Progress 10/01/11 to 09/30/16
Outputs
Progress Report Objectives (from AD-416): Objective 1: Develop a recombinant herpesvirus expressing OvHV-2 proteins that stimulate a neutralizing antibody response. Subobjective 1.A. Develop an infectious AlHV-2 BAC clone. Subobjective 1.B. Construct infectious recombinant AlHV-2 BACs containing OvHV-2 genes encoding proteins that can stimulate a neutralizing antibody response. Subobjective 1.C. Determine neutralizing activity of hyper immune sera against individual OvHV-2 proteins expressed by recombinant AlHV-2. Objective 2: Develop an efficacious vaccine for protection of clinically-susceptible species from sheep-associated MCF. Subobjective 2.A. Vaccinate bison with recombinant AlHV-2 containing OvHV-2 genes to stimulate a neutralizing antibody response against OvHV-2. Subobjective 2.B. Determine the MCF protection rates of bison vaccinated with recombinant AlHV-2 upon challenge with a lethal dose of OvHV-2. Subobjective 2.C. Determine if vaccination prevents bison from developing MCF when exposed to OvHV-2 infected sheep. Approach (from AD-416): The proposed research will develop a recombinant herpesvirus expressing OvHV-2 proteins by utilizing AlHV-2, a non-pathogenic MCFV carried by African antelopes (hartebeest and topi) that can grow in cell culture, as a vaccine. AlHV-2 has been isolated from clinically normal topi antelope and hartebeest in Africa and the U.S. (61, 79, 84). There has been no report of AlHV-2-induced MCF in cattle under natural transmission conditions, although an MCF case in red deer reported from the San Diego Wildlife Park was associated with an AlHV-2-like virus from Jackson Hartebeest (36). Experimental inoculation of cattle with cell-free AlHV-2 isolates can result in infection, but does not induce clinical disease. Moreover, inoculation of cattle with AlHV-2 does not elicit antibodies protective against subsequent AlHV-1 challenge (66). We will use recombination-mediated genetic engineering to generate recombinant AlHV-2 (rAlHV-2) containing relevant OvHV-2 genes (rAlHV-2OvHV-2g). The rAlHV- 2OvHV-2g will be tested as a vaccine to stimulate local immune responses in the respiratory tract to protect clinically susceptible hosts from SA- MCF. Our main hypothesis is: immunization with rAlHV-2OvHV-2g will stimulate neutralizing antibodies at the viral entry site in bison, which will correlate with reduced viral load and protection against lethal OvHV- 2 challenge. The hypothesis will be tested through the previous two objectives. This is the final report for this project which expired September 30, 2016. Substantial progress was made on all objectives and their subobjectives over the 5 years of the project. The sequencing and annotation of the AlHV-2 genome were successfully completed. Construction of an infectious AlHV-2 BAC clone was near completion at that time, however when the infectivity of AlHV-2 was tested in experimental animals (rabbits and bison), unexpected results revealed that AlHV-2 was capable of inducing malignant catarrhal fever (MCF) in bison, while it failed to infect rabbits, indicating that AlHV-2 was not an appropriate virus to use as a vaccine backbone. These results promptly redirected our research plan to use an attenuated AlHV-1 as an OvHV-2 vaccine backbone. As AlHV-1 was removed from the Select Agent list in 2013 and an attenuated AlHV-1 mutant with deletion of the ORF73 gene was generated by our collaborators at the University of Liege, Belgium, we were able to import the attenuated AlHV-1 mutant to use as the vaccine backbone. In order to identify OvHV-2 glycoproteins that can stimulate neutralizing antibody responses to be used as vaccine targets, hyper immune sera against selected OvHV-2 glycoproteins were produced following DNA immunization of rabbits using a gene gun. All rabbits developed high antibody titers against the glycoproteins and their sera were critical to identify vaccine targets. Since there is no cell culture system available to propagate OvHV-2, an alternative in vivo virus neutralization system was developed to assess neutralizing activity of antibodies to OvHV-2. The system made it possible to measure the ability of antibodies to block OvHV-2 at the entry site, which is of great significance for OvHV-2 vaccine development, especially in identifying vaccine targets. Using this animal system, we confirmed that serum antibodies against three OvHV- 2 glycoproteins (B, H and L) completely block OvHV-2 infection and prevent MCF in rabbits. The data indicate glycoproteins are promising targets for a vaccine aimed at stimulating protective immune responses against OvHV-2. As a proof of concept that mucosal immunity, especially a high level of neutralizing antibodies in lung, is key to protect animals from developing MCF, a study to evaluate the role of passively transferred neutralizing antibodies against these OvHV-2 glycoproteins in the respiratory track of rabbits was conducted. Additionally, a vaccine challenge study using plasmid DNA expressing OvHV-2 genes delivered directly to the lung prior to OvHV-2 inoculation was initiated to evaluate antibody responses and protection from MCF in rabbits. Both experiments failed to protect rabbits from the challenge, therefore new approaches are being evaluated to test the hypothesis that specific neutralizing antibodies present in the respiratory tract can block the virus and prevent MCF. In order to test the attenuated AlHV-1 as a vaccine backbone to express and deliver the identified OvHV-2 glycoproteins, several chimeric viruses with one or more AlHV-1 