV isolate is able to limit its own replication to a level where the innate immune system is not alerted. Further, ex vivoHK leucocytes derived from fish infected with the two isolates were stimulated with CpG DNA. Significantly, higher IFN levels were found in the LV compared to the HV group in the freshwater phase. This suggests that the viruses attenuate the antiviral host immune response at different levels which may contribute to the observed differences in disease outcome. PMID:25557127

  3. Transcriptome analysis of rainbow trout infected with high and low virulence strains of Infectious hematopoietic necrosis virus

    USGS Publications Warehouse

    Purcell, Maureen K.; Marjara, Inderjit Singh; Batts, William; Kurath, Gael; Hansen, John D.

    2010-01-01

    There are three main genetic lineages or genogroups of Infectious hematopoietic necrosis virus (IHNV) in N. America. Strains representing the M genogroup are more virulent in rainbow trout relative to the U genogroup. In this study, we used microarray analysis to evaluate potential mechanisms responsible for host-specific virulence in rainbow trout that were given intraperitoneal injections of buffer or a representative M or U type virus strain. Reverse transcriptase quantitative PCR (RT-qPCR) was used to assess viral load and gene expression of select immune genes. Viral load was significantly higher in trout infected with the M virus starting at 24 h post-infection (p.i.) and continuing until 72 h p.i. Microarray analysis of the 48 h time point revealed 153 up-regulated and 248 down-regulated features in response to M virus infection but only 62 up-regulated and 49 down-regulated features following U virus infection. Translation and transcription features were among the most frequent down-regulated features in response to M virus infection and may be associated with the host cell shutoff phenomenon. A greater host cell shutoff response by the M virus may facilitate subversion of the host cell transcriptional machinery and enhance viral replication, suggesting the M virus may be better optimized to manipulate the rainbow trout transcriptional and translational machinery. Anti-viral associated features were the most commonly up-regulated features. A common set of features were up-regulated in both the M and U infection groups, but were induced to a higher magnitude in the M infection group. Gene expression of the anti-viral genes Mx-1 and Vig-1 was correlated but not entirely dependent on viral load in the anterior kidney. Slower replication of the U virus may allow the host more time to induce protective anti-viral immune mechanisms.

  4. A universal next generation sequencing protocol to generate non-infectious barcoded cDNA libraries from high containment RNA viruses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Several biosafety level (BSL)-3/4 pathogens are high consequence, single-stranded RNA viruses and their genomes, when introduced into permissive cells, are infectious. Moreover many of these viruses are Select Agents (SAs), and their genomes are also considered SAs. For this reason cDNAs and/or th...

  5. Using mean infectious dose of wild duck-and poultry-origin high and low pathogenicity avian influenza viruses as one measure of infectivity and adaptation to poultry

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The mean infectious doses of selected avian influenza virus (AIV) isolates, determined in domestic poultry under experimental conditions, were shown to be both host and virus dependent and could be considered one measure of the infectivity and adaptation to a specific host. As such, the mean infect...

  6. Evaluation of bivalent Newcastle disease virus (NDV) vectored infectious laryngotracheitis vaccines in broiler chickens in the presence of NDV maternally derived antibody

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Previously we have demonstrated that Newcastle disease virus (NDV) recombinants expressing the infectious laryngotracheitis virus (ILTV) glycoproteins B (gB) or D (gD) protein conferred complete clinical protection against ILTV and NDV challenges in specific pathogen free (SPF) and 3 week old commer...

  7. Identification and characterization of two distinct bursal B-cell subpopulations following infectious bursal disease virus infection of White Leghorn chickens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Infectious bursal disease virus (IBDV) is an immunosuppressive virus which primarily infects IgM, B-cells in the bursa of Fabricius. Flow cytometric analysis was used to phenotype B-cell populations in the bursa and spleen following IBDV infection. In the bursa, two IgM B-cell subpopulations, desig...

  8. Complete sequence and development of a full-length infectious clone of an Ohio isolate of Maize dwarf mosaic virus (MDMV)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Maize dwarf mosaic virus (MDMV) is an important and widespread aphid-transmitted virus of corn. It is a member of the genus Potyvirus in the family Potyviridae. Here we report the complete genome sequence of, and construction of an infectious clone of an Ohio isolate of MDMV-A. MDMV sequences from i...

  9. Immature Dengue Virus Is Infectious in Human Immature Dendritic Cells via Interaction with the Receptor Molecule DC-SIGN

    PubMed Central

    Torres Pedraza, Silvia; Hoornweg, Tabitha E.; van de Pol, Denise P. I.; Rodenhuis-Zybert, Izabela A.; Wilschut, Jan; Smit, Jolanda M.

    2014-01-01

    Background Dengue Virus (DENV) is the most common mosquito-borne viral infection worldwide. Important target cells during DENV infection are macrophages, monocytes, and immature dendritic cells (imDCs). DENV-infected cells are known to secrete a large number of partially immature and fully immature particles alongside mature virions. Fully immature DENV particles are considered non-infectious, but antibodies have been shown to rescue their infectious properties. This suggests that immature DENV particles only contribute to the viral load observed in patients with a heterologous DENV re-infection. Methodology/Principal findings In this study, we re-evaluated the infectious properties of fully immature particles in absence and presence of anti-DENV human serum. We show that immature DENV is infectious in cells expressing DC-SIGN. Furthermore, we demonstrate that immature dendritic cells, in contrast to macrophage-like cells, do not support antibody-dependent enhancement of immature DENV. Conclusions/Significance Our data shows that immature DENV can infect imDCs through interaction with DC-SIGN, suggesting that immature and partially immature DENV particles may contribute to dengue pathogenesis during primary infection. Furthermore, since antibodies do not further stimulate DENV infectivity on imDCs we propose that macrophages/monocytes rather than imDCs contribute to the increased viral load observed during severe heterotypic DENV re-infections. PMID:24886790

  10. [A Case of Acute Acalculous Cholecystitis During Infectious Mononucleosis Caused by the Epstein-Barr Virus in a Young Woman].

    PubMed

    Ono, Shiro; Kobayashi, Tadanao; Nishio, Kenji

    2016-05-01

    Infection with the Epstein-Barr virus (EBV) is a common disease and is mainly asymptomatic during childhood, whereas infectious mononucleosis with clinical signs such as fever, pharyngitis, lymphadenopathy and hepatosplenomegaly often occurs in adolescents and adults with primary infection. Acalculous cholecystitis has been reported as a rare complication. We report herein a case of acalculous cholecystitis accompanied by infectious mononucleosis by EBV, which was treated successfully by medical treatment. A 33-year-old woman who had been admitted by fever, pharyngitis and lymphadenopathy developed a right upper quadrant pain, that was diagnosed as acalculous cholecystitis based on an imaging study. Antibiotic treatment did not resolve the symptoms, and surgical intervention was considered. We diagnosed her as having infectious mononucleosis based on a typical physical presentation and seropositivity for the EBV viral capsid antigen, suggesting that the acalculous cholecystatis might have been a complication of the EBV infection. After the administration of glucocorticoid and acyclovir, the patient became afebrile and the abdominal pain disappeared. Though acalculous cholecystitis rarely accompanies infectious mononucleosis caused by EBV, clinicians should be aware of this complication to avoid unnecessary cholecystectomy. PMID:27529970

  11. Soil-borne wheat mosaic virus infectious clone and manipulation for gene-carrying capacity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Soilborne wheat mosaic virus (SBWMV) is a bipartite single stranded positive sense RNA virus with rigid-rod shaped virions. Taxonomically the virus is in the family Viragviridae, as are commonly used gene silencing or expression viral vectors, Tobacco rattle virus (TRV) and Barley stripe mosaic viru...

  12. Generation of serotype 1/serotype 2 reassortant viruses of the infectious bursal disease virus and their investigation in vitro and in vivo.

    PubMed

    Zierenberg, Kati; Raue, Rüdiger; Nieper, Hermann; Islam, Md Rafiqul; Eterradossi, Nicolas; Toquin, Didier; Müller, Hermann

    2004-09-15

    Infectious bursal disease virus (IBDV) is the causative agent of acute or immunosuppressive disease in chickens. Serotype 1 strains are pathogenic whereas serotype 2 strains neither cause disease nor protect against infection with the serotype 1 strains. The target organ of serotype 1 strains is the bursa Fabricii (BF). The molecular determinants of this tropism, and therefore pathogenicity, are poorly understood. IBDV is a non-enveloped icosahedral virus particle of 60 nm in diameter, which contains two genome segments of double-stranded RNA. Here, the generation of interserotypic reassortants using the reverse genetics approach is reported. The results of in vitro and in vivo investigations show that genome segment A determines the bursa tropism of IBDV, whereas segment B is involved in the efficiency of viral replication; they further indicate the significance of the interaction of the polymerase (segment B) with the structural protein VP3 (segment A) or the viral genome for efficient virus formation and replication. PMID:15325078

  13. A reassortment vaccine candidate as the improved formulation to induce protection against very virulent infectious bursal disease virus.

    PubMed

    Qi, Xiaole; Chen, Yuming; Ren, Xiangang; Zhang, Lizhou; Gao, Li; Wang, Nian; Qin, Liting; Wang, Yongqiang; Gao, Yulong; Wang, Xiaomei

    2014-03-14

    Infectious bursal disease (IBD) is a highly contagious immunosuppressive disease affecting all major poultry producing areas of the world. Infectious bursal disease virus (IBDV) is genetically prone to mutation so that vaccines have to be changed accordingly. However, the traditional method of vaccine development with blind passage could not fit the style of the emergency prevention of IBDV. In this study, for the first time, a segment-reassortment attenuated IBDV rXATB, consisting of modified segment A of a prevalent strain and segment B of an attenuated strain, was designed and rescued; rXATB was stable and could induce good humoral and cellular immune responses which resulted in excellent protection against the lethal challenge of vvIBDV without obvious immunosuppression in chicken. This study revolutionarily provides a new formulation based on reverse genetics to develop new vaccine against prevalent IBDV. PMID:24486309

  14. Atlantic salmon, Salmo salar L. are broadly susceptible to isolates representing the North American genogroups of infectious hematopoietic necrosis virus

    USGS Publications Warehouse

    Kurath, Gael; Winton, James R.; Dale, Ole Bendik; Purcell, Maureen K.; Falk, Knut; Busch, Robert D.

    2016-01-01

    Beginning in 1992, three epidemic waves of infectious hematopoietic necrosis, often with high mortality, occurred in farmed Atlantic salmon Salmo salar L. on the west coast of North America. We compared the virulence of eleven strains of infectious hematopoietic necrosis virus (IHNV), representing the U, M and L genogroups, in experimental challenges of juvenile Atlantic salmon in freshwater. All strains caused mortality and there was wide variation within genogroups: cumulative mortality for five U-group strains ranged from 20 to 100%, four M-group strains ranged 30-63% and two L-group strains varied from 41 to 81%. Thus, unlike Pacific salmonids, there was no apparent correlation of virulence in a particular host species with virus genogroup. The mortality patterns indicated two different phenotypes in terms of kinetics of disease progression and final per cent mortality, with nine strains having moderate virulence and two strains (from the U and L genogroups) having high virulence. These phenotypes were investigated by histopathology and immunohistochemistry to describe the variation in the course of IHNV disease in Atlantic salmon. The results from this study demonstrate that IHNV may become a major threat to farmed Atlantic salmon in other regions of the world where the virus has been, or may be, introduced.

  15. Concentration of infectious hematopoietic necrosis virus from water samples by tangential flow filtration and polyethylene glycol precipitation

    USGS Publications Warehouse

    Batts, W.N.; Winton, J.R.

    1989-01-01

    Infectious hematopoietic necrosis virus (IHNV) was concentrated from water samples by polyethylene glycol (PEG) precipitation, tangential flow filtration (TFF), and by a combination of TFF followed by PEG precipitation of the retentate. Used alone, PEG increased virus titers more than 200-fold, and the efficiency of recovery was as great as 100%. Used alone, TFF concentrated IHNV more than 20-fold, and average recovery was 70%. When the two techniques were combined, 10-L water samples were reduced to about 300 mL by TFF and the virus was precipitated with PEG into a 1 to 2 g pellet; total recovery was as great as 100%. The combined techniques were used to isolate IHNV from water samples taken from a river containing adult sockeye salmon (Oncorhynchus nerka) and from a hatchery pond containing adult spring chinook salmon (O. tshawytscha). The combination of these methods was effective in concentrating and detecting IHNV from water containing only three infectious particles per 10-L sample.

  16. Construction of Recombinant Baculoviruses Expressing Infectious Bursal Disease Virus Main Protective Antigen and Their Immune Effects on Chickens.

    PubMed

    Ge, Jingping; An, Qi; Song, Shanshan; Gao, Dongni; Ping, Wenxiang

    2015-01-01

    In order to overcome the limitations of conventional vaccines for infectious bursal disease virus (IBDV), we constructed recombinant dual expression system baculoviruses with VP2 and VP2/4/3, the main protective antigens of IBDV. We compared the immune effects of the baculoviruses in avian cells and detected their control effects on chickens with infectious bursal disease. We used Western blot analysis to measure VP2 protein and VP2/4/3 polyprotein expression in avian cells infected using the Bac-to-Bac baculovirus expression system. The recombinant baculoviruses were used to vaccinate specific pathogen-free chickens, which produced specific protective antibodies and strong cellular immune responses. The results of the virus challenge experiment revealed that the protective efficiency of VP2 and VP2/4/3 virus vaccines were 95.8% and 100%, respectively, both of which were higher than the vaccine group (87.5%), and significantly higher than the control group (50%). The results demonstrated that the immune effect of BV-S-ITRs-VP2/4/3 was superior to that of BV-S-ITRs-VP2. Compared with traditional attenuated vaccine and genetically engineered live vector vaccine, the dual expression viral vector vaccine has good bio-safety. The results of this study provide a foundation for the further development of poultry vaccines, in addition to providing a useful reference for developing non-replicating live vaccines against other viral diseases. PMID:26167907

  17. Protective vaccination against infectious bursal disease virus with whole recombinant Kluyveromyces lactis yeast expressing the viral VP2 subunit.

