2013 Annual Report
1a.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.
1b.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.
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.
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.
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.