genes encoding glycoproteins B, H, and/or L replaced by its OvHV-2 counterpart gene were constructed. One of the chimeric viruses was able to infect and replicate in cell culture and in rabbits. This chimeric virus was nonpathogenic for rabbits and induced production of antibodies, which is a significant step towards MCF vaccine development. Furthermore, we demonstrated that the chimeric virus can be used in an in vitro neutralization assay to measure neutralizing antibodies to OvHV-2. This is not only critical to measure protective antibody responses, but also provides a valuable tool for OvHV-2 biology studies. Accomplishments 01 Developed a recombinant virus as a vaccine candidate for sheep- associated malignant catarrhal fever (SA-MCF). The progress towards vaccine development for sheep-associated MCF has been hindered by the absence of methods to attenuate or modify the causative agent � ovine herpesvirus 2 (OvHV-2), which does not grow in cell culture. As an alternative, ARS researchers at Pullman, Washington, in collaboration with faculties at University of Li�ge, Belgium, developed a recombinant virus by replacing the glycoprotein B (gB) gene in a non-pathogenic alcelaphine herpesvirus 1 (AlHV-1) by the OvHV-2 counterpart and evaluated the potential of the AlHV-1/OvHV-2 chimera to be used as a vaccine. We have demonstrated that the chimeric virus can infect rabbits and induce an antibody response to OvHV-2 without causing disease. The chimeric virus can also grow in cell culture and its infection can be blocked by antibodies against OvHV-2 gB, meaning that it can be used in a neutralization assay to detect and measure neutralizing antibodies to OvHV-2. Development of the AlHV-1/OvHV-2 chimeric virus that is nonpathogenic and expresses an OvHV-2 vaccine target is a significant step towards the development of a vaccine for SA-MCF. In addition, because the chimeric virus can be used in a neutralization assay, it is also a valuable diagnostic tool for detection of MCF viral neutralizing antibodies in infected animals.
Impacts
(N/A)
Publications
- Taus, N.S., Marquard, J., Cunha, C.W., O'Toole, D., Li, H. 2015. Cross- reactivity of neutralizing antibodies among malignant catarrhal fever viruses. PLoS One. 10(12):30145073.
- Borca, M.V., Gay, C.G., Risatti, G., O'Toole, G., Li, H., Kuhn, J., Lewis, C., Loiacono, C., White, D.M. 2015. Viral hemorrhagic fevers of animals caused by DNA viruses. In:Shapshak, P., Sinnott, J.T., Somboonwit, C., Kuhn, J., editors. Global Virology-Identifying and Investigating Viral Diseases. New York, NY:Springer. p. 319-343.
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Progress 10/01/14 to 09/30/15
Outputs
Progress Report Objectives (from AD-416): Objective 1: Develop a recombinant herpesvirus expressing OvHV-2 proteins that stimulate a neutralizing antibody response. Subobjective 1.A. Develop an infectious AlHV-2 BAC clone. Subobjective 1.B. Construct infectious recombinant AlHV-2 BACs containing OvHV-2 genes encoding proteins that can stimulate a neutralizing antibody response. Subobjective 1.C. Determine neutralizing activity of hyper immune sera against individual OvHV-2 proteins expressed by recombinant AlHV-2. Objective 2: Develop an efficacious vaccine for protection of clinically-susceptible species from sheep-associated MCF. Subobjective 2.A. Vaccinate bison with recombinant AlHV-2 containing OvHV-2 genes to stimulate a neutralizing antibody response against OvHV-2. Subobjective 2.B. Determine the MCF protection rates of bison vaccinated with recombinant AlHV-2 upon challenge with a lethal dose of OvHV-2. Subobjective 2.C. Determine if vaccination prevents bison from developing MCF when exposed to OvHV-2 infected sheep. Approach (from AD-416): The proposed research will develop a recombinant herpesvirus expressing OvHV-2 proteins by utilizing AlHV-2, a non-pathogenic MCFV carried by African antelopes (hartebeest and topi) that can grow in cell culture, as a vaccine. AlHV-2 has been isolated from clinically normal topi antelope and hartebeest in Africa and the U.S. (61, 79, 84). There has been no report of AlHV-2-induced MCF in cattle under natural transmission conditions, although an MCF case in red deer reported from the San Diego Wildlife Park was associated with an AlHV-2-like virus from Jackson Hartebeest (36). Experimental inoculation of cattle with cell-free AlHV-2 isolates can result in infection, but does not induce clinical disease. Moreover, inoculation of cattle with AlHV-2 does not elicit antibodies protective against subsequent AlHV-1 challenge (66). We will use recombination-mediated genetic engineering to generate recombinant AlHV-2 (rAlHV-2) containing relevant OvHV-2 genes (rAlHV-2OvHV-2g). The rAlHV- 2OvHV-2g will be tested as a vaccine to stimulate local immune responses in the respiratory tract to protect clinically susceptible hosts from SA- MCF. Our main hypothesis is: immunization with rAlHV-2OvHV-2g will stimulate neutralizing antibodies at the viral entry site in bison, which will correlate with reduced viral load and protection against lethal OvHV- 2 challenge. The hypothesis will be tested through the previous two objectives. After the research plan was redirected to use an attenuated alcelaphine herpesvirus 1 (AlHV-1) as the vaccine vector due to AlHV-2 causing disease in bison, we have completed several projects during FY 2015 related to Objective 1 (Develop a recombinant herpesvirus expressing ovine herpes virus-2 [OvHV-2] proteins that stimulate a neutralizing antibody response) and Objective 2 (Develop an efficacious vaccine for