    PubMed

    Arnold, Marina; Durairaj, Vijay; Mundt, Egbert; Schulze, Katja; Breunig, Karin D; Behrens, Sven-Erik

    2012-01-01

    Here we report on vaccination approaches against infectious bursal disease (IBD) of poultry that were performed with complete yeast of the species Kluyveromyces lactis (K. lactis). Employing a genetic system that enables the rapid production of stably transfected recombinant K. lactis, we generated yeast strains that expressed defined quantities of the virus capsid forming protein VP2 of infectious bursal disease virus (IBDV). Both, subcutaneous as well as oral vaccination regiments with the heat-inactivated but otherwise untreated yeast induced IBDV-neutralizing antibodies in mice and chickens. A full protection against a subsequent IBDV infection was achieved by subcutaneous inoculation of only milligram amounts of yeast per chicken. Oral vaccination also generated protection: while mortality was observed in control animals after virus challenge, none of the vaccinees died and ca. one-tenth were protected as indicated by the absence of lesions in the bursa of Fabricius. Recombinant K. lactis was thus indicated as a potent tool for the induction of a protective immune response by different applications. Subcutaneously applied K. lactis that expresses the IBDV VP2 was shown to function as an efficacious anti-IBD subunit vaccine. PMID:23024743

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

    PubMed

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

    2013-06-01

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

  19. Persistence of the tissue culture origin vaccine for infectious laryngotracheitis virus in commercial chicken flocks in Brazil.

    PubMed

    Parra, Silvana H Santander; Nuñez, Luis F; Astolfi-Ferreira, Claudete S; Ferreira, Antonio J Piantino

    2015-11-01

    Infectious laryngotracheitis (ILT) is a respiratory disease of great importance that causes serious economic losses in the poultry industry. Its control is based on biosecurity procedures and vaccination programs that use live attenuated vaccines such as tissue culture origin (TCO), chicken embryo origin (CEO), and vectored vaccines. However, problems have been reported, such as the reversion of virulence, virus latency, and field virus outbreaks. Several molecular techniques have been developed to differentiate between the field and vaccine strains. This study was conducted to determine the presence of infectious laryngotracheitis virus (ILTV) in Brazil from 2012 to 2014. PCR-RFLP (restriction fragment length polymorphism) was used to detect and differentiate ILTV strains; DNA sequencing and predictive RFLP analysis were also used for this purpose. Molecular analysis detected the presence of ILTV in 15 samples that were characterized as strains of TCO vaccine origin. This study showed that the ILTV TCO vaccine strain has been circulating in commercial chicken flocks in Brazil since its introduction during the 2002 outbreak. PMID:26500264

  20. Protective Vaccination against Infectious Bursal Disease Virus with Whole Recombinant Kluyveromyces lactis Yeast Expressing the Viral VP2 Subunit

    PubMed Central

    Arnold, Marina; Durairaj, Vijay; Mundt, Egbert; Schulze, Katja; Breunig, Karin D.; Behrens, Sven-Erik

    2012-01-01

    Here we report on vaccination approaches against infectious bursal disease (IBD) of poultry that were performed with complete yeast of the species Kluyveromyces lactis (K. lactis). Employing a genetic system that enables the rapid production of stably transfected recombinant K. lactis, we generated yeast strains that expressed defined quantities of the virus capsid forming protein VP2 of infectious bursal disease virus (IBDV). Both, subcutaneous as well as oral vaccination regiments with the heat-inactivated but otherwise untreated yeast induced IBDV-neutralizing antibodies in mice and chickens. A full protection against a subsequent IBDV infection was achieved by subcutaneous inoculation of only milligram amounts of yeast per chicken. Oral vaccination also generated protection: while mortality was observed in control animals after virus challenge, none of the vaccinees died and ca. one-tenth were protected as indicated by the absence of lesions in the bursa of Fabricius. Recombinant K. lactis was thus indicated as a potent tool for the induction of a protective immune response by different applications. Subcutaneously applied K. lactis that expresses the IBDV VP2 was shown to function as an efficacious anti-IBD subunit vaccine. PMID:23024743

  1. Construction of Recombinant Baculoviruses Expressing Infectious Bursal Disease Virus Main Protective Antigen and Their Immune Effects on Chickens

    PubMed Central

    Song, Shanshan; Gao, Dongni; Ping, Wenxiang

    2015-01-01

    In order to overcome the limitations of conventional vaccines for infectious bursal disease virus (IBDV), we constructed recombinant dual expression system baculoviruses with VP2 and VP2/4/3, the main protective antigens of IBDV. We compared the immune effects of the baculoviruses in avian cells and detected their control effects on chickens with infectious bursal disease. We used Western blot analysis to measure VP2 protein and VP2/4/3 polyprotein expression in avian cells infected using the Bac-to-Bac baculovirus expression system. The recombinant baculoviruses were used to vaccinate specific pathogen-free chickens, which produced specific protective antibodies and strong cellular immune responses. The results of the virus challenge experiment revealed that the protective efficiency of VP2 and VP2/4/3 virus vaccines were 95.8% and 100%, respectively, both of which were higher than the vaccine group (87.5%), and significantly higher than the control group (50%). The results demonstrated that the immune effect of BV-S-ITRs-VP2/4/3 was superior to that of BV-S-ITRs-VP2. Compared with traditional attenuated vaccine and genetically engineered live vector vaccine, the dual expression viral vector vaccine has good bio-safety. The results of this study provide a foundation for the further development of poultry vaccines, in addition to providing a useful reference for developing non-replicating live vaccines against other viral diseases. PMID:26167907

  2. Development of a Reverse Genetic System for Infectious Salmon Anemia Virus: Rescue of Recombinant Fluorescent Virus by Using Salmon Internal Transcribed Spacer Region 1 as a Novel Promoter

    PubMed Central

    Toro-Ascuy, Daniela; Tambley, Carolina; Beltran, Carolina; Mascayano, Carolina; Sandoval, Nicolas; Olivares, Eduardo; Medina, Rafael A.; Spencer, Eugenio

    2014-01-01

    Infectious salmon anemia (ISA) is a serious disease of marine-farmed Atlantic salmon (Salmo salar) caused by ISA virus (ISAV), belonging to the genus Isavirus, family Orthomyxoviridae. There is an urgent need to understand the virulence factors and pathogenic mechanisms of ISAV and to develop new vaccine approaches. Using a recombinant molecular biology approach, we report the development of a plasmid-based reverse genetic system for ISAV, which includes the use of a novel fish promoter, the Atlantic salmon internal transcribed spacer region 1 (ITS-1). Salmon cells cotransfected with pSS-URG-based vectors expressing the eight viral RNA segments and four cytomegalovirus (CMV)-based vectors that express the four proteins of the ISAV ribonucleoprotein complex allowed the generation of infectious recombinant ISAV (rISAV). We generated three recombinant viruses, wild-type rISAV901_09 and rISAVrS6-NotI-HPR containing a NotI restriction site and rISAVS6/EGFP-HPR harboring the open reading frame of enhanced green fluorescent protein (EGFP), both within the highly polymorphic region (HPR) of segment 6. All rescued viruses showed replication activity and cytopathic effect in Atlantic salmon kidney-infected cells. The fluorescent recombinant viruses also showed a characteristic cytopathic effect in salmon cells, and the viruses replicated to a titer of 6.5 × 105 PFU/ml, similar to that of the wild-type virus. This novel reverse genetics system offers a powerful tool to study the molecular biology of ISAV and to develop a new generation of ISAV vaccines to prevent and mitigate ISAV infection, which has had a profound effect on the salmon industry. PMID:25480750

  3. An analysis of the complete sequence of a sugarcane bacilliform virus genome infectious to banana and rice.

    PubMed

    Bouhida, M; Lockhart, B E; Olszewski, N E

    1993-01-01

    The genome of sugarcane bacilliform virus (ScBV), a badnavirus, consists of a circular dsDNA. The complete sequence of a cloned infective ScBV genome is reported here. The genome is 7568 bp in size and possesses a number of features suggesting that ScBV is a pararetrovirus. A tRNA(Met)-binding site that may serve as a primer for minus-strand synthesis is present. The plus-strand of the ScBV genome contains three open reading frames (ORFs) which are capable of encoding proteins with calculated M(r) values of 22K, 13K and 215K. The 215K protein has regions with similarity to the RNA-binding domains, aspartic proteases and replicases of retro-elements. In addition, the 215K protein also has a region with restricted similarity to the intercellular transport proteins of plant viruses. Comparisons with the other sequenced badnaviruses, Commelina yellow mottle (CoYMV) and rice tungro bacilliform (RTBV) viruses, indicate that the arrangement of the ORFs in these viruses is conserved. Located next to the putative RNA-binding domain is a cysteine-rich region that is unique to the badnaviruses. When the molecular relationships of a portion of the reverse transcriptases of plant pararetroviruses were determined, two badnaviruses, CoYMV and ScBV, form one distinct cluster, whereas three caulimoviruses, cauliflower mosaic virus, carnation etched ring virus and figwort mosaic virus, form a second cluster. The badnavirus RTBV and the caulimovirus soybean chlorotic mottle virus occupy intermediate positions between the clusters. When introduced by Agrobacterium-mediated inoculation, a construct containing 1.1 copies of the cloned ScBV genome is infectious to both rice and banana. PMID:8423447

  4. Newly incriminated anopheline vectors of human malaria parasites in Junin Department, Peru.

    PubMed

    Hayes, J; Calderon, G; Falcon, R; Zambrano, V

    1987-09-01

    Sporozoite data from salivary gland dissections are presented that clearly incriminate Anopheles trinkae, An. pseudopunctipennis, An. sp. near fluminensis, An. oswaldoi, An. nuneztovari and An. rangeli as vectors of malaria parasites in the Rio Ene Valley, a hyperendemic malarious area in Junin Department, eastern Peru. Anopheles trinkae is considered the most important vector based on dissections, abundance and man-vector contact. Other notes are presented on the relative abundance, bionomics and previous records of these species in Peru and in the study sites. PMID:3333060

  5. Occurrence of Newcastle Disease and Infectious Bursal Disease Virus Antibodies in Double-Spurred Francolins in Nigeria

    PubMed Central

    Oluwayelu, Daniel Oladimeji; Adebiyi, Adebowale Idris; Olaniyan, Ibukunoluwa; Ezewele, Phyllis; Aina, Oluwasanmi

    2014-01-01

    The double-spurred francolin Francolinus bicalcaratus has been identified as a good candidate for future domestication due to the universal acceptability of its meat and its adaptability to anthropogenically altered environments. Therefore, in investigating the diseases to which they are susceptible, serum samples from 56 francolins in a major live-bird market (LBM) in Ibadan, southwestern Nigeria, were screened for antibodies against Newcastle disease (ND) and infectious bursal disease (IBD) viruses. Haemagglutination inhibition (HI) test and enzyme-linked immunosorbent assay (ELISA) revealed 25.0% and 35.7% prevalence of ND virus (NDV) antibodies, respectively, while 5.4% and 57.1% prevalence of IBD virus (IBDV) antibodies was detected by agar gel precipitation test (AGPT) and ELISA, respectively. This first report on the occurrence of NDV and IBDV antibodies in apparently healthy, unvaccinated double-spurred francolins from a LBM suggests that they were subclinically infected with either field or vaccine viruses and could thus serve as possible reservoirs of these viruses to domestic poultry. Furthermore, if they are to be domesticated for intensive rearing, a vaccination plan including ND and IBD should be developed and implemented. PMID:26464918

  6. The effect of Allium sativum (Garlic) extract on infectious bronchitis virus in specific pathogen free embryonic egg

    PubMed Central

    Mohajer Shojai, Tabassom; Ghalyanchi Langeroudi, Arash; Karimi, Vahid; Barin, Abbas; Sadri, Naser

    2016-01-01

    Objective: Garlic is a plant has been used as a flavor, and anti-microbial and anti-diarrheal agent. Infectious bronchitis virus (IBV) is a coronavirus. The available vaccines against IBV cannot cover new variants. This study evaluated the inhibitory effects of garlic extract on IBV. Materials and Methods: The constituents of garlic extract were detected by gas chromatography. This study was done in four groups of embryonic SPF eggs; first group was used for virus titration; second group received the mixture of different virus titration and constant amount of garlic extract; third group received 10-3 titration of virus and after 8 hr received garlic extract and the last group received different dilutions of garlic extract. Results: Based on our results, in the second group, IBV vaccine strain (4/91) at all titration and M41 in 10-2 and 10-3 titration and in the third group both variants of virus the embryonic Index (EI) was significantly increased. Conclusion: The garlic extract had inhibitory effects on IBV in the chickens embryo. PMID:27516987

  7. Occurrence of Newcastle Disease and Infectious Bursal Disease Virus Antibodies in Double-Spurred Francolins in Nigeria.

    PubMed

    Oluwayelu, Daniel Oladimeji; Adebiyi, Adebowale Idris; Olaniyan, Ibukunoluwa; Ezewele, Phyllis; Aina, Oluwasanmi

    2014-01-01

    The double-spurred francolin Francolinus bicalcaratus has been identified as a good candidate for future domestication due to the universal acceptability of its meat and its adaptability to anthropogenically altered environments. Therefore, in investigating the diseases to which they are susceptible, serum samples from 56 francolins in a major live-bird market (LBM) in Ibadan, southwestern Nigeria, were screened for antibodies against Newcastle disease (ND) and infectious bursal disease (IBD) viruses. Haemagglutination inhibition (HI) test and enzyme-linked immunosorbent assay (ELISA) revealed 25.0% and 35.7% prevalence of ND virus (NDV) antibodies, respectively, while 5.4% and 57.1% prevalence of IBD virus (IBDV) antibodies was detected by agar gel precipitation test (AGPT) and ELISA, respectively. This first report on the occurrence of NDV and IBDV antibodies in apparently healthy, unvaccinated double-spurred francolins from a LBM suggests that they were subclinically infected with either field or vaccine viruses and could thus serve as possible reservoirs of these viruses to domestic poultry. Furthermore, if they are to be domesticated for intensive rearing, a vaccination plan including ND and IBD should be developed and implemented. PMID:26464918

  8. Equine Tetherin Blocks Retrovirus Release and Its Activity Is Antagonized by Equine Infectious Anemia Virus Envelope Protein

    PubMed Central

    Yin, Xin; Hu, Zhe; Gu, Qinyong; Wu, Xingliang; Zheng, Yong-Hui; Wei, Ping

    2014-01-01

    Human tetherin is a host restriction factor that inhibits replication of enveloped viruses by blocking viral release. Tetherin has an unusual topology that includes an N-terminal cytoplasmic tail, a single transmembrane domain, an extracellular domain, and a C-terminal glycosylphosphatidylinositol anchor. Tetherin is not well conserved across species, so it inhibits viral replication in a species-specific manner. Thus, studies of tetherin activities from different species provide an important tool for understanding its antiviral mechanism. Here, we report cloning of equine tetherin and characterization of its antiviral activity. Equine tetherin shares 53%, 40%, 36%, and 34% amino acid sequence identity with feline, human, simian, and murine tetherins, respectively. Like the feline tetherin, equine tetherin has a shorter N-terminal domain than human tetherin. Equine tetherin is localized on the cell surface and strongly blocks human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus (SIV), and equine infectious anemia virus (EIAV) release from virus-producing cells. The antiviral activity of equine tetherin is neutralized by EIAV envelope protein, but not by the HIV-1 accessory protein Vpu, which is a human tetherin antagonist, and EIAV envelope protein does not counteract human tetherin. These results shed new light on our understanding of the species-specific tetherin antiviral mechanism. PMID:24227834

  9. Bioengineering of Tobacco Mosaic Virus to Create a Non-Infectious Positive Control for Ebola Diagnostic Assays

    NASA Astrophysics Data System (ADS)

    Lam, Patricia; Gulati, Neetu M.; Stewart, Phoebe L.; Keri, Ruth A.; Steinmetz, Nicole F.

