Location: Animal Disease Research2012 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).
3. Progress Report:
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.
1. Developed an animal system to measure antibody blocking of ovine herpesvirus 2 entry. Ovine herpesvirus 2 (OvHV-2), the causative virus of sheep-associated malignant catarrhal fever (SA-MCF), has never been grown in cell culture; thus, an alternative to a cell culture-based virus neutralization test is needed to assess neutralizing antibody activity to OvHV-2 in SA-MCF vaccine development. ARS scientists at the Animal Disease 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 in animals by measuring infection parameters in sheep or survival rates in rabbits. This is the first system developed that can be used to determine 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.
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.