protection of clinically-susceptible species from sheep-associated MCF). Objective 1: We are evaluating an attenuated AlHV-1 as a vaccine backbone to deliver and express OvHV-2 gB and gH/gL, viral glycoproteins capable of stimulating protective antibody response. The attenuated AlHV-1 with the gene ORF73 removed (AlHV-1 ORF73-null virus) was cloned as a bacterial artificial chromosome (BAC) at the University of Liege, Belgium, and obtained from our collaborators. Using homologous recombination and the galK selection system, we have constructed the following chimeric viruses: 1) AlHV-1 ORF73-null/OVHV-2 ORF8, 2) AlHV-1 ORF73-null/OVHV-2 ORF22, 3) AlHV-1 ORF73-null/OVHV-2 ORF47, 4) AlHV-1 ORF73-null/OVHV-2 ORF22-ORF47, 5) AlHV-1 ORF73-null/OVHV-2 ORF8-ORF22-ORF47. In each chimera, one or more AlHV-1 genes encoding gB (ORF8), gH (ORF22) and gL (ORF47) were replaced by its OvHV-2 counterpart gene(s). The AlHV-1 ORF73-null/OvHV-2 ORF8 virus was able to infect Madin-Darby bovine kidney (MDBK) and fetal mouflon sheep kidney (FMSK) cells, where it replicated at similar levels to the parental virus (AlHV-1 ORF73-null). This virus has been also inoculated by intranasal nebulization in rabbits to test its infectivity in vivo and the experiment is currently in progress. The AlHV-1 ORF73- null/OVHV-2 ORF22 virus was unable to infect cells in vitro. Since the product of ORF22, gH, forms an heterodimer with gL, it is possible that proteins from the same organism have to be co-expressed. The AlHV-1 ORF73- null/OVHV-2 ORF47, AlHV-1 ORF73-null/OVHV-2 ORF22-ORF47, and AlHV-1 ORF73- null/OVHV-2 ORF8-ORF22-ORF47 viruses are currently under evaluation for infectivity and growth kinetics in vitro. FMSK cells, a primary cell line with a limited life-span in culture, were immortalized by stable transfection with a plasmid encoding human telomerase reverse transcriptase (hTERT). Immortalized cells can be grown indefinitely in culture, which provides a constant source of cells for AlHV-1 propagation. Fetal sheep lung cells were isolated and primary cells were stably transfected with hTERT. The cells will be characterized. Epithelial- type cells will be tested for the ability to support lytic OvHV-2 infection and the production of infectious virus. Objective 2: A vaccine challenge study was completed to evaluate antibody responses and protection from malignant catarrhal fever (MCF) in rabbits immunized with plasmid DNA expressing OvHV-2 gB, gH and gL genes delivered directly into lung cells by intra-tracheal inoculation. The experiment was unsuccessful likely due to low transfection efficiency and insufficient expression of the proteins of interest, and will have to be repeated using a different transfection reagent and higher amounts of DNA. These experiments are critical to prove that mucosal immunity, especially high levels of neutralizing antibodies in lung, is the key to protecting animals from development of MCF as initially proposed. Based on phylogenetic analysis, the MCF viruses cluster into two subgroups corresponding to the reservoir hosts� subfamilies: Alcelaphinae/ Hippotraginae and Caprinae, and AlHV-1 and OvHV-2 are the prototypes in these two subgroups, respectively. We evaluated the relatedness of neutralizing antigens shared among MCF viruses. Neutralizing antibody activity against AlHV-1 was detected in samples from infected hosts in the Alcelaphinae and Hippotraginae subfamilies, but not from hosts in the Caprinae subfamily. OvHV-2 neutralizing activity was demonstrated in samples from goats (Caprinae) but not from wildebeest (Alcelaphinae). These results indicate that neutralizing antibody cross-reactivity exists among MCF viruses within a virus subgroup but not between subgroups. This information is important for diagnosis of MCF and vaccine development. OvHV-2 fusion glycoproteins, including gB, gH and gL, were expressed in mammalian cells and a cell-cell fusion assay was developed. This will allow OvHV-2 fusion to be studied in the absence of cell culture to propagate OvHV-2. The assay is currently being optimized and preliminary results indicate that the OvHV-2 putative receptor binding protein pOv8 increases fusion. Besides virus entry studies, the cell-cell fusion assay can potentially be used for analysis of antibody binding functions, which is also important for diagnostic and vaccine evaluation. Accomplishments 01 Malignant catarrhal fever (MCF) viruses do not share neutralizing antigens between the subgroups. Stimulating neutralizing antibodies to block MCF virus entry at the initial replication site is the proof of concept for the successful development of vaccines; therefore, it is critical to understand whether MCF viruses share neutralizing antigens. ARS researchers at Pullman, Washington, in collaboration with researchers at Washington State University and the University of Wyoming evaluated neutralizing antibody cross-reactivity between these two MCF virus subgroups using AlHV-1 in culture and OvHV-2 in animals. Neutralizing antibody activity against AlHV-1 was detected in samples from infected African antelopes (in the Alcelaphinae and Hippotraginae subfamilies), but not from sheep, goats and their relatives (in the Caprinae subfamily). OvHV-2 neutralizing activity was demonstrated in samples from goats (Caprinae subfamily), but not from wildebeest (Alcelaphinae subfamily). These results indicate that neutralizing antibody cross-reactivity exists among MCF viruses within a virus subgroup but not between subgroups. This information is of great importance for diagnosis of MCF and vaccine development.