    2016-03-01

    The 2014 Ebola epidemic is the largest to date. There is no cure or treatment for this deadly disease; therefore there is an urgent need to develop new diagnostics to accurately detect Ebola. Current RT-PCR assays lack sensitive and reliable positive controls. To address this critical need, we devised a bio-inspired positive control for use in RT-PCR diagnostics: we encapsulated scrambled Ebola RNA sequences inside of tobacco mosaic virus to create a biomimicry that is non-infectious, but stable, and could therefore serve as a positive control in Ebola diagnostic assays. Here, we report the bioengineering and validation of this probe.

  10. Genomic sequence analysis of a new reassortant infectious bursal disease virus from commercial broiler flocks in Central China.

    PubMed

    Cui, Pei; Ma, Shi-Jie; Zhang, Yu-Geng; Li, Xin-Sheng; Gao, Xiao-Yun; Cui, Bao-An; Chen, Hong-Ying

    2013-09-01

    We report the complete nucleotide sequence of a reassortant infectious bursal disease (IBD) virus (IBDV) HN isolate from commercial broiler flocks in central China. The genome consisted of 3,232 and 2,652 nucleotides in the coding regions of segments A and B, respectively. Alignment of both nucleotide and deduced amino acid sequences and phylogenetic analysis revealed that the genome segments A and B of HN were derived from the attenuated strain B87 and the VV strain OKYM. This is a new reassortant IBDV strain that has emerged in nature, involving segment A of a cell-culture-adapted attenuated vaccine strain B87. PMID:23543159

  11. Bioengineering of Tobacco Mosaic Virus to Create a Non-Infectious Positive Control for Ebola Diagnostic Assays

    PubMed Central

    Lam, Patricia; Gulati, Neetu M.; Stewart, Phoebe L.; Keri, Ruth A.; Steinmetz, Nicole F.

    2016-01-01

    The 2014 Ebola epidemic is the largest to date. There is no cure or treatment for this deadly disease; therefore there is an urgent need to develop new diagnostics to accurately detect Ebola. Current RT-PCR assays lack sensitive and reliable positive controls. To address this critical need, we devised a bio-inspired positive control for use in RT-PCR diagnostics: we encapsulated scrambled Ebola RNA sequences inside of tobacco mosaic virus to create a biomimicry that is non-infectious, but stable, and could therefore serve as a positive control in Ebola diagnostic assays. Here, we report the bioengineering and validation of this probe. PMID:27030058

  12. Bioengineering of Tobacco Mosaic Virus to Create a Non-Infectious Positive Control for Ebola Diagnostic Assays.

    PubMed

    Lam, Patricia; Gulati, Neetu M; Stewart, Phoebe L; Keri, Ruth A; Steinmetz, Nicole F

    2016-01-01

    The 2014 Ebola epidemic is the largest to date. There is no cure or treatment for this deadly disease; therefore there is an urgent need to develop new diagnostics to accurately detect Ebola. Current RT-PCR assays lack sensitive and reliable positive controls. To address this critical need, we devised a bio-inspired positive control for use in RT-PCR diagnostics: we encapsulated scrambled Ebola RNA sequences inside of tobacco mosaic virus to create a biomimicry that is non-infectious, but stable, and could therefore serve as a positive control in Ebola diagnostic assays. Here, we report the bioengineering and validation of this probe. PMID:27030058

  13. Seroprevalence of infectious bursal disease virus in local chickens in Udu Local Government Area of Delta State, South East Nigeria.

    PubMed

    Abraham-Oyiguh, J; Adewumi, M O; Onoja, A B; Suleiman, I; Sulaiman, L K; Ahmed, S J; Jagboro, S T

    2015-01-01

    Infectious Bursal Disease Virus (IBDV) poses a great global threat to the poultry industry. Knowledge of the occurrence of the disease is important in the design and implementation of a control program, therefore this study determines the seroprevalence of IBDV in local chickens in Udu Local Government Area of Delta State. 250 chickens were bled by exsanguination and sera obtained were screened using Agar Gel Immunodiffusion (AGID) test. The seropositivity was 51.6%, which is indicates endemicity of the disease. Biosecurity and good sanitary measures are recommended. Molecular characterization of the strains should be carried out for inclusion in generic vaccines. PMID:25331185

  14. Molecular characterization of the virulent infectious hematopoietic necrosis virus (IHNV) strain 220-90

    PubMed Central

    2010-01-01

    Background Infectious hematopoietic necrosis virus (IHNV) is the type species of the genus Novirhabdovirus, within the family Rhabdoviridae, infecting several species of wild and hatchery reared salmonids. Similar to other rhabdoviruses, IHNV has a linear single-stranded, negative-sense RNA genome of approximately 11,000 nucleotides. The IHNV genome encodes six genes; the nucleocapsid, phosphoprotein, matrix protein, glycoprotein, non-virion protein and polymerase protein genes, respectively. This study describes molecular characterization of the virulent IHNV strain 220-90, belonging to the M genogroup, and its phylogenetic relationships with available sequences of IHNV isolates worldwide. Results The complete genomic sequence of IHNV strain 220-90 was determined from the DNA of six overlapping clones obtained by RT-PCR amplification of genomic RNA. The complete genome sequence of 220-90 comprises 11,133 nucleotides (GenBank GQ413939) with the gene order of 3'-N-P-M-G-NV-L-5'. These genes are separated by conserved gene junctions, with di-nucleotide gene spacers. An additional uracil nucleotide was found at the end of the 5'-trailer region, which was not reported before in other IHNV strains. The first 15 of the 16 nucleotides at the 3'- and 5'-termini of the genome are complementary, and the first 4 nucleotides at 3'-ends of the IHNV are identical to other novirhadoviruses. Sequence homology and phylogenetic analysis of the glycoprotein genes show that 220-90 strain is 97% identical to most of the IHNV strains. Comparison of the virulent 220-90 genomic sequences with less virulent WRAC isolate shows more than 300 nucleotides changes in the genome, which doesn't allow one to speculate putative residues involved in the virulence of IHNV. Conclusion We have molecularly characterized one of the well studied IHNV isolates, 220-90 of genogroup M, which is virulent for rainbow trout, and compared phylogenetic relationship with North American and other strains

  15. Genetic Characterization of the Belgian Nephropathogenic Infectious Bronchitis Virus (NIBV) Reference Strain B1648.

    PubMed

    Reddy, Vishwanatha R A P; Theuns, Sebastiaan; Roukaerts, Inge D M; Zeller, Mark; Matthijnssens, Jelle; Nauwynck, Hans J

    2015-08-01

    The virulent nephropathogenic infectious bronchitis virus (NIBV) strain B1648 was first isolated in 1984, in Flanders, Belgium. Despite intensive vaccination, B1648 and its variants are still circulating in Europe and North Africa. Here, the full-length genome of this Belgian NIBV reference strain was determined by next generation sequencing (NGS) to understand its evolutionary relationship with other IBV strains, and to identify possible genetic factors that may be associated with the nephropathogenicity. Thirteen open reading frames (ORFs) were predicted in the B1648 strain (51UTR-1a-1b-S-3a-3b-E-M-4b-4c-5a-5b-N-6b-31UTR). ORFs 4b, 4c and 6b, which have been rarely reported in literature, were present in B1648 and most of the other IBV complete genomes. According to phylogenetic analysis of the full-length genome, replicase transcriptase complex, spike protein, partial S1 gene and M protein, B1648 strain clustered with the non-Massachusetts type strains NGA/A116E7/2006, UKr 27-11, QX-like ITA/90254/2005, QX-like CK/SWE/0658946/10, TN20/00, RF-27/99, RF/06/2007 and SLO/266/05. Based on the partial S1 fragment, B1648 clustered with the strains TN20/00, RF-27/99, RF/06/2007 and SLO/266/05 and, further designated as B1648 genotype. The full-length genome of B1648 shared the highest sequence homology with UKr 27-11, Gray, JMK, and NGA/A116E7/2006 (91.2% to 91.6%) and was least related with the reference Beaudette and Massachusetts strains (89.7%). Nucleotide and amino acid sequence analyses indicated that B1648 strain may have played an important role in the evolution of IBV in Europe and North Africa. Further, the nephropathogenicity determinants might be located on the 1a, spike, M and accessory proteins (3a, 3b, 4b, 4c, 5a, 5b and 6b). Overall, strain B1648 is distinct from all the strains reported so far in Europe and other parts of the world. PMID:26262637

  16. Genetic Characterization of the Belgian Nephropathogenic Infectious Bronchitis Virus (NIBV) Reference Strain B1648

    PubMed Central

    Reddy, Vishwanatha R.A.P.; Theuns, Sebastiaan; Roukaerts, Inge D.M.; Zeller, Mark; Matthijnssens, Jelle; Nauwynck, Hans J.

    2015-01-01

    The virulent nephropathogenic infectious bronchitis virus (NIBV) strain B1648 was first isolated in 1984, in Flanders, Belgium. Despite intensive vaccination, B1648 and its variants are still circulating in Europe and North Africa. Here, the full-length genome of this Belgian NIBV reference strain was determined by next generation sequencing (NGS) to understand its evolutionary relationship with other IBV strains, and to identify possible genetic factors that may be associated with the nephropathogenicity. Thirteen open reading frames (ORFs) were predicted in the B1648 strain (5′UTR-1a-1b-S-3a-3b-E-M-4b-4c-5a-5b-N-6b-3′UTR). ORFs 4b, 4c and 6b, which have been rarely reported in literature, were present in B1648 and most of the other IBV complete genomes. According to phylogenetic analysis of the full-length genome, replicase transcriptase complex, spike protein, partial S1 gene and M protein, B1648 strain clustered with the non-Massachusetts type strains NGA/A116E7/2006, UKr 27-11, QX-like ITA/90254/2005, QX-like CK/SWE/0658946/10, TN20/00, RF-27/99, RF/06/2007 and SLO/266/05. Based on the partial S1 fragment, B1648 clustered with the strains TN20/00, RF-27/99, RF/06/2007 and SLO/266/05 and, further designated as B1648 genotype. The full-length genome of B1648 shared the highest sequence homology with UKr 27-11, Gray, JMK, and NGA/A116E7/2006 (91.2% to 91.6%) and was least related with the reference Beaudette and Massachusetts strains (89.7%). Nucleotide and amino acid sequence analyses indicated that B1648 strain may have played an important role in the evolution of IBV in Europe and North Africa. Further, the nephropathogenicity determinants might be located on the 1a, spike, M and accessory proteins (3a, 3b, 4b, 4c, 5a, 5b and 6b). Overall, strain B1648 is distinct from all the strains reported so far in Europe and other parts of the world. PMID:26262637

  17. Viral competition and maternal immunity influence the clinical disease caused by very virulent infectious bursal disease virus.

    PubMed

    Jackwood, Daral J

    2011-09-01

    The very virulent form of infectious bursal disease virus (vvIBDV) causes an immunosuppressive disease that is further characterized by the rapid onset of morbidity and high mortality in susceptible chickens. In 2009, vvIBDV was first reported in California, U. S. A., and since that time only a few cases of acute infectious bursal disease attributed to vvIBDV have been recognized in California. In other countries where vvIBDV has become established, it rapidly spreads to most poultry-producing regions. Two factors that may be involved in limiting the spread or reducing the severity of the clinical disease caused by vvIBDV in the U. S. A. are maternal immunity and competition with endemic variant strains of the virus. In this study, the ability of vvIBDV to infect and cause disease in maternally immune layer chickens was examined at weekly intervals over a 5-wk period during which their neutralizing maternal antibodies waned. Birds inoculated with vvIBDV at 2, 3, and 4 wk of age seemed healthy throughout the duration of the experiment, but macroscopic and microscopic lesions were observed in their bursa tissues. A real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay also confirmed the presence of vvIBDV RNA in their bursa tissues, indicating this virus was infecting the birds even at 2 wk of age when neutralizing maternal antibodies to infectious bursal disease virus were still relatively high (> 2000 geometric mean antibody titer). No mortality was observed in any birds when inoculated at 2, 3, or 4 wk of age; however, inoculation at 5 and 6 wk of age resulted in 10% and 20% mortality, respectively. Three experiments on the competition between vvIBDV and the two variant viruses T1 and FF6 were conducted. In all three experiments, specific-pathogen-free (SPF) birds that were inoculated with only the vvIBDV became acutely moribund, and except for Experiment 1 (62% mortality) all succumbed to the infection within 4 days of being exposed. When the

  18. From glanders to Hendra virus: 125 years of equine infectious diseases.

    PubMed

    Slater, Josh

    2013-08-31

    Josh Slater looks back at the past 125 years of developments in equine infectious disease, including landmark discoveries in microbiology and genomics, and considers what the future may hold. PMID:23997164

  19. Classical swine fever virus: recovery of infectious viruses from cDNA constructs and generation of recombinant cytopathogenic defective interfering particles.

    PubMed Central

    Meyers, G; Thiel, H J; Rümenapf, T

    1996-01-01

    The 5'- and 3'-terminal sequences of the genomic RNA from classical swine fever virus (CSFV) were determined, and the resulting information was used for construction of full-length CSFV cDNA clones. After transfection of in vitro-transcribed RNA derived from a cDNA construct, infectious CSFV was recovered from porcine cells. To confirm the de novo generation of infectious CSFV from cloned DNA, a genetically tagged CSFV was constructed. In comparison with parental CSFV, the recombinant viruses were retarded in growth by about 1 order of magnitude. Introduction of a deletion by exchange of part of the full-length construct for corresponding cDNA fragments derived from the genomes of cytopathogenic CSFV defective interfering particles (DIs) (G. Meyers and H.-J. Thiel, J. Virol. 69:3683-3689. 1995) resulted in recovery of cytopathogenic DIs in the DI genomes is responsible for their cytopathogenicity. The established system will allow novel approaches to analysis of pestiviral molecular biology and in particular to elucidation of the molecular basis of attenuation and cytopathogenicity of these viruses. PMID:8627678

  20. Gag Protein Epitopes Recognized by CD4+ T-Helper Lymphocytes from Equine Infectious Anemia Virus-Infected Carrier Horses

    PubMed Central

    Lonning, S. M.; Zhang, W.; McGuire, T. C.