Impacts
(N/A)
Publications
- Wudiri, G., Pritchard, S., Li, H., Liu, J., Aguilar, H., Gilk, S., Nicola, A. 2014. Molecular requirement for sterols in herpes simplex virus entry and infectivity. Journal of Virology. 88(23):13918-13922.
- Cunha, C.W., Knowles Jr, D.P., Taus, N.S., O'Toole, D., Nicola, A.V., Aguilar, H.C., Li, H. 2014. Antibodies to ovine herpesvirus 2 glycoprotein antibodies decrease virus infectivity and prevent malignant catarrhal fever in rabbits. Veterinary Microbiology. 175(2-4):349-355.
- Vik�ren, T., Klevar, S., Li, H., Germundsson, A. 2015. A geographical cluster of malignant catarrhal fever in Moose (Alces alces)in Norway. Journal of Wildlife Diseases. doi: 10.7589/2014-04-097.
- Boutier, M., Ronsmans, M., Ouyang, P., Fournier, G., Reschner, A., Rakus, K., Wilkie, G., Farnir, F., Bayrou, C., Lieffrig, F., Li, H., Desmecht, D., Davison, A., Vanderplasschen, A. 2015. Rational development of an attenuated recombinant cyprinid herpesvirus 3 vaccine using prokaryotic mutagenesis and in vivo bioluminescent imaging. PLoS Pathogens. doi: 10. 1371/journal.ppat.1004690.
- Frontoso, R., Autorino, G., Friedrich, K., Li, H., Eleni, C., Cocumelli, C. , Di Cerbo, P., Manna, G., Scicluna, M. 2015. An acute multispecies episode of sheep-associated malignant catarrhal fever in captive wild animals in an Italian zoo. Transboundary and Emerging Diseases. doi: 10. 1111/tbed.12321.
- Nelson, D.D., Taus, N.S., Schneider, D.A., Cunha, C.W., Davis, W.C., Brown, W.C., Li, H., O'Toole, D., Oaks, L.J. 2013. Fibroblasts express OvHV-2 capsid protein in vasculitis lesions of American bison (Bison bison) with experimental sheep-associated malignant catarrhal fever. Veterinary Microbiology. doi: 10.1016/j.vetmic.2013.07.021.
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Progress 10/01/13 to 09/30/14
Outputs
Progress Report Objectives (from AD-416): Objective 1: Develop a recombinant herpesvirus expressing OvHV-2 proteins that stimulate a neutralizing antibody response. Subobjective 1.A. Develop an infectious AlHV-2 BAC clone. Subobjective 1.B. Construct infectious recombinant AlHV-2 BACs containing OvHV-2 genes encoding proteins that can stimulate a neutralizing antibody response. Subobjective 1.C. Determine neutralizing activity of hyper immune sera against individual OvHV-2 proteins expressed by recombinant AlHV-2. Objective 2: Develop an efficacious vaccine for protection of clinically-susceptible species from sheep-associated MCF. Subobjective 2.A. Vaccinate bison with recombinant AlHV-2 containing OvHV-2 genes to stimulate a neutralizing antibody response against OvHV-2. Subobjective 2.B. Determine the MCF protection rates of bison vaccinated with recombinant AlHV-2 upon challenge with a lethal dose of OvHV-2. Subobjective 2.C. Determine if vaccination prevents bison from developing MCF when exposed to OvHV-2 infected sheep. Approach (from AD-416): The proposed research will develop a recombinant herpesvirus expressing OvHV-2 proteins by utilizing AlHV-2, a non-pathogenic MCFV carried by African antelopes (hartebeest and topi) that can grow in cell culture, as a vaccine. AlHV-2 has been isolated from clinically normal topi antelope and hartebeest in Africa and the U.S. (61, 79, 84). There has been no report of AlHV-2-induced MCF in cattle under natural transmission conditions, although an MCF case in red deer reported from the San Diego Wildlife Park was associated with an AlHV-2-like virus from Jackson Hartebeest (36). Experimental inoculation of cattle with cell-free AlHV-2 isolates can result in infection, but does not induce clinical disease. Moreover, inoculation of cattle with AlHV-2 does not elicit antibodies protective against subsequent AlHV-1 challenge (66). We will use recombination-mediated genetic engineering to generate recombinant AlHV-2 (rAlHV-2) containing relevant OvHV-2 genes (rAlHV-2OvHV-2g). The rAlHV- 2OvHV-2g will be tested as a vaccine to stimulate local immune responses in the respiratory tract to protect clinically susceptible hosts from SA- MCF. Our main hypothesis is: immunization with rAlHV-2OvHV-2g will stimulate neutralizing antibodies at the viral entry site in bison, which will correlate with reduced viral load and protection against lethal OvHV- 2 challenge. The hypothesis will be tested through the previous two objectives. Since 2013, the research plan has been redirected to using the acephaline herpes virus 1 with gene ORF73 removed (AlHV-1 ORF73 null virus), a gene- deletion mutant, as the vaccine vector due to the ability of AlHV-2 to cause disease in bison. Several projects were completed during FY 2014 related to the research plan- Objective 1 (Develop a recombinant herpesvirus expressing ovine herpes virus-2 [OvHV-2] proteins that stimulates a neutralizing antibody response) and Objective 2 (Develop an efficacious vaccine for protection of clinically-susceptible species from sheep-associated MCF). Using the recently developed rabbit infection model as an alternative for a cell culture neutralization test, we evaluated OvHV-2 glycoprotein-specific antibodies for the ability to block OvHV-2 entry. The results showed that serum antibodies against three glycoproteins, gB, gH, and gL, completely blocked viral infection and prevented disease development in rabbits. These data indicate that OvHV-2 gB, gH, and gL are promising targets for a vaccine aimed at stimulating protective immune responses against OvHV-2. In order to further understand the mechanism of OvHV-2 entry and determine the critical viral components involved in viral entry, we have established monoclonal antibody libraries against OvHV-2 gB, gH and gL and characterization of these monoclonal antibodies is in progress. Initial manipulation of the AlHV-1 ORF73-null bacterial artificial chromosome (BAC) DNA has been done. The insertion of OvHV-2 genes encoding gB, gH and gL into the AlHV-1 ORF73-null BAC has been initiated and the experiments are in progress to confirm the presence of both AlHV1 and OvHV-2 gB genes in the BAC genome with correct protein expression. A vaccine challenge study has been initiated to evaluate antibody responses and protection from malignant catarrhal fever (MCF) in rabbits immunized with plasmid DNA expressing OvHV-2 gB, gH and gL genes delivered into the skin by a gene gun or directly into lung cells by intra-tracheal inoculation. These experiments are critical to proving that mucosal immunity, especially a high level of neutralizing antibodies in lung, is the key to protecting animals from development of MCF as initially proposed. Since all MCF viruses are closely related and AlHV-1 will be used as the OvHV-2 vaccine backbone, we examined whether neutralizing antigens are shared among these viruses. Our results showed that the anti- AlHV-1 serum failed to block OvHV-2 entry in rabbits (all rabbits developed MCF) indicating that AlHV-1 and OvHV-2 do not share protective antigens. On the other hand, when the rabbits were inoculated with OvHV-2 treated with anti-caprine (goat) herpesvirus 2 (CpHV-2) serum, 83% of the rabbits were protected from MCF. These data suggest that CpHV-2 shares protective antigens with OvHV-2 and the vaccine developed for OvHV-2- induced MCF can potentially be used for protection of animals susceptible to both sheep and goat-associated MCF. In addition, we have made progress in developing a cell culture system by isolating cells from sheep lungs and inoculating these cells with OvHV-2-containing nasal secretions collected from infected sheep. Preliminary results showed that these cells were infected with OvHV-2 and further work is on-going to evaluate the infection outcome. A commercially available calf cell line was also inoculated with OvHV-2 and determined to be infected, although the virus did not appear to undergo a complete replication cycle. Work is on-going to determine if there is a method to stimulate the virus to undergo a complete replication cycle thus making this a usable culture system. Accomplishments 01 Antibodies specific for ovine herpes virus 2 (OvHV-2) proteins block virus infection and prevent malignant catarrhal fever (MCF) in rabbits. Identification of OvHV-2 proteins that can stimulate a protective immune response is a prerequisite to the development of a vaccine for sheep-associated MCF. ARS scientists in Pullman, Washington, in collaboration with researchers from Washington State University, demonstrated that antibodies against OvHV-2 glycoproteins (gB, gH, and gL) are capable of blocking virus entry into rabbit cells. Antibodies specific for OvHV-2 proteins were produced in rabbits following DNA immunization using a gene gun and OvHV-2 was treated with anti-sera prior to inoculation into rabbits. The results showed that antibodies against gB, gH/gL completely blocked viral infection and prevented disease development in rabbits, indicating that OvHV-2 gB and gH/gL are suitable targets for a vaccine aimed at stimulating protective immune responses. 02 Caprine herpesvirus 2 (CpHV-2) shares protective antigens with OvHV-2. CpHV-2, a malignant catarrhal fever (MCF) virus carried by domestic and exotic goats, is one of the causes of MCF in zoos and game farms. There is no vaccine or treatment available for goat-associated MCF at this time. ARS scientists in Pullman, Washington, in collaboration with researchers from Washington State University, analyzed neutralizing (protective) antigens shared among MCF viruses by testing the ability of antisera to acephaline herpes virus 1 (AlHV-1) or CpHV-2 to block OvHV-2 entry in rabbits. The results showed that the anti-AlHV-1 serum failed to block OvHV-2 entry in rabbits resulting in development of MCF in all rabbits, while anti-CpHV-2 serum protected 83% of rabbits from the disease. The data suggest that a vaccine developed for OvHV-2- induced MCF can potentially be used in zoos and game farms for protection against both sheep and goat-associated MCF.
Impacts
(N/A)
Publications
- Li, H., Cunha, C.W., Taus, N.S., Knowles Jr, D.P. 2014. Malignant catarrhal fever: inching towards understanding. Annual Review of Animal and Veterinary Biosciences. 2:209-33.
- O'Toole, D., Li, H. 2014. The pathology of malignant catarrhal fever, with an emphasis on ovine herpesvirus 2. Veterinary Pathology. 51(2):437-52.