    1999-01-01

    Antigen-specific T-helper (Th) lymphocytes are critical for the development of antiviral humoral responses and the expansion of cytotoxic T lymphocytes (CTL). Identification of relevant Th lymphocyte epitopes remains an important step in the development of an efficacious subunit peptide vaccine against equine infectious anemia virus (EIAV), a naturally occurring lentivirus of horses. This study describes Th lymphocyte reactivity in EIAV carrier horses to two proteins, p26 and p15, encoded by the relatively conserved EIAV gag gene. Using partially overlapping peptides, multideterminant and possibly promiscuous epitopes were identified within p26. One peptide was identified which reacted with peripheral blood mononuclear cells (PBMC) from all five EIAV-infected horses, and three other peptides were identified which reacted with PBMC from four of five EIAV-infected horses. Four additional peptides containing both CTL and Th lymphocyte epitopes were also identified. Multiple epitopes were recognized in a region corresponding to the major homology region of the human immunodeficiency virus, a region with significant sequence similarity to other lentiviruses including simian immunodeficiency virus, puma lentivirus, feline immunodeficiency virus, Jembrana disease virus, visna virus, and caprine arthritis encephalitis virus. PBMC reactivity to p15 peptides from EIAV carrier horses also occurred. Multiple p15 peptides were shown to be reactive, but not all infected horses had Th lymphocytes recognizing p15 epitopes. The identification of peptides reactive with PBMC from outbred horses, some of which encoded both CTL and Th lymphocyte epitopes, should contribute to the design of synthetic peptide or recombinant vector vaccines for EIAV. PMID:10196322

  1. Detection and Quantification of Infectious Hypodermal and Hematopoietic Necrosis Virus and White Spot Virus in Shrimp Using Real-Time Quantitative PCR and SYBR Green Chemistry

    PubMed Central

    Dhar, Arun K.; Roux, Michelle M.; Klimpel, Kurt R.

    2001-01-01

    A rapid and highly sensitive real-time PCR detection and quantification method for infectious hypodermal and hematopoietic necrosis virus (IHHNV), a single-stranded DNA virus, and white spot virus (WSV), a double-stranded DNA (dsDNA) virus infecting penaeid shrimp (Penaeus sp.), was developed using the GeneAmp 5700 sequence detection system coupled with SYBR Green chemistry. The PCR mixture contains a fluorescence dye, SYBR Green, which upon binding to dsDNA exhibits fluorescence enhancement. The enhancement of fluorescence was proportional to the initial concentration of the template DNA. A linear relationship was observed between the amount of input plasmid DNA and cycle threshold (CT) values over a range of 1 to 105 copies of the viral genome. To control the variation in sampling and processing among samples, the shrimp β-actin gene was amplified in parallel with the viral DNA. The CT values of IHHNV- and WSV-infected samples were used to determine absolute viral copy numbers from the standard CT curves of these viruses. For each virus and its β-actin control, the specificity of amplification was monitored by using the dissociation curve of the amplified product. Using genomic DNA as a template, SYBR Green PCR was found to be 100- to 2000-fold more sensitive than conventional PCR, depending on the virus, for the samples tested. The results demonstrate that SYBR Green PCR can be used as a rapid and highly sensitive detection and quantification method for shrimp viruses and that it is amenable to high-throughout assay. PMID:11474000

  2. Molecular detection and characterization of infectious laryngotracheitis virus (Gallid herpesvirus-1) from clinical samples of commercial poultry flocks in India.

    PubMed

    Gowthaman, Vasudevan; Singh, Sambhu Dayal; Dhama, Kuldeep; Barathidasan, Rajamani; Mathapati, Basavaraj S; Srinivasan, Palani; Saravanan, Sellappan; Ramakrishnan, Muthannan Andavar

    2014-01-01

    Although the existence of infectious laryngotracheitis virus (ILTV) in India was first reported in 1964, no reports are available regarding its molecular detection and characterization. The present study was aimed to detect and characterize ILTV from recent respiratory disease complex (RDC) outbreaks of commercial poultry flocks in different parts of the country by using envelope glycoprotein G gene (US4 gene) based PCR and sequencing. A total of thirty two flocks with a history of RDC were investigated. Overall, all the strains/breeds of birds and all ages of birds are equally susceptible and depending on the severity, the clinical signs and gross lesions were varied. Out of 32 flocks investigated 10 were found positive for ILTV infection by PCR. The phylogenetic analyses of eight representative sequences in the present study deciphered that Indian ILT viruses are closely related to chicken embryo origin vaccine strains of Italy, USA, China and Brazil. PMID:25674602

  3. Virulence correlates with fitness in vivo for two M group genotypes of Infectious hematopoietic necrosis virus (IHNV).

    USGS Publications Warehouse

    Wargo, Andrew R.; Garver, Kyle A.; Kurath, Gael

    2010-01-01

    The nature of the association between viral fitness and virulence remains elusive in vertebrate virus systems, partly due to a lack of in vivo experiments using statistically sufficient numbers of replicate hosts. We examined the relationship between virulence and fitness in Infectious hematopoietic necrosis virus (IHNV), in vivo, in intact living rainbow trout. Trout were infected with a high or low virulence genotype of M genogroup IHNV, or a mixture of the two genotypes, so as to calculate relative fitness and the effect of a competition environment on fitness. Fitness was measured as total viral load in the host at time of peak viral density, quantified by genotype-specific quantitative RT-PCR (qRT-PCR). The more virulent IHNV genotype reached higher densities in both single and mixed infections. There was no effect of competition on the performance of either genotype. Our results suggest a positive link between IHNV genotype fitness and virulence.

  4. Rapid Detection of Infectious Flacherie Virus of the Silkworm, Bombyx mori, using RT-PCR and Nested PCR

    PubMed Central

    Vootla, Shyam Kumar; Lu, Xing Meng; Kari, Neetha; Gadwala, Mallikarjun; Lu, Qineng

    2013-01-01

    In this study, a method for detection of an ssRNA viral pathogen that causes viral flacherie in the silkworm, Bombyx mori (L.) (Lepidoptera: Bombycidae), was used for the detection of B. mori infectious flacherie virus (BmIFV). A combination of nested and reverse transcriptase polymerase chain reaction was used for detection. Although BmIFV has been reported in almost all the sericultural regions of the world, there had been no reports of BmIFV incidence in India. Therefore, the confirmation of the presence of BmIFV in Karnataka, India, is of great significance. The present method is advantageous because it can be used to detect the virus by using samples from infected midgut tissues, thus simplifying and avoiding laborious genome isolation procedures. This method could help in early detection of BmIFV disease pathogens and help reduce crop losses. PMID:24785655

  5. Measurement of the Infection and Dissemination of Bluetongue Virus in Culicoides Biting Midges Using a Semi-Quantitative RT-PCR Assay and Isolation of Infectious Virus

    PubMed Central

    Veronesi, Eva; Antony, Frank; Gubbins, Simon; Golding, Nick; Blackwell, Alison; Mertens, Peter PC.; Brownlie, Joe; Darpel, Karin E.; Mellor, Philip S.; Carpenter, Simon

    2013-01-01

    Background Culicoides biting midges (Diptera: Ceratopogonidae) are the biological vectors of globally significant arboviruses of livestock including bluetongue virus (BTV), African horse sickness virus (AHSV) and the recently emerging Schmallenberg virus (SBV). From 2006–2009 outbreaks of BTV in northern Europe inflicted major disruption and economic losses to farmers and several attempts were made to implicate Palaearctic Culicoides species as vectors. Results from these studies were difficult to interpret as they used semi-quantitative RT-PCR (sqPCR) assays as the major diagnostic tool, a technique that had not been validated for use in this role. In this study we validate the use of these assays by carrying out time-series detection of BTV RNA in two colony species of Culicoides and compare the results with the more traditional isolation of infectious BTV on cell culture. Methodology/Principal Findings A BTV serotype 1 strain mixed with horse blood was fed to several hundred individuals of Culicoides sonorensis (Wirth & Jones) and C. nubeculosus (Mg.) using a membrane-based assay and replete individuals were then incubated at 25°C. At daily intervals 25 Culicoides of each species were removed from incubation, homogenised and BTV quantified in each individual using sqPCR (Cq values) and virus isolation on a KC-C. sonorensis embryonic cell line, followed by antigen enzyme-linked immunosorbent assay (ELISA). In addition, comparisons were also drawn between the results obtained with whole C. sonorensis and with individually dissected individuals to determine the level of BTV dissemination. Conclusions/Significance Cq values generated from time-series infection experiments in both C. sonorensis and C. nubeculosus confirmed previous studies that relied upon the isolation and detection of infectious BTV. Implications on the testing of field-collected Culicoides as potential virus vectors by PCR assays and the use of such assays as front-line tools for use in

  6. Infectious Uveitis

    PubMed Central

    2015-01-01

    Infectious uveitis is one of the most common and visually devastating causes of uveitis in the US and worldwide. This review provides a summary of the identification, treatment, and complications associated with certain forms of viral, bacterial, fungal, helminthic, and parasitic uveitis. In particular, this article reviews the literature on identification and treatment of acute retinal necrosis due to herpes simplex virus, varicella virus, and cytomegalovirus. While no agreed-upon treatment has been identified, the characteristics of Ebola virus panuveitis is also reviewed. In addition, forms of parasitic infection such as Toxoplasmosis and Toxocariasis are summarized, as well as spirochetal uveitis. Syphilitic retinitis is reviewed given its increase in prevalence over the last decade. The importance of early identification and treatment of infectious uveitis is emphasized. Early identification can be achieved with a combination of maintaining a high suspicion, recognizing certain clinical features, utilizing multi-modal imaging, and obtaining specimens for molecular diagnostic testing. PMID:26618074

  7. Development of an Equine-Tropic Replication-Competent Lentivirus Assay for Equine Infectious Anemia Virus-Based Lentiviral Vectors

    PubMed Central

    Bannister, Richard; Leroux-Carlucci, Marie A.; Evans, Nerys E.; Miskin, James E.; Mitrophanous, Kyriacos A.

    2012-01-01

    Abstract The release of lentiviral vectors for clinical use requires the testing of vector material, production cells, and, if applicable, ex vivo-transduced cells for the presence of replication-competent lentivirus (RCL). Vectors derived from the nonprimate lentivirus equine infectious anemia virus (EIAV) have been directly administered to patients in several clinical trials, with no toxicity observed to date. Because EIAV does not replicate in human cells, and because putative RCLs derived from vector components within human vector production cells would most likely be human cell-tropic, we previously developed an RCL assay using amphotropic murine leukemia virus (MLV) as a surrogate positive control and human cells as RCL amplification/indicator cells. Here we report an additional RCL assay that tests for the presence of theoretical “equine-tropic” RCLs. This approach provides further assurance of safety by detecting putative RCLs with an equine cell-specific tropism that might not be efficiently amplified by the human cell-based RCL assay. We tested the ability of accessory gene-deficient EIAV mutant viruses to replicate in a highly permissive equine cell line to direct our choice of a suitable EIAV-derived positive control. In addition, we report for the first time the mathematical rationale for use of the Poisson distribution to calculate minimal infectious dose of positive control virus and for use in monitoring assay positive/spike control failures in accumulating data sets. No RCLs have been detected in Good Manufacturing Practice (GMP)-compliant RCL assays to date, further demonstrating that RCL formation is highly unlikely in contemporary minimal lentiviral vector systems. PMID:23121195

  8. Construction and characterization of a full-length infectious simian T-cell lymphotropic virus type 3 molecular clone.

    PubMed

    Chevalier, Sébastien Alain; Walic, Marine; Calattini, Sara; Mallet, Adeline; Prévost, Marie-Christine; Gessain, Antoine; Mahieux, Renaud

    2007-06-01

    Together with their simian T-cell lymphotropic virus (STLV) equivalent, human T-cell lymphotropic virus type 1 (HTLV-1), HTLV-2, and HTLV-3 form the primate T-cell lymphotropic virus (PTLV) group. Over the years, understanding the biology and pathogenesis of HTLV-1 and HTLV-2 has been widely improved by the creation of molecular clones. In contrast, so far, PTLV-3 experimental studies have been restricted to the overexpression of the tax gene using reporter assays. We have therefore decided to construct an STLV-3 molecular clone. We generated a full-length STLV-3 proviral clone (8,891 bp) by PCR amplification of overlapping fragments. This STLV-3 molecular clone was then transfected into 293T cells. Reverse transcriptase PCR experiments followed by sequence analysis of the amplified products allowed us to establish that both gag and tax/rex mRNAs were transcribed. Western blotting further demonstrated the presence of the STLV-3 p24gag protein in the cell culture supernatant from transfected cells. Transient transfection of 293T cells and of 293T-long terminal repeat-green fluorescent protein cells with the STLV-3 clone promoted syncytium formation, a hallmark of PTLV Env expression, as well as the appearance of fluorescent cells, also demonstrating that the Tax3 protein was expressed. Virus particles were visible by electron microscopy. These particles are infectious, as demonstrated by our cell-free-infection experiments with purified virions. All together, our data demonstrate that the STLV-3 molecular clone is functional and infectious. This clone will give us a unique opportunity to study in vitro the different pX transcripts and the putative presence of antisense transcripts and to evaluate the PTLV-3 pathogenicity in vivo. PMID:17428869

  9. Induction of protective immunity in chickens immunized with plant-made chimeric Bamboo mosaic virus particles expressing very virulent Infectious bursal disease virus antigen.