- Tri Komala, S., Pritchard, S.M., Cunha, C.W., Wudiri, G.A., Aguilar, H.C., Taus, N.S., Nicola, A.V. 2013. Contributions of herpes simplex virus type 1 envelope proteins to entry by endocytosis. Journal of Virology. 87(24) :13922-13926.
- Taus, N.S., O'Toole, D., Herndon, D.R., Cunha, C.W., Warg, J.V., Seal, B.S. , Brooking, A., Li, H. 2014. Malignant catarrhal fever in American bison (Bison bison) experimentally infected with alcelaphine herpesvirus 2. Veterinary Microbiology. DOI:10.1016/j.vetmic.2014.04.003.
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Progress 10/01/12 to 09/30/13
Outputs
Progress Report Objectives (from AD-416): Objective 1: Develop a recombinant herpesvirus expressing OvHV-2 proteins that stimulate a neutralizing antibody response. Subobjective 1.A. Develop an infectious AlHV-2 BAC clone. Subobjective 1.B. Construct infectious recombinant AlHV-2 BACs containing OvHV-2 genes encoding proteins that can stimulate a neutralizing antibody response. Subobjective 1.C. Determine neutralizing activity of hyper immune sera against individual OvHV-2 proteins expressed by recombinant AlHV-2. Objective 2: Develop an efficacious vaccine for protection of clinically-susceptible species from sheep-associated MCF. Subobjective 2.A. Vaccinate bison with recombinant AlHV-2 containing OvHV-2 genes to stimulate a neutralizing antibody response against OvHV-2. Subobjective 2.B. Determine the MCF protection rates of bison vaccinated with recombinant AlHV-2 upon challenge with a lethal dose of OvHV-2. Subobjective 2.C. Determine if vaccination prevents bison from developing MCF when exposed to OvHV-2 infected sheep. Approach (from AD-416): The proposed research will develop a recombinant herpesvirus expressing OvHV-2 proteins by utilizing AlHV-2, a non-pathogenic MCFV carried by African antelopes (hartebeest and topi) that can grow in cell culture, as a vaccine. AlHV-2 has been isolated from clinically normal topi antelope and hartebeest in Africa and the U.S. (61, 79, 84). There has been no report of AlHV-2-induced MCF in cattle under natural transmission conditions, although an MCF case in red deer reported from the San Diego Wildlife Park was associated with an AlHV-2-like virus from Jackson Hartebeest (36). Experimental inoculation of cattle with cell-free AlHV-2 isolates can result in infection, but does not induce clinical disease. Moreover, inoculation of cattle with AlHV-2 does not elicit antibodies protective against subsequent AlHV-1 challenge (66). We will use recombination-mediated genetic engineering to generate recombinant AlHV-2 (rAlHV-2) containing relevant OvHV-2 genes (rAlHV-2OvHV-2g). The rAlHV- 2OvHV-2g will be tested as a vaccine to stimulate local immune responses in the respiratory tract to protect clinically susceptible hosts from SA- MCF. Our main hypothesis is: immunization with rAlHV-2OvHV-2g will stimulate neutralizing antibodies at the viral entry site in bison, which will correlate with reduced viral load and protection against lethal OvHV- 2 challenge. The hypothesis will be tested through the previous two objectives. Replacing 5348-32000-024-00D (October 2011). Several projects were completed during FY 2013 related to the research plan- Objective 1 (Develop a recombinant herpesvirus expressing OvHV-2 proteins that stimulates a neutralizing antibody response). A study characterizing AlHV-2 infectivity in experimental animals revealed that AlHV-2 is capable of inducing MCF in bison; while it failed to infect rabbits, suggesting AlHV-2 is not an appropriate virus as a vaccine backbone. These data prompted us to redirect our research plan to use the ORF73-null AlHV-1 as the vaccine backbone since AlHV-1 has been recently removed from the Select Agent list and the virulence of ORF73-null AlHV-1 is diminished. Importation of the ORF73-null AlHV-1 from our collaborators at the University of Liege, Belgium, is underway. In order to identify glycoproteins that can stimulate a neutralizing antibody response, we have successfully completed the production of hyper immune sera against OvHV-2 glycoproteins. Analysis of antibody titers against individual OvHV-2 glycoproteins showed that all rabbits developed high antibody titers against the proteins, indicating that DNA immunization using a gene gun is an excellent approach to generate antibodies against individual proteins for research. The ability of the hyper immune sera to block experimental OvHV-2 infection in rabbits is being assessed. Since OvHV-2 and AlHV-1 are closely related viruses and AlHV-1 will be used as the OvHV-2 vaccine backbone, we examined whether antibody against AlHV-1 can block OvHV-2 entry in rabbits. The study showed that antibodies against AlHV-1 failed to block OvHV-2 entry, while antibodies against OvHV-2 completely prevented OvHV-2 infection in rabbits, suggesting that there is no cross-protection between the two viruses despite the fact that they share significant antibody cross-reactivity. In addition, we confirmed that the herpesvirus termed MCFV-WTD, which causes MCF in white- tailed deer with an unknown origin, is capable of causing disease in other species of deer and identified that goats are a potential reservoir for the virus. Accomplishments 01 DNA immunization using a gene gun efficiently induced antibody responses against OvHV-2 glycoproteins in rabbits. Identification of OvHV-2 proteins that can stimulate a neutralizing antibody response is a prerequisite to the development of a vaccine for sheep-associated MCF. The ARS scientists in Pullman, Washington, in collaboration with researchers from Washington State University developed an efficient platform to generate hyper immune sera against individual OvHV-2 glycoproteins in rabbits by gene immunization. Individual OvHV-2 genes of interest were codon-optimized, synthesized, and constructed in plasmids. Rabbits were immunized with the plasmid constructions using a biolistic DNA delivery system referred to as a gene gun. The results showed that all rabbits immunized with individual or combined OvHV-2 genes developed a strong antibody response. Development of this efficient immunization protocol in combination with recently developed �in vivo OvHV-2 blocking system� is of great significance in identification of vaccine targets for sheep-associated MCF. 02 Goats are a source for causing herpesvirus in deer. Malignant Catarrhal Fever Virus � White Tailed Deer, (MCFV-WTD) was first identified to cause malignant catarrhal fever (MCF), a deadly disease in white tailed deer in 2000, but natural source(s) for the causal virus had not been determined. ARS scientists in Pullman, Washington, worked collaboratively with veterinarians and diagnosticians from several state veterinary diagnostic laboratories and confirmed that MCFV-WTD is capable of causing MCF not only in white-tailed deer, but also in other species of deer. Goats were identified as one source of this virus MCFV-WTD. Identification of goats as a source of this virus is critical for MCF disease in mixed species operations, especially in zoological gardens, game farms, and petting zoos, since goats are commonly housed with a variety of other species, including animals that are highly susceptible to MCF, such as deer.