    PubMed

    Chen, Tsung-Hsien; Chen, Ten-Hong; Hu, Chung-Chi; Liao, Jia-Teh; Lee, Chin-Wei; Liao, Jiunn-Wang; Lin, Maw-Yeong; Liu, Hung-Jen; Wang, Min-Ying; Lin, Na-Sheng; Hsu, Yau-Heiu

    2012-06-01

    Very virulent Infectious bursal disease virus (vvIBDV) causes a highly contagious disease in young chickens and leads to significant economic loss in the poultry industry. Effective new vaccines are urgently needed. Autonomously replicating plant virus-based vector provides attractive means for producing chimeric virus particles (CVPs) in plants that can be developed into vaccines. In this study, we demonstrate the potential for vaccine development of Bamboo mosaic virus (BaMV) epitope-presentation system, where the antigen from vvIBDV VP2 was fused to the N-terminus of BaMV coat protein. Accordingly, an infections plasmid, pBIBD2, was constructed. Inoculation of the recombinant BaMV clone pBIBD2 enabled the generation of chimeric virus, BIBD2, and stable expression of IBDV VP2 antigen on its coat protein. After intramuscular immunization with BIBD2 CVPs, chickens produced antibodies against IBDV and were protected from vvIBDV (V263/TW strain) challenges. These results corroborate the feasibility of BaMV-based CVP platform in plants for the development and production of vaccines against IBDV. PMID:22406128

  10. 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

  11. Restriction fragment length polymorphism analysis of multiple genome regions of Korean isolates of infectious laryngotracheitis virus collected from chickens.

    PubMed

    Kim, Hye-Ryoung; Kang, Min-Su; Kim, Mi-Jin; Lee, Hee-Soo; Kwon, Yong-Kuk

    2013-08-01

    This study was conducted to characterize infectious laryngotracheitis (ILT) viruses isolated from poultry in South Korea using RFLP analysis of PCR products. Seven wild-type Korean isolates from commercial chicken farms collected between 1986 and 2012 were compared with 3 imported commercial vaccine strains [LT Blen (Hudson strain, United States), Laryngo Vac (Cover strain, United States), and Nobilis ILT (Serva strain, France)] and a Korean chicken embryo origin (CEO) vaccine strain [ILT-VAC (Gyeonggi97 strain, Korea)]. Six of the field isolates were highly virulent viruses, and the Kr12AD37 isolate was considered an attenuated type according to Han's RFLP method. These virulent Korean ILT viruses were divided into 3 classes (class I, II, and III). The Kr12AD37 isolate was found to have the same RFLP pattern as the Korean CEO vaccine strain, and both of these strains were different from the 3 foreign vaccine strains. The results suggest that the Korean CEO vaccine strain has been responsible for recent outbreaks, and the characterization of ILT viruses by RFLP was useful for diagnosis by providing epidemiological information. PMID:23873552

  12. Production of infectious ferret hepatitis E virus in a human hepatocarcinoma cell line PLC/PRF/5.

    PubMed

    Li, Tian-Cheng; Yoshizaki, Sayaka; Yang, Tingting; Kataoka, Michiyo; Nakamura, Tomofumi; Ami, Yasushi; Yuriko, Suzaki; Takeda, Naokazu; Wakita, Takaji

    2016-02-01

    A strain of ferret hepatitis E virus (HEV), sF4370, isolated from an imported ferret was used to inoculate a human hepatocarcinoma cell line, PLC/PRF/5. The virus genome and capsid protein were detected in the cell culture supernatant. Immunofluorescence microscopy indicated that the capsid protein was located in the cytoplasm. The virus particles were purified from the culture supernatant by sucrose gradient ultracentrifugation. The capsid protein with molecular mass of ∼72 kDa was detected in fractions with density of 1.150-1.162 g/cm(3), and particles of ferret HEV was associated with cell membrane. The virus recovered from the supernatant was serially passaged with PLC/PRF/5 cells and had the ability to infect ferrets by oral inoculation, indicating that the ferret HEV grown in PLC/PRF/5 was infectious. The establishment of ferret HEV cell culture system might be useful to understand the life cycle, mechanism of infection and replication of ferret HEV. PMID:26763355

  13. Computational and molecular analysis of conserved influenza A virus RNA secondary structures involved in infectious virion production.

    PubMed

    Kobayashi, Yuki; Dadonaite, Bernadeta; van Doremalen, Neeltje; Suzuki, Yoshiyuki; Barclay, Wendy S; Pybus, Oliver G

    2016-09-01

    As well as encoding viral proteins, genomes of RNA viruses harbor secondary and tertiary RNA structures that have been associated with functions essential for successful replication and propagation. Here, we identified stem-loop structures that are extremely conserved among 1,884 M segment sequences of influenza A virus (IAV) strains from various subtypes and host species using computational and evolutionary methods. These structures were predicted within the 3' and 5' ends of the coding regions of M1 and M2, respectively, where packaging signals have been previously proposed to exist. These signals are thought to be required for the incorporation of a single copy of 8 different negative-strand RNA segments (vRNAs) into an IAV particle. To directly test the functionality of conserved stem-loop structures, we undertook reverse genetic experiments to introduce synonymous mutations designed to disrupt secondary structures predicted at 3 locations and found them to attenuate infectivity of recombinant virus. In one mutant, predicted to disrupt stem loop structure at nucleotide positions 219-240, attenuation was more evident at increased temperature and was accompanied by an increase in the production of defective virus particles. Our results suggest that the conserved secondary structures predicted in the M segment are involved in the production of infectious viral particles during IAV replication. PMID:27399914

  14. Neutralization Analysis of a Chicken Single-Chain Variable Fragment Derived from an Immune Antibody Library Against Infectious Bronchitis Virus.

    PubMed

    Lin, Yuan; Li, Benqiang; Ye, Jiaxin; Wang, Man; Wang, Jianhua; Zhang, Ying; Zhu, Jianguo

    2015-09-01

    Avian infectious bronchitis virus (IBV), which is prevalent in many countries causing severe economic loss to the poultry industry, causes infectious bronchitis (IB) in birds. Recombinant single-chain variable fragments (scFvs) have been proven to effectively inhibit many viruses, both in vitro and in vivo, and they could be a potential diagnostic and therapeutic reagent to control IB. In this study, six anti-IBV chicken scFvs, ZL.10, ZL.64, ZL.78, ZL.80, ZL.138, and ZL.256, were obtained by screening random clones from an immune antibody library. An analysis of nucleotide sequences revealed that they represented distinctive genetic sequences and greatly varied in complementarity-determining region three of the heavy chain. Neutralization tests showed that ZL.10, which bound the S1 protein in western blots, inhibited the formation of syncytia in Vero cells 48 h post IBV infection and decreased the transcriptional level of nucleoprotein mRNA to 17.2%, while the other five scFvs, including ZL.78 and ZL.256, that bound the N protein did not. In conclusion, the results suggested that specific and neutralizing chicken scFvs against IBV, which can be safe and economical antibody reagents, can be produced in vitro through prokaryotic expression. PMID:26090700

  15. A practical validation approach for virus titer testing of avian infectious bursal disease live vaccine according to current regulatory guidelines.

    PubMed

    Weber Sušanj, Mirta; Košiček, Miljenko; Krnić, Ela Kosor; Ballarin-Perharić, Alenka; Terzić, Svjetlana

    2012-01-01

    The method for virus titer determination of avian infectious bursal disease (IBD) live vaccine, developed long before regulatory validation guidelines is a cell culture based biological assay intended for use in vaccine release testing. The aim of our study was to perform a validation, based on fit-for-purpose principle, of an old 50% tissue culture infectious dose (TCID(50)) method according to Guidelines of the International Cooperation on Harmonization of Technical Requirements for Registration of Veterinary Medicinal Products (VICH). This paper addresses challenges and discusses some key aspects that should be considered when validating biological methods. A different statistical approach and non-parametric statistics was introduced in validation protocol in order to derive useful information from experimental data. This approach is applicable for a wide range of methods. In conclusion, the previous virus titration method had showed to be precise, accurate, linear, robust and in accordance with current regulatory standards, which indicates that there is no need for additional re-development or upgrades of the method for its suitability for intended use. PMID:21996052

  16. Activation of the immune response against Infectious Bursal Disease Virus after intramuscular inoculation of an intermediate strain.

    PubMed

    Carballeda, Juan Manuel; Zoth, Silvina Chimeno; Gómez, Evangelina; Gravisaco, María José; Berinstein, Analía

    2011-09-01

    Infectious bursal disesase is a highly contagious, wide spread immunosuppressive chicken disease caused by the Infectious Bursal Disease Virus (IBDV). IBDV is a two segmented double-strand RNA virus, member of the Birnaviridae family. In order to study the interaction between IBDV and the immune system, chickens were exposed to an intermediate IBDV strain by intramuscular route, and using Real Time PCR the expression of a panel of avian cytokines and chemokines in duodenum, spleen and bursa of Fabricius was analyzed. Also, splenic nitrite (NO₂) production and the frequencies of different mononuclear cell populations were evaluated by Griess reaction and flow cytometry, respectively. Intramuscular (i.m.) IBDV inoculation promoted an over expression of proinflammatory cytokines IL-6, IL-15 and gIFN in spleen, which correlated with an increase of gIFN plasma concentration measured by ELISA, together with an increment of NO₂ concentration in splenocyte supernatants at 1dpi. Results obtained in the present work showed that IBDV of intermediate virulence, given i.m., induced similar effects to those previously described for highly virulent IBDV in early innate immune responses. Considering that the i.m. route is the route of choice for the delivery of new generation vaccines, and that the use of recombinant antigens also requires the addition of adjuvants for proper immune stimulation, results presented here could contribute to identify suitable cytokines to be used or to be stimulated when utilizing subunit vaccines, for the improvement of prevention tools for avian health. PMID:21514000

  17. Reverse restriction fragment length polymorphism (RRFLP): A novel technique for genotyping infectious laryngotracheitis virus (ILTV) live attenuated vaccines.

    PubMed

    Callison, Scott A; Riblet, Sylva M; Rodríguez-Avila, Andres; García, Maricarmen

    2009-09-01

    A novel technique, the reverse restriction fragment length polymorphism (RRFLP) assay, was developed as a means of detecting specific informative polymorphic sites in the infectious laryngotracheitis virus (ILTV) genome. During the RRFLP procedure, DNA is digested with restriction enzymes targeting an informative polymorphic site and then used as template in a real-time polymerase chain reaction (PCR) with primers flanking the informative region. The analysis of the DeltaC(t) values obtained from digested and undigested template DNA provides the genotype of the DNA. In this study, the RRFLP assay was applied as a method to differentiate between the two types of infectious laryngotracheitis virus attenuated live vaccines. Sequence analysis of ILTV vaccines revealed an informative polymorphic site in the 5'-non-coding region of the infected cell protein (ICP4) gene. Unique AvaI and AlwI restriction enzyme sites were identified in the tissue culture origin and chicken embryo origin attenuated vaccines, respectively. These two informative polymorphic sites were used in a RRFLP assay to genotype rapidly and reproducibly ILTV attenuated live vaccines. PMID:19433109

  18. Development of a SYBR Green quantitative polymerase chain reaction assay for rapid detection and quantification of infectious laryngotracheitis virus.

    PubMed

    Mahmoudian, Alireza; Kirkpatrick, Naomi C; Coppo, Mauricio; Lee, Sang-Won; Devlin, Joanne M; Markham, Philip F; Browning, Glenn F; Noormohammadi, Amir H

    2011-06-01

    Infectious laryngotracheitis is an acute viral respiratory disease of chickens with a worldwide distribution. Sensitive detection of the causative herpesvirus is particularly important because it can persist in the host at a very low copy number and be transmitted to other birds. Quantification of viral genome copy number is also useful for clinical investigations and experimental studies. In the study presented here, a quantitative polymerase chain reaction (qPCR) assay was developed using SYBR Green chemistry and the viral gene UL15a to detect and quantify infectious laryngotracheitis virus (ILTV) in ILTV-inoculated chicken embryos or naturally infected birds. The specificity of the assay was confirmed using a panel of viral and bacterial pathogens of poultry. The sensitivity of the assay was compared with two conventional PCR assays, virus titration and an antigen-detecting enzyme-linked immunosorbent assay. The qPCR developed in this study was highly sensitive and specific, and has potential for quantification of ILTV in tissues from naturally and experimentally infected birds and embryos. PMID:21711182

  19. Detection of wild- and vaccine-type avian infectious laryngotracheitis virus in clinical samples and feather shafts of commercial chickens.

    PubMed

    Davidson, Irit; Nagar, Sagit; Ribshtein, Israel; Shkoda, Irena; Perk, Shimon; Garcia, Maricarmen

    2009-12-01

    Infectious laryngotracheitis (ILT) is a respiratory disease of poultry caused by an alphaherpesvirus (ILTV). To evaluate differential detection of ILTVs belonging to the two types, wild-type or vaccine-type, both causing clinical signs, five PCRs were evaluated to detect wild-type and vaccine-type ILTV in clinical samples. By directly sampling the organs, we aimed to avoid changes in the virus genome and to facilitate a fast diagnosis. The samples were tracheal and spleen homogenates and feather shafts. The latter are easy to collect, nonlethal for the bird, and advantageous for monitoring purposes. We investigated the time interval for vaccine virus detection following commercial vaccination by the vent application, which is successfully practiced in Israel. The study indicated that ILTV amplification from feather shafts was possible in clinical cases for about a one-month period after vaccination. Vaccine strains were identified by nested PCR for the ILTV-gE gene and differed from wild-type ILTV strains by two criteria: (1) While avirulent vaccines could be detected for about a month after the vent application, wild-type virus could be detected, in conjunction with clinical signs, for an unlimited time period; and (2) The ILTV vaccine was present in the bird in minute quantities compared to the wild-type virus. We assessed the virus type that appeared in conjunction with the clinical signs and determined that the clinical signs appeared in conjunction with both molecular forms of ILTV. The vaccine virus-type and the wild-type ILTV differed by their distinct restriction pattern when using the HaeIII restriction enzyme digestion of the nested amplification product. PMID:20095166

  20. Quantitative expression profiling of immune response genes in rainbow trout following infectious haematopoietic necrosis virus (IHNV) infection or DNA vaccination

    USGS Publications Warehouse

    Purcell, Maureen K.; Kurath, Gael; Garver, Kyle A.; Herwig, Russell P.; Winton, James R.

    2004-01-01

    Infectious haematopoietic necrosis virus (IHNV) is a well-studied virus of salmonid fishes. A highly efficacious DNA vaccine has been developed against this virus and studies have demonstrated that this vaccine induces both an early and transient non-specific anti-viral phase as well as long-term specific protection. The mechanisms of the early anti-viral phase are not known, but previous studies noted changes in Mx gene expression, suggesting a role for type I interferon. This study used quantitative real-time reverse transcriptase PCR methodology to compare expression changes over time of a number of cytokine or cytokine-related genes in the spleen of rainbow trout following injection with poly I:C, live IHNV, the IHNV DNA vaccine or a control plasmid encoding the non-antigenic luciferase gene. The target genes included Mx-1, viral haemorrhagic septicaemia virus induced gene 8 (Vig-8), TNF-α1, TNF-α2, IL-1β1, IL-8, TGF-β1 and Hsp70. Poly I:C stimulation induced several genes but the strongest and significant response was observed in the Mx-1 and Vig-8 genes. The live IHN virus induced a significant response in all genes examined except TGF-β1. The control plasmid construct and the IHNV DNA vaccine marginally induced a number of genes, but the main difference between these two groups was a statistically significant induction of the Mx-1 and Vig-8 genes by the IHNV vaccine only. The gene expression profiles elicited by the live virus and the IHNV DNA vaccine differed in a number of aspects but this study confirms the clear role for a type I interferon-like response in early anti-viral defence.