Impacts
(N/A)
Publications
- Des Neves, C.G., Ihlebaek, H.M., Skjerve, E., Hemmingsen, W., Li, H., Tryland, M. 2013. Gammaherpesvirus infection in semidomesticated reindeer (Rangifer tarandus tarandus): a cross-sectional serological study in northern Norway. Journal of Wildlife Diseases. doi: 10.7589/WD.2012-07-185.
- Vikoren, T., Klevar, S., Li, H., Germundsson, A. 2013. Malignant catarrhal fever virus identified in free-ranging musk ox (Ovibos moschatus) in Norway . Journal of Wildlife Diseases. doi:10.7589/WD.2012-10-259.
- Cunha, C.W., O'Toole, D., Taus, N.S., Knowles Jr, D.P., Li, H. 2013. Are rabbits a suitable model to study sheep-associated malignant catarrhal in susceptible hosts? Veterinary Microbiology. 163(3-4):358-363.
- Li, H., Cunha, C.W., Abbitt, B., Demaar, T.W., Lenz, S., Hayes, J.R., Taus, N.S. 2013. Goats are a potential reservoir for the herpesvirus (MCFV-WTD), causing malignant catarrhal fever in deer. Journal of Zoo and Wildlife Medicine. 44(2):484-486.
- Li, H., Cunha, C.W., O'Toole, D., Nicola, A.V., Knowles Jr, D.P., Taus, N. S. 2013. Development of an in vivo system to measure antibody-blocking of ovine herpesvirus 2 entry. Journal of Virological Methods. 188(1-2):104- 107.
- Palmer, M.V., Thacker, T.C., Madison, R.J., Koster, L., Swenson, S., Li, H. 2013. Active and latent ovine Herpesvirus-2 (OvHV-2) infection in a herd of captive white-tailed deer (Odocoileus virginianus). Journal of Comparative Pathology. 149(2-3):162-166.
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Progress 10/01/11 to 09/30/12
Outputs
Progress Report Objectives (from AD-416): Objective 1: Develop a recombinant herpesvirus expressing OvHV-2 proteins that stimulate a neutralizing antibody response. Subobjective 1.A. Develop an infectious AlHV-2 BAC clone. Subobjective 1.B. Construct infectious recombinant AlHV-2 BACs containing OvHV-2 genes encoding proteins that can stimulate a neutralizing antibody response. Subobjective 1.C. Determine neutralizing activity of hyper immune sera against individual OvHV-2 proteins expressed by recombinant AlHV-2. Objective 2: Develop an efficacious vaccine for protection of clinically-susceptible species from sheep-associated MCF. Subobjective 2.A. Vaccinate bison with recombinant AlHV-2 containing OvHV-2 genes to stimulate a neutralizing antibody response against OvHV-2. Subobjective 2.B. Determine the MCF protection rates of bison vaccinated with recombinant AlHV-2 upon challenge with a lethal dose of OvHV-2. Subobjective 2.C. Determine if vaccination prevents bison from developing MCF when exposed to OvHV-2 infected sheep. Approach (from AD-416): The proposed research will develop a recombinant herpesvirus expressing OvHV-2 proteins by utilizing AlHV-2, a non-pathogenic MCFV carried by African antelopes (hartebeest and topi) that can grow in cell culture, as a vaccine. AlHV-2 has been isolated from clinically normal topi antelope and hartebeest in Africa and the U.S. (61, 79, 84). There has been no report of AlHV-2-induced MCF in cattle under natural transmission conditions, although an MCF case in red deer reported from the San Diego Wildlife Park was associated with an AlHV-2-like virus from Jackson Hartebeest (36). Experimental inoculation of cattle with cell-free AlHV-2 isolates can result in infection, but does not induce clinical disease. Moreover, inoculation of cattle with AlHV-2 does not elicit antibodies protective against subsequent AlHV-1 challenge (66). We will use recombination-mediated genetic engineering to generate recombinant AlHV-2 (rAlHV-2) containing relevant OvHV-2 genes (rAlHV-2OvHV-2g). The rAlHV- 2OvHV-2g will be tested as a vaccine to stimulate local immune responses in the respiratory tract to protect clinically susceptible hosts from SA- MCF. Our main hypothesis is: immunization with rAlHV-2OvHV-2g will stimulate neutralizing antibodies at the viral entry site in bison, which will correlate with reduced viral load and protection against lethal OvHV- 2 challenge. The hypothesis will be tested through the previous two objectives. Replacing 5348-32000-024-00D (October 2011). The following details the progress we have made during 2012 related to objective 1 (Develop a recombinant herpesvirus expressing OvHV-2 proteins that stimulates a neutralizing antibody response): 1) We have completed the sequencing and annotation of the AlHV-2 genome. Sequence analysis of AlHV-2 genes revealed that the AlHV-2 gene structure is similar to a close relative virus, AlHV-1, carried by wildebeest, suggesting a similar strategy for generating an infectious AlHV-1 artificial bacterial chromosome (BAC) clone could be applied for AlHV-2. 