  1. Dengue Virus Non-structural Protein 1 Modulates Infectious Particle Production via Interaction with the Structural Proteins

    PubMed Central

    Scaturro, Pietro; Cortese, Mirko; Chatel-Chaix, Laurent; Fischl, Wolfgang; Bartenschlager, Ralf

    2015-01-01

    Non-structural protein 1 (NS1) is one of the most enigmatic proteins of the Dengue virus (DENV), playing distinct functions in immune evasion, pathogenesis and viral replication. The recently reported crystal structure of DENV NS1 revealed its peculiar three-dimensional fold; however, detailed information on NS1 function at different steps of the viral replication cycle is still missing. By using the recently reported crystal structure, as well as amino acid sequence conservation, as a guide for a comprehensive site-directed mutagenesis study, we discovered that in addition to being essential for RNA replication, DENV NS1 is also critically required for the production of infectious virus particles. Taking advantage of a trans-complementation approach based on fully functional epitope-tagged NS1 variants, we identified previously unreported interactions between NS1 and the structural proteins Envelope (E) and precursor Membrane (prM). Interestingly, coimmunoprecipitation revealed an additional association with capsid, arguing that NS1 interacts via the structural glycoproteins with DENV particles. Results obtained with mutations residing either in the NS1 Wing domain or in the β-ladder domain suggest that NS1 might have two distinct functions in the assembly of DENV particles. By using a trans-complementation approach with a C-terminally KDEL-tagged ER-resident NS1, we demonstrate that the secretion of NS1 is dispensable for both RNA replication and infectious particle production. In conclusion, our results provide an extensive genetic map of NS1 determinants essential for viral RNA replication and identify a novel role of NS1 in virion production that is mediated via interaction with the structural proteins. These studies extend the list of NS1 functions and argue for a central role in coordinating replication and assembly/release of infectious DENV particles. PMID:26562291

  2. A microRNA from infectious spleen and kidney necrosis virus modulates expression of the virus-mock basement membrane component VP08R.

    PubMed

    Yan, Muting; He, Jianhui; Zhu, Weibin; Zhang, Jing; Xia, Qiong; Weng, Shaoping; He, Jianguo; Xu, Xiaopeng

    2016-05-01

    Infectious spleen and kidney necrosis virus (ISKNV) is the type species of the genus Megalocytivirus, family Iridoviridae. Infection of ISKNV is characterized by a unique pathological phenomenon in that the infected cells are attached by lymphatic endothelial cells (LECs). ISKNV mediates the formation of a virus-mock basement membrane (VMBM) structure on the surface of infected cells to provide attaching sites for LECs. The viral protein VP08R is an important component of VMBM. In this study, a novel ISKNV-encoded microRNA, temporarily named ISKNV-miR-1, was identified. ISKNV-miR-1 is complementary to the VP08R-coding sequence and can modulate VP08R expression through reducing its mRNA level. This suggests that formation of VMBM may be under fine regulation by ISKNV. PMID:26896933

  3. Infection of Broilers with Two Virulent Strains of Infectious Laryngotracheitis Virus: Criteria for Evaluation of Experimental Infections.

    PubMed

    Vagnozzi, Ariel; Riblet, Sylva M; Williams, Susan M; Zavala, Guillermo; García, Maricarmen

    2015-09-01

    Infectious laryngotracheitis (ILT) is a highly contagious disease of chickens and is responsible for significant economic losses in the poultry industry worldwide; it is caused by Gallid herpesvirus-1 (GaHV-1), commonly known as infectious laryngotracheitis virus (ILTV). Experimental evaluation of ILTV strains is fundamental to identify changes in virulence that can contribute to the severity and spread of outbreaks and consequently influence the efficacy of vaccination. Several criteria had been utilized to determine the degree of virulence associated with ILTV strains. The objectives of this study were to compare the levels of virulence of the standard United States Department of Agriculture (USDA) challenge strain with a contemporary outbreak-related strain (63140) and to evaluate the efficacy of individual criteria to identify changes in virulence. Broilers were inoculated with increasing infectious doses of each strain. The criteria utilized to evaluate virulence were clinical signs of the disease, mortality, microscopic tracheal lesions, trachea genome viral loads, and antibody titers. Clinical signs scores were a useful parameter to define the peak of clinical disease but did not reveal differences in virulence between strains. Similarly, trachea microscopic lesion scores or levels of serum antibody titers were parameters that did not reveal obvious differences in virulence between strains. However, mortalities and increased viral genome loads in trachea of chickens inoculated with lower (log10 1 to 2) infectious doses clearly differentiated 63140 as a more-virulent ILTV strain. This study provides the framework to compare the virulence level of emerging ILTV isolates to the now-characterized USDA and 63140 strains. PMID:26478158

  4. Virus-induced gene silencing and transient gene expression in soybean using Bean pod mottle virus infectious clones

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Virus-induced gene silencing (VIGS) is a powerful and rapid approach for determining the functions of plant genes. The basis of VIGS is that a viral genome is engineered so that it can carry fragments of plant genes, typically in the 200-300 base pair size range. The recombinant viruses are used to ...

  5. Infectious haematopoietic necrosis virus genogroup-specific virulence mechanisms in sockeye salmon, Oncorhynchus nerka (Walbaum), from Redfish Lake, Idaho

    USGS Publications Warehouse

    Purcell, M.K.; Garver, K.A.; Conway, C.; Elliott, D.G.; Kurath, G.

    2009-01-01

    Characterization of infectious haematopoietic necrosis virus (IHNV) field isolates from North America has established three main genogroups (U, M and L) that differ in host-specific virulence. In sockeye salmon, Oncorhynchus nerka, the U genogroup is highly virulent, whereas the M genogroup is nearly non-pathogenic. In this study, we sought to characterize the virus-host dynamics that contribute to genogroup-specific virulence in a captive stock of sockeye salmon from Redfish Lake in Idaho. Juvenile sockeye salmon were challenged by immersion and injection with either a representative U or M viral strain and sampled periodically until 14 days post-infection (p.i.). Fish challenged with each strain had positive viral titre by day 3, regardless of challenge route, but the fish exposed to the M genogroup virus had significantly lower virus titres than fish exposed to the U genogroup virus. Gene expression analysis by quantitative reverse transcriptase PCR was used to simultaneously assess viral load and host interferon (IFN) response in the anterior kidney. Viral load was significantly higher in the U-challenged fish relative to M-challenged fish. Both viruses induced expression of the IFN-stimulated genes (ISGs), but expression was usually significantly lower in the M-challenged group, particularly at later time points (7 and 14 days p.i.). However, ISG expression was comparable with 3 days post-immersion challenge despite a significant difference in viral load. Our data indicated that the M genogroup virus entered the host, replicated and spread in the sockeye salmon tissues, but to a lesser extent than the U genogroup. Both virus types induced a host IFN response, but the high virulence strain (U) continued to replicate in the presence of this response, whereas the low virulence strain (M) was cleared below detectable levels. We hypothesize that high virulence is associated with early in vivo replication allowing the virus to achieve a threshold level, which the

  6. Combined use of the ASK and SHK-1 cell lines to enhance the detection of infectious salmon anemia virus

    USGS Publications Warehouse

    Rolland, J.B.; Bouchard, D.; Coll, J.; Winton, J.R.

    2005-01-01

    Infectious salmon anemia (ISA) is a severe disease primarily affecting commercially farmed Atlantic salmon (Salmo salar) in seawater. The disease has been reported in portions of Canada, the United Kingdom, the Faroe Islands, and the United States. Infectious salmon anemia virus (ISAV), the causative agent of ISA, has also been isolated from several asymptomatic marine and salmonid fish species. Diagnostic assays for the detection of ISAV include virus isolation in cell culture, a reverse transcriptase-PCR, an enzyme-linked immunosorbent assay, and an indirect fluorescent antibody test. Virus isolation is considered the gold standard, and 5 salmonid cell lines are known to support growth of ISAV. In this study, the relative performance of the salmon head kidney 1 (SHK-1), Atlantic salmon kidney (ASK), and CHSE-214 cell lines in detecting ISAV was evaluated using samples from both experimentally and naturally infected Atlantic salmon. Interlaboratory comparisons were conducted using a quality control-quality assurance ring test. Both the ASK and SHK-1 cell lines performed well in detecting ISAV, although the SHK-1 line was more variable in its sensitivity to infection and somewhat slower in the appearance of cytopathic effect. Relative to the SHK-1 and ASK lines, the CHSE-214 cell line performed poorly. Although the ASK line appeared to represent a good alternative to the more commonly used SHK-1 line, use of a single cell line for diagnostic assays may increase the potential for false-negative results. Thus, the SHK-1 and ASK cell lines can be used in combination to provide enhanced ability to detect ISAV.

  7. A Multiplex PCR/LDR Assay for the Simultaneous Identification of Category A Infectious Pathogens: Agents of Viral Hemorrhagic Fever and Variola Virus.

    PubMed

    Das, Sanchita; Rundell, Mark S; Mirza, Aashiq H; Pingle, Maneesh R; Shigyo, Kristi; Garrison, Aura R; Paragas, Jason; Smith, Scott K; Olson, Victoria A; Larone, Davise H; Spitzer, Eric D; Barany, Francis; Golightly, Linnie M

    2015-01-01

    CDC designated category A infectious agents pose a major risk to national security and require special action for public health preparedness. They include viruses that cause viral hemorrhagic fever (VHF) syndrome as well as variola virus, the agent of smallpox. VHF is characterized by hemorrhage and fever with multi-organ failure leading to high morbidity and mortality. Smallpox, a prior scourge, has been eradicated for decades, making it a particularly serious threat if released nefariously in the essentially non-immune world population. Early detection of the causative agents, and the ability to distinguish them from other pathogens, is essential to contain outbreaks, implement proper control measures, and prevent morbidity and mortality. We have developed a multiplex detection assay that uses several species-specific PCR primers to generate amplicons from multiple pathogens; these are then targeted in a ligase detection reaction (LDR). The resultant fluorescently-labeled ligation products are detected on a universal array enabling simultaneous identification of the pathogens. The assay was evaluated on 32 different isolates associated with VHF (ebolavirus, marburgvirus, Crimean Congo hemorrhagic fever virus, Lassa fever virus, Rift Valley fever virus, Dengue virus, and Yellow fever virus) as well as variola virus and vaccinia virus (the agent of smallpox and its vaccine strain, respectively). The assay was able to detect all viruses tested, including 8 sequences representative of different variola virus strains from the CDC repository. It does not cross react with other emerging zoonoses such as monkeypox virus or cowpox virus, or six flaviviruses tested (St. Louis encephalitis virus, Murray Valley encephalitis virus, Powassan virus, Tick-borne encephalitis virus, West Nile virus and Japanese encephalitis virus). PMID:26381398

  8. Recombinant infectious bursal disease virus expressing Newcastle disease virus (NDV) neutralizing epitope confers partial protection against virulent NDV challenge in chickens.

    PubMed

    Li, Kai; Gao, Li; Gao, Honglei; Qi, Xiaole; Gao, Yulong; Qin, Liting; Wang, Yongqiang; Wang, Xiaomei

    2014-01-01

    In this study, the regions in the infectious bursal disease virus (IBDV) genome that are amenable to the introduction of a sequence encoding a virus-neutralizing epitope of Newcastle disease virus (NDV) hemagglutinin-neuraminidase (HN) protein were identified. By using the reverse genetics approach, insertions or substitutions of sequences encoding the NDV epitope were engineered in the exposed loops (PBC, PHI and [Formula: see text] ) of the VP2 capsid protein and the N terminus of the nonstructural VP5 protein as well as the pep7a and pep7b regions of the pVP2 precursor of a commonly used IBDV vaccine strain, Gt. Three recombinant IBDVs expressing the NDV epitopes were successfully rescued in the PBC, pep7b and VP5 regions and the expressed epitope was recognized by anti-HN antibodies. Genetic analysis showed that the IBDV recombinants carrying the NDV epitopes were stable in cell cultures and in chickens. Animal studies demonstrated that the IBDV recombinants were innocuous in chickens. Vaccination with the recombinant viruses generated antibody responses against both IBDV and NDV, and provided 70-80% protection against IBDV and 50-60% protection against NDV. These results indicate that the recombinant IBDV has the potential to serve as a novel vaccine vector for other pathogens. In future studies, it is worth considering research to improve IBDV vector vaccine to get complete protection and safety of animals and humans. PMID:24200519

  9. Immunohistochemical Detection of Rift Valley Fever Virus with Non-Infectious, Recombinant Viral Protein Antibodies

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Rift Valley fever virus (RVFV) causes re-emerging disease outbreaks and abortion storms in mature cattle, sheep, and goats, and can cause 100% mortality in young animals. The spread of this exotic, insect transmitted virus is of particular concern because of its widely recognized potential for being...

  10. Hatchery Spray Cabinet Administration Does Not Damage Avian Coronavirus Infectious Bronchitis Virus Vaccine Based on Analysis by Electron Microscopy and Virus Titration.

    PubMed

    Roh, Ha-Jung; Jordan, Brian J; Hilt, Deborah A; Ard, Mary B; Jackwood, Mark W

    2015-03-01

    studies in our laboratory showed that the Arkansas-Delmarva Poultry Industry (Ark-DPI) vaccine given to 1-day-old chickens by hatchery spray cabinet replicated poorly and failed to adequately protect broilers against homologous virus challenge, whereas the same vaccine given by eye-drop did replicate and the birds were protected following homologous virus challenge. To determine if mechanical damage following spray application plays a role in failure of the Ark-DPI vaccine, we examined the morphology of three Ark-DPI vaccines from different manufacturers using an electron microscope and included a Massachusetts (Mass) vaccine as control. One of the Ark-DPI vaccines (vaccine A) and the Mass vaccine had significantly (P < 0.005) fewer spikes than the other two Ark-DPI vaccines. We also found that the Ark-DPI and Mass vaccines had significantly (P < 0.005) fewer spike proteins per virus particle when compared to their respective challenge viruses. This observation is interesting and may provide some insight into the mechanism behind infectious bronchitis virus attenuation. No obvious differences were observed in virus morphology and no consistent trend in the number of spikes per virion was found in before- and after-spray samples. We also determined the vaccine titer before and after spray in embryonated eggs and found that both Ark-DPI and Mass vaccines had a similar drop in titer, 0.40 logi and 0.310 logi, respec10ively. Based on these data, it appears that mechanical damage to the Ark-DPI vaccine is not occurring when delivered by a hatchery spray cabinet, suggesting that some other factor is contributing to the failure of that vaccine when given by that method. PMID:26292549

  11. Protection induced by commercially available live-attenuated and recombinant viral vector vaccines against infectious laryngotracheitis virus in broiler chickens.