2) Construction of infectious AlHV-2 BAC clone is near completion. The recombination plasmid containing the BAC cassette flanked by AlHV-2 sequences has been constructed and transfection conditions for FMSK cells have been determined. 3) We have determined whether rabbits can be infected with AlHV-2. Unfortunately, it was not successful to infect rabbits with AlHV-2, which prompted the immediate question of whether or not bison can be infected with AlHV-2. An experiment to infect bison with AlHV-2 is underway. Whether AlHV-2 is capable of infecting bison becomes a significant issue to direct the next phase of the project plan. 4) We have implemented an alternative strategy to immunize rabbits for immune serum against OvHV-2 glycoproteins. The alternative strategy was to immunize rabbits with OvHV-2 glycoprotein genes by using a gene gun in order to identify protective antigens for the vaccine. This approach will speed up the research progress. All five candidate genes (ORF 8, ORF 22, ORF 47, Ov 7, and Ov 8) have been synthesized, subcloned into mammalian expression vectors, and are ready to immunize rabbits. 5) We have developed animal models to measure antibody blocking of OvHV-2 entry. Since there is no cell culture system available to grow OvHV-2, an alternative virus neutralization test is needed to assess neutralizing antibody activity to OvHV-2 in SA-MCF vaccine development. We evaluated both sheep and rabbits to determine whether they could be used as an animal system to measure the ability of antibodies to block OvHV-2 at the entry site. Virus and anti-OvHV-2 serum were mixed before intranasal infection of the animals, and infection parameters and survival rates were evaluated to assess reduction in virus infectivity. The data from the study indicate that both sheep and rabbits can be used as a system to measure antibody's ability to block OvHV-2 entry in animals, which is of significance in OvHV-2 vaccine development, especially focusing on evaluation of protective antibody responses. Accomplishments 01 Developed an animal system to measure antibody blocking of ovine herpesvirus 2 entry. Ovine herpesvirus 2 (OvHV-2), the causative virus sheep-associated malignant catarrhal fever (SA-MCF), has never been grow in cell culture; thus, an alternative to a cell culture-based virus neutralization test is needed to assess neutralizing antibody activity OvHV-2 in SA-MCF vaccine development. ARS scientists at the Animal Disea Research Unit, Pullman, Washington, evaluated both sheep and rabbits to determine whether they could be used as an animal system to measure the ability of antibodies to block OvHV-2 at the entry site by mixing virus and anti-OvHV-2 serum before intranasal inoculation of the animals. Experimental results indicate that both sheep and rabbits can be used as an animal system to determine antibody's ability to block OvHV-2 entry i animals by measuring infection parameters in sheep or survival rates in rabbits. This is the first system developed that can be used to determin antibody's ability to block OvHV-2 at entry, which is of significance in OvHV-2 vaccine development, especially focusing on evaluation of protective antibody responses.
Impacts
(N/A)
Publications
- Cunha, C.W., Gailbreath, K.L., O'Toole, D., Knowles Jr, D.P., Schneider, D. A., White, S.N., Taus, N.S., Davis, C.J., Davis, W.C., Li, H. 2012. Ovine herpesvirus 2 infection in american bison: virus and host dynamics in the development of sheep-associated malignant catarrhal fever. Veterinary Microbiology. 159:307-319.
- Li, H., Brooking, .A., Cunha, C.W., Highland, M.A., O'Toole, .D., Knowles Jr, D.P., Taus, N.S. 2012. Experimental induction of malignant catarrhal fever in pigs with ovine herpesvirus 2 by intranasal nebulization. Veterinary Microbiology. 10.1016/j.vetmic.2012.04.016.
- Li, H., Cunha, C.W., Taus, N.S. 2011. Malignant catarrhal fever: understanding molecular diagnostics in context of epidemiology. International Journal of Molecular Sciences. 10:6881-6893.
- Gasper, D., Barr, B., Li, H., Taus, N.S., Peterson, R., Benjamin, G., Hunt, T., Pesavento, P. 2012. Ibex-associated malignant catarrhal fever-like disease in a group of bongo antelope (Tragelaphus euryceros). Veterinary Pathology. 3:492-7.
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