    PubMed

    Vagnozzi, Ariel; Zavala, Guillermo; Riblet, Sylva M; Mundt, Alice; García, Maricarmen

    2012-01-01

    Viral vector vaccines using fowl poxvirus (FPV) and herpesvirus of turkey (HVT) as vectors and carrying infectious laryngotracheitis virus (ILTV) genes are commercially available to the poultry industry in the USA. Different sectors of the broiler industry have used these vaccines in ovo or subcutaneously, achieving variable results. The objective of the present study was to determine the efficacy of protection induced by viral vector vaccines as compared with live-attenuated ILTV vaccines. The HVT-LT vaccine was more effective than the FPV-LT vaccine in mitigating the disease and reducing levels of challenge virus when applied in ovo or subcutaneously, particularly when the challenge was performed at 57 days rather than 35 days of age. While the FPV-LT vaccine mitigated clinical signs more effectively when administered subcutaneously than in ovo, it did not reduce the concentration of challenge virus in the trachea by either application route. Detection of antibodies against ILTV glycoproteins expressed by the viral vectors was a useful criterion to assess the immunogenicity of the vectors. The presence of glycoprotein I antibodies detected pre-challenge and post challenge in chickens vaccinated with HVT-LT indicated that the vaccine induced a robust antibody response, which was paralleled by significant reduction of clinical signs. The chicken embryo origin vaccine provided optimal protection by significantly mitigating the disease and reducing the challenge virus in chickens vaccinated via eye drop. The viral vector vaccines, applied in ovo and subcutaneously, provided partial protection, reducing to some degree clinical signs, and challenge VIRUS replication in the trachea. PMID:22845318

  12. The Last C-Terminal Residue of VP3, Glutamic Acid 257, Controls Capsid Assembly of Infectious Bursal Disease Virus

    PubMed Central

    Chevalier, Christophe; Lepault, Jean; Da Costa, Bruno; Delmas, Bernard

    2004-01-01

    Infectious bursal disease virus (IBDV) is a nonenveloped virus with an icosahedral capsid composed of two proteins, VP2 and VP3, that derive from the processing of the polyprotein NH2-pVP2-VP4-VP3-COOH. The virion contains VP1, the viral polymerase, which is both free and covalently linked to the two double-stranded RNA (dsRNA) genomic segments. In this study, the virus assembly process was studied further with the baculovirus expression system. While expression of the wild-type polyprotein was not found to be self-sufficient to give rise to virus-like particles (VLPs), deletion or replacement of the five C-terminal residues of VP3 was observed to promote capsid assembly. Indeed, the single deletion of the C-terminal glutamic acid was sufficient to induce VLP formation. Moreover, fusion of various peptides or small proteins (a green fluorescent protein or a truncated form of ovalbumin) at the C terminus of VP3 also promoted capsid assembly, suggesting that assembly required screening of the negative charges at the C terminus of VP3. The fused polypeptides mimicked the effect of VP1, which interacts with VP3 to promote VLP assembly. The C-terminal segment of VP3 was found to contain two functional domains. While the very last five residues of VP3 mainly controlled both assembly and capsid architecture, the five preceding residues constituted the VP1 (and possibly the pVP2/VP2) binding domain. Finally, we showed that capsid formation is associated with VP2 maturation, demonstrating that the protease VP4 is involved in the virus assembly process. PMID:15016850

  13. Phylogeny and S1 Gene Variation of Infectious Bronchitis Virus Detected in Broilers and Layers in Turkey.

    PubMed

    Yilmaz, Huseyin; Altan, Eda; Cizmecigil, Utku Y; Gurel, Aydin; Ozturk, Gulay Yuzbasioglu; Bamac, Ozge Erdogan; Aydin, Ozge; Britton, Paul; Monne, Isabella; Cetinkaya, Burhan; Morgan, Kenton L; Faburay, Bonto; Richt, Juergen A; Turan, Nuri

    2016-09-01

    The avian coronavirus infectious bronchitis virus (AvCoV-IBV) is recognized as an important global pathogen because new variants are a continuous threat to the poultry industry worldwide. This study investigates the genetic origin and diversity of AvCoV-IBV by analysis of the S1 sequence derived from 49 broiler flocks and 14 layer flocks in different regions of Turkey. AvCoV-IBV RNA was detected in 41 (83.6%) broiler flocks and nine (64.2%) of the layer flocks by TaqMan real-time RT-PCR. In addition, AvCoV-IBV RNA was detected in the tracheas 27/30 (90%), lungs 31/49 (62.2%), caecal tonsils 7/22 (31.8%), and kidneys 4/49 (8.1%) of broiler flocks examined. Pathologic lesions, hemorrhages, and mononuclear infiltrations were predominantly observed in tracheas and to a lesser extent in the lungs and a few in kidneys. A phylogenetic tree based on partial S1 sequences of the detected AvCoV-IBVs (including isolates) revealed that 1) viruses detected in five broiler flocks were similar to the IBV vaccines Ma5, H120, M41; 2) viruses detected in 24 broiler flocks were similar to those previously reported from Turkey and to Israel variant-2 strains; 3) viruses detected in seven layer flocks were different from those found in any of the broiler flocks but similar to viruses previously reported from Iran, India, and China (similar to Israel variant-1 and 4/91 serotypes); and 4) that the AVCoV-IBV, Israeli variant-2 strain, found to be circulating in Turkey appears to be undergoing molecular evolution. In conclusion, genetically different AvCoV-IBV strains, including vaccine-like strains, based on their partial S1 sequence, are circulating in broiler and layer chicken flocks in Turkey and the Israeli variant-2 strain is undergoing evolution. PMID:27610718

  14. Protective oral vaccination against infectious bursal disease virus using the major viral antigenic protein VP2 produced in Pichia pastoris.

    PubMed

    Taghavian, Omid; Spiegel, Holger; Hauck, Rüdiger; Hafez, Hafez M; Fischer, Rainer; Schillberg, Stefan

    2013-01-01

    Infectious bursal disease virus (IBDV) causes economically important immunosuppressive disease in young chickens. The self-assembling capsid protein (VP2) from IBDV strain IR01 was expressed in Pichia pastoris resulting in the formation of homomeric, 23-nm infectious bursal disease subviral particles (IBD-SVPs) with a yield of 76 mg/l before and 38 mg/l after purification. Anti-IBDV antibodies were detected in chickens injected with purified IBD-SVPs or fed with either purified IBD-SVPs or inactivated P. pastoris cells containing IBD-VP2 (cell-encapsulated). Challenge studies using the heterologous classical IBDV strain (MB3) showed that intramuscular vaccination with 20 µg purified IBD-SVPs conferred full protection, achieved complete virus clearance and prevented bursal damage and atrophy, compared with only 40% protection, 0-10% virus clearance accompanied by severe atrophy and substantial bursal damage in mock-vaccinated and challenge controls. The commercial IBDV vaccine also conferred full protection and achieved complete virus clearance, albeit with partial bursal atrophy. Oral administration of 500 µg purified IBD-SVPs with and without adjuvant conferred 100% protection but achieved only 60% virus clearance with adjuvant and none without it. Moderate bursal damage was observed in both cases but the inclusion of adjuvant resulted in bursal atrophy similar to that observed with live-attenuated vaccine and parenteral administration of 20 µg purified IBD-SVPs. The oral administration of 250 mg P. pastoris cells containing IBD-VP2 resulted in 100% protection with adjuvant and 60% without, accompanied by moderate bursal damage and atrophy in both groups, whereas 25 mg P. pastoris cells containing IBD-VP2 resulted in 90-100% protection with moderate bursal lesions and severe atrophy. Finally, the oral delivery of 50 µg purified IBD-SVPs achieved 40-60% protection with severe bursal lesions and atrophy. Both oral and parenteral administration of yeast

  15. Protective Oral Vaccination against Infectious bursal disease virus Using the Major Viral Antigenic Protein VP2 Produced in Pichia pastoris

    PubMed Central

    Taghavian, Omid; Spiegel, Holger; Hauck, Rüdiger; Hafez, Hafez M.; Fischer, Rainer; Schillberg, Stefan

    2013-01-01

    Infectious bursal disease virus (IBDV) causes economically important immunosuppressive disease in young chickens. The self-assembling capsid protein (VP2) from IBDV strain IR01 was expressed in Pichia pastoris resulting in the formation of homomeric, 23-nm infectious bursal disease subviral particles (IBD-SVPs) with a yield of 76 mg/l before and 38 mg/l after purification. Anti-IBDV antibodies were detected in chickens injected with purified IBD-SVPs or fed with either purified IBD-SVPs or inactivated P. pastoris cells containing IBD-VP2 (cell-encapsulated). Challenge studies using the heterologous classical IBDV strain (MB3) showed that intramuscular vaccination with 20 µg purified IBD-SVPs conferred full protection, achieved complete virus clearance and prevented bursal damage and atrophy, compared with only 40% protection, 0–10% virus clearance accompanied by severe atrophy and substantial bursal damage in mock-vaccinated and challenge controls. The commercial IBDV vaccine also conferred full protection and achieved complete virus clearance, albeit with partial bursal atrophy. Oral administration of 500 µg purified IBD-SVPs with and without adjuvant conferred 100% protection but achieved only 60% virus clearance with adjuvant and none without it. Moderate bursal damage was observed in both cases but the inclusion of adjuvant resulted in bursal atrophy similar to that observed with live-attenuated vaccine and parenteral administration of 20 µg purified IBD-SVPs. The oral administration of 250 mg P. pastoris cells containing IBD-VP2 resulted in 100% protection with adjuvant and 60% without, accompanied by moderate bursal damage and atrophy in both groups, whereas 25 mg P. pastoris cells containing IBD-VP2 resulted in 90–100% protection with moderate bursal lesions and severe atrophy. Finally, the oral delivery of 50 µg purified IBD-SVPs achieved 40–60% protection with severe bursal lesions and atrophy. Both oral and parenteral administration of yeast

  16. Nucleotide sequence of the 3' region of an infectious human T-cell leukemia virus type II genome.

    PubMed Central

    Shimotohno, K; Wachsman, W; Takahashi, Y; Golde, D W; Miwa, M; Sugimura, T; Chen, I S

    1984-01-01

    The nucleic acid sequence of the 3' region of human T-cell leukemia virus type II (HTLV-II) proviral DNA was determined using a HTLV-II proviral clone that could be recovered as infectious, transforming virus. The sequence data indicate a region of unknown function of approximately equal to 1.6 kilobase pairs in the 3' region, analogous to the X region previously identified in human T-cell leukemia virus type I (HTLV-I). Three overlapping open reading frames are present in the X region of HTLV-II. One of these open reading frames, Xc, is most likely to encode a protein product, because it has greater predicted amino acid sequence homology (78%) with the X-IV region of HTLV-I and a greater percentage of its base differences with X-IV at the third nucleotide position of codons than do the other open reading frames. Sequences of the X-region that include the open reading frames are conserved in two deletion mutants of HTLV-II, which are associated with a subline of Mo cells with a decreased dependence on fetal bovine serum. Images PMID:6093110

  17. Genetic Signature of Rapid IHHNV (Infectious Hypodermal and Hematopoietic Necrosis Virus) Expansion in Wild Penaeus Shrimp Populations

    PubMed Central

    Robles-Sikisaka, Refugio; Bohonak, Andrew J.; McClenaghan, Leroy R.; Dhar, Arun K.

    2010-01-01

    Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is a widely distributed single-stranded DNA parvovirus that has been responsible for major losses in wild and farmed penaeid shrimp populations on the northwestern Pacific coast of Mexico since the early 1990's. IHHNV has been considered a slow-evolving, stable virus because shrimp populations in this region have recovered to pre-epizootic levels, and limited nucleotide variation has been found in a small number of IHHNV isolates studied from this region. To gain insight into IHHNV evolutionary and population dynamics, we analyzed IHHNV capsid protein gene sequences from 89 Penaeus shrimp, along with 14 previously published sequences. Using Bayesian coalescent approaches, we calculated a mean rate of nucleotide substitution for IHHNV that was unexpectedly high (1.39×10−4 substitutions/site/year) and comparable to that reported for RNA viruses. We found more genetic diversity than previously reported for IHHNV isolates and highly significant subdivision among the viral populations in Mexican waters. Past changes in effective number of infections that we infer from Bayesian skyline plots closely correspond to IHHNV epizootiological historical records. Given the high evolutionary rate and the observed regional isolation of IHHNV in shrimp populations in the Gulf of California, we suggest regular monitoring of wild and farmed shrimp and restriction of shrimp movement as preventative measures for future viral outbreaks. PMID:20668694

  18. Infectious bursal disease virus rescued efficiently with 3' authentic RNA sequence induces humoral immunity without bursal atrophy.

    PubMed

    Mosley, Yung-Yi C; Wu, Ching Ching; Lin, Tsang Long

    2013-01-11

    A reverse genetics infectious bursal disease virus (RG-IBDV) that contained authentic 3' RNA sequence was characterized both in vitro and in vivo. LP1-IBDV, a cell line-adapted IBDV strain variant E (VE) was used as the parent virus for constructing viral cDNA clones. Authentic 3' RNA sequence was generated by using cis-acting hepatitis delta virus ribozyme (HDR). The absence of HDR in the clones did not affect viral protein expression, but the obtained viral titers were reduced by 200-folds when compared to the clones with HDR sequence. RG-IBDV generated from clones with HDR sequence was similar to LP1-IBDV by plaque size, growth kinetics and electron microscopic morphology. RG-IBDV did not cause bursal atrophy in 3-week-old chickens at 3, 7 and 17 days post infection (DPI) and induced high ELISA and neutralizing antibody titers to IBDV at 7 and 17 DPI. The results indicated that RG-IBDV can be generated efficiently with an authentic 3' RNA terminus and the obtained RG-IBDV is non-pathogenic and immunogenic with the potential as a vaccine strain that can be further genetically modified to broaden its application. PMID:23196210

  19. Inhibitory activity of the equine infectious anemia virus major 5' splice site in the absence of Rev.

    PubMed Central

    Tan, W; Schalling, M; Zhao, C; Luukkonen, M; Nilsson, M; Fenyö, E M; Pavlakis, G N; Schwartz, S

    1996-01-01

    The major 5' splice site of equine infectious anemia virus (EIAV) conforms to the consensus 5' splice site in eight consecutive positions and is located immediately upstream of the gag AUG. Our results show that the presence of this 5' splice site on the EIAV gag mRNA decreases Gag production 30- to 60-fold. This is caused by inefficient nuclear mRNA export and inefficient mRNA utilization. Inhibition could be overcome by providing human immunodeficiency virus type 1 Rev/Rev-responsive element, human T-cell leukemia virus type 1 Rex/Rex-responsive element, or simian retrovirus type 1 constitutive transport element. In addition, inhibition could be abolished by introducing single point mutations in the 5' splice site or by moving the 5' splice site away from its natural position immediately upstream of the gag AUG. This demonstrates that both maintenance of a perfect consensus 5' splice site and its proper location on the mRNA are important for inhibitory activity of the EIAV major 5' splice site. PMID:8648699

  20. Toll-like receptor 2 ligand, lipoteichoic acid is inhibitory against infectious laryngotracheitis virus infection in vitro and in vivo.

    PubMed

    Haddadi, S; Thapa, S; Kameka, A M; Hui, J; Czub, M; Nagy, E; Muench, G; Abdul-Careem, M F

    2015-01-01

    Lipoteichoic acid (LTA) is one of the pathogen associated molecular patterns (PAMPs) that activates toll-like receptor (TLR)2-cluster of differentiation (CD)14 signalling pathway. This recognition elicits antiviral responses that have been recorded against viruses of mammals although such responses have not been characterized adequately against avian viruses. In this investigation, we characterized the LTA induced antiviral responses against infectious laryntotracheitis virus (ILTV) infection in vitro and in vivo. We found that LTA is capable of up regulating mRNA expression of innate proteins in macrophages such as MyD88, iNOS and IL-1β and reduces the ILTV plaques in vitro. Similarly, we found that LTA treatment of embryonic day 18 (ED18) eggs can lead to the antiviral response against pre-hatch ILTV infection in vivo and is associated with expansion of macrophage populations and expression of IL-1β and MyD88 in the lung. The data highlight that LTA can be a potential innate immune stimulant that can be used against ILTV infection in chickens. PMID:25195716

  1. Antigenic properties and diagnostic potential of baculovirus-expressed infectious bursal disease virus proteins VPX and VP3.

    PubMed

    Martínez-Torrecuadrada, J L; Lázaro, B; Rodriguez, J F; Casal, J I

    2000-07-01

    The routine technique for detecting antibodies specific to infectious bursal disease virus (IBDV) is a serological evaluation by enzyme-linked immunosorbent assay (ELISA) with preparations of whole virions as the antigens. To avoid using complete virus in the standard technique, we have developed two new antigens through the expression of the VPX and VP3 genes in insect cells. VPX and especially VP3 were expressed at high levels in insect cells and simple to purify. The immunogenicity of both proteins was similar to that of the native virus. VPX was able to elicit neutralizing antibodies but VP3 was not. Purified VPX and VP3 were tested in an indirect ELISA with more than 300 chicken sera. There was an excellent correlation between the results of the ELISA using VPX and those of the two commercial kits. VP3 did not perform as well as VPX, and the linear correlation was significantly lower. A comparison with the standard reference technique, seroneutralization, showed that the indirect ELISA was more sensitive. Therefore, VPX-based ELISA is a good alternative to conventional ELISAs that use whole virions. PMID:10882666

  2. Development of RT-qPCR assays for the specific identification of two major genotypes of avian infectious bronchitis virus.

    PubMed

    Marandino, Ana; Tomás, Gonzalo; Hernández, Martín; Panzera, Yanina; Craig, María Isabel; Vagnozzi, Ariel; Vera, Federico; Techera, Claudia; Grecco, Sofía; Banda, Alejandro; Hernández, Diego; Pérez, Ruben

    2016-09-01

    Infectious bronchitis virus (Gammacoronavirus, Coronaviridae) is a genetically variable RNA virus (27.6kb) that causes one of the most persistent respiratory disease in poultry. The virus is classified in genotypes with different epidemiological relevance and clinical implications. The present study reports the development and validation of specific RT-qPCR assays for the detection of two major IBV genotypes: South America I (SAI) and Asia/South America II (A/SAII). The SAI genotype is an exclusive and widespread South American lineage while the A/SAII genotype is distributed in Asia, Europe and South America. Both identification assays employ TaqMan probes that hybridize with unique sequences in the spike glycoprotein gene. The assays successfully detected all the assessed strains belonging to both genotypes, showing high specificity and absence of cross-reactivity. Using serial dilutions of in vitro-transcribed RNA we obtained acceptable determination coefficients, PCR efficiencies and relatively small intra- and inter-assay variability. The assays demonstrated a wide dynamic range between 10(1)-10(7) and 10(2)-10(7) RNA copies/reaction for SAI and A/SAII strains, respectively. The possibility to characterize a large number of samples in a rapid, sensitive and reproducible way makes these techniques suitable tools for routine testing, IBV control, and epidemiological research in poultry. PMID:27181213

  3. Survey for antibodies to infectious bursal disease virus serotype 2 in wild turkeys and Sandhill Cranes of Florida, USA.

    PubMed

    Candelora, Kristen L; Spalding, Marilyn G; Sellers, Holly S

    2010-07-01

    Captive-reared Whooping Cranes (Grus americana) released into Florida for the resident reintroduction project experienced unusually high mortality and morbidity during the 1997-98 and 2001-02 release seasons. Exposure to infectious bursal disease virus (IBDV) serotype 2 as evidenced by seroconversion was suspected to be the factor that precipitated these mortality events. Very little is known about the incidence of IBD in wild bird populations. Before this study, natural exposure had not been documented in wild birds of North America having no contact with captive-reared cranes, and the prevalence and transmission mechanisms of the virus in wild birds were unknown. Sentinel chickens (Gallus gallus) monitored on two Whooping Crane release sites in central Florida, USA, during the 2003-04 and 2004-05 release seasons seroconverted, demonstrating natural exposure to IBDV serotype 2. Blood samples collected from Wild Turkeys (Meleagris gallopavo) and Sandhill Cranes (Grus canadensis) in eight of 21 counties in Florida, USA, and one of two counties in southern Georgia, USA, were antibody-positive for IBDV serotype 2, indicating that exposure from wild birds sharing habitat with Whooping Cranes is possible. The presence of this virus in wild birds in these areas is a concern for the resident flock of Whooping Cranes because they nest and raise their chicks in Florida, USA. However, passively transferred antibodies may protect them at this otherwise vulnerable period in their lives. PMID:20688680

  4. Spreading of infectious materials from the laser interaction zone: viruses and bacteria

    NASA Astrophysics Data System (ADS)

    Weber, Lothar W.

    1996-12-01

    Actual occupational infections of medical staff is dominated by HBV, HIV and HCV-infections by dermal blood inoculation like needle injuries. What amount of these blood borne infections was possibly done via the aerosol pathway is unknown today. Looking at the laser generated aerodynamic particle sizes and the particle size of human pathogen viruses as circulating or cell fixed units shows common transmission abilities to the human respiratory system. In cell tissue monolayer model systems and contaminated serum systems with virus infections this mechanics were demonstrated as viable. For safety evaluation, the lifetime, spreading behavior and infection potential by viruses and bacterias of contaminated human laser aerosol must be further characterized.

  5. DEVELOPMENT AND VALIDATION OF A REAL-TIME TAQMAN(R) PCR ASSAY FOR THE DETECTION AND QUANTITATION OF INFECTIOUS LARYNGOTRACHEITIS VIRUS IN POULTRY

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In this study, we report the development and validation of a real-time PCR assay using a Taqman(R) labeled probe (ILTV assay) for the detection and quantification of infectious larygotracheitis virus (ILTV). The ILTV assay was highly specific with a detection limit of 25 viral template copies/amplif...

  6. Putative Promoters Isolated From Infectious Hypodermal and Hematopoietic Necrosis Virus of Shrimp Drive Expression of a Reporter Gene in Bacteria, Insect Cells, Fish Cells, and Shrimp

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Infectious hypodermal and hematopoietic necrosis virus (IHHNV) of shrimp contains a linear single-stranded DNA genome of approximately 4.1 kb with three putative open reading frames (ORFs) namely, the left ORF, middle ORF and the right ORF on the same DNA strand. Whereas the left ORF codes for non-s...

  7. Effects of dietary source and intake of energy on immune competence and the response to an infectious bovine rhinotracheitis virus (IBRV) challenge in cattle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Objectives were to evaluate how dietary energy intake and source affect immune competence and response to an infectious bovine rhinotracheitis virus (IBRV) challenge in cattle. Forty-eight crossbred beef steers were stratified by body weight within 2 periods and randomized to 1 of 3 dietary treatmen...

  8. Very virulent infectious bursal disease virus produces more-severe disease and lesions in specific pathogen free (SPF) Leghorn than in SPF broiler chickens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Infectious bursal disease virus (IBDV) is an important pathogen of chickens causing negative economic impacts in poultry industries worldwide. IBDV has a variable range of virulence, with very virulent (vvIBDV) strains being responsible for the greatest losses from mortality and decreased performanc...

  9. Live vaccination with an H5-hemagglutinin-expressing infectious laryngotracheitis virus recombinant protects chickens against different highly pathogenic avian influenza viruses of the H5 subtype.

    PubMed

    Pavlova, Sophia P; Veits, Jutta; Mettenleiter, Thomas C; Fuchs, Walter

    2009-08-13

    Recently, we described an infectious laryngotracheitis virus (ILTV, gallid herpesvirus 1) recombinant, which had been attenuated by deletion of the viral dUTPase gene UL50, and abundantly expressed the hemagglutinin (HA) gene of a H5N1 type highly pathogenic avian influenza virus (HPAIV) of Vietnamese origin. In the present study, efficacy of this vectored vaccine (ILTV-DeltaUL50IH5V) against different H5 HPAIV was evaluated in 6-week-old chickens. After a single ocular immunization all animals developed HA-specific antibodies, and were protected against lethal infection not only with the homologous HPAIV isolate A/duck/Vietnam/TG24-01/2005 (H5N1, clade 1, hemagglutinin amino acid sequence identity 100%), but also with heterologous HPAIV A/swan/Germany/R65/2006 (H5N1, clade 2.2, identity 96.1%) or HPAIV A/chicken/Italy/8/98 (H5N2, identity 93.8%). No symptoms of disease were observed after challenge with the H5N1 viruses, and only 20% of H5N2 challenged animals developed minimal clinical signs. Real-time RT-PCR analyses of oropharyngeal swabs revealed limited challenge virus replication, but the almost complete absence of HPAIV RNA from cloacal swabs indicated that no generalized infections occurred. Thus, unlike several previous vectors, ILTV-DeltaUL50IH5V was able to protect chickens against different HPAIV isolates of the H5 subtype. Vaccination with HA-expressing ILTV also allowed differentiation of immunized from AIV-infected animals by serological tests for antibodies against influenza virus nucleoprotein. PMID:19573638

  10. Crystallization and preliminary crystallographic study of Feline infectious peritonitis virus main protease in complex with an inhibitor.

    PubMed

    Wang, Jinshan; Wang, Fenghua; Tan, Yusheng; Chen, Xia; Zhao, Qi; Fu, Sheng; Li, Shuang; Chen, Cheng; Yang, Haitao

    2014-12-01

    Feline infectious peritonitis virus (FIPV) causes a lethal systemic granulomatous disease in wild and domestic cats around the world. Currently, no effective vaccines or drugs have been developed against it. As a member of the genus Alphacoronavirus, FIPV encodes two polyprotein precursors required for genome replication and transcription. Each polyprotein undergoes extensive proteolytic processing, resulting in functional subunits. This process is mainly mediated by its genome-encoded main protease, which is an attractive target for antiviral drug design. In this study, the main protease of FIPV in complex with a Michael acceptor-type inhibitor was crystallized. The complex crystals diffracted to 2.5 Å resolution and belonged to space group I422, with unit-cell parameters a = 112.3, b = 112.3, c = 102.1 Å. There is one molecule per asymmetric unit. PMID:25484209

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

    USGS Publications Warehouse

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

    2016-01-01

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

  12. Epidemic of infectious laryngotracheitis in Italy: characterization of virus isolates by PCR-restriction fragment length polymorphism and sequence analysis.

    PubMed

    Moreno, Ana; Piccirillo, Alessandra; Mondin, Alessandra; Morandini, Emilio; Gavazzi, Luigi; Cordioli, Paolo

    2010-12-01

    Between May 2007 and October 2008, 34 outbreaks of mild to moderate forms of infectious laryngotracheitis (ILT) occurred in commercial broiler flocks in Italy. Affected birds showed watery eyes, conjunctivitis, nasal discharge, reduction of feed and water consumption, and gasping with expectoration of blood-stained mucus. The mortality rate was < 10%. Gross lesions consisted of conjunctivitis, excess of mucus, blood, or presence of diphtheritic membranes in trachea. A real-time PCR assay was performed to confirm the presence of ILT virus (ILTV) DNA in tracheal tissue homogenates. Twenty-three ILTV isolates were propagated on the chorion-allantoic membrane of embryonated chicken eggs showing typical plaques. PCR combined with restriction fragment length polymorphism and gene sequencing of isolates showed a high genetic correlation between field strains and chicken embryo origin vaccines. PMID:21313836

  13. Titre distribution patterns of infectious haematopoietic necrosis virus in ovarian fluids of hatchery and feral salmon populations

    USGS Publications Warehouse

    Mulcahy, D.; Pascho, R.J.; Jenes, C.K.

    1983-01-01

    Infectious haematopoietic mecrosis virus (IHNV) is enzootic in virtually all populations of sockeye salmon, Oncorhynchus nerka (Walbaum), and in populations of chinook salmon, O. 1shawytscha (Walbaum), of the Sacramento River drainage in California. This disease is an obstacle in hatcheries using brood stocks from these populations. However, naturally spawning sockeye salmon are highly successful and are the most important commercially fished salmon species in the United States. Most of the commercial landings of sockeye salmon are of feral fish originating in Alaska. The success of natural populations of salmon in which IHNV is enzootic, and the recurrent outbreaks of the disease in hatchery fish, led us to compare IHNW prevalence rates in hatchery and feral salmon populations.

  14. Phylogenetic r