Location: Animal Disease Research
2020 Annual Report
Objectives
Malignant catarrhal fever (MCF) is an often fatal viral disease that primarily affects ruminants. Several gammaherpesviruses in the Macavirus genus can cause MCF. These viruses are carried asymptomatically by certain animal species but can cause disease when transmitted to clinically susceptible species. Ovine herpesvirus 2 (OvHV-2) is globally distributed and the most frequent cause of MCF worldwide. In North America, OvHV-2-induced MCF is the leading cause of death in American bison, which are highly susceptible to the disease. MCF results in significant economic impact on agriculture not only due to the loss of animals but also because it imposes restrictions on multispecies grazing. The development of an effective vaccine to MCF is a top priority for the agricultural industry. However, because OvHV-2 cannot be cultured in vitro, conventional methods cannot be utilized to attenuate or modify viruses to be used as a vaccine. In this project, we propose a novel strategy to develop a vaccine against MCF using recombinant non-pathogenic gammaherpesviruses expressing key OvHV-2 glycoproteins to induce immune responses capable of protecting animals from disease. Importantly, we also plan to develop a modified virus neutralization assay to assess vaccine efficacy.
Objective 1: Develop and evaluate neutralizing antibody assays for OvHV-2 using in vitro and rabbit models with the goal of assessing vaccine efficacy and protection from MCF.
Subobjective 1A. Develop a recombinant AlHV-1 expressing OvHV-2 glycoproteins for an in vitro neutralization assay.
Subobjective 1B. Develop and validate an in vitro neutralization assay for detection of OvHV-2 antibodies.
Subobjective 1C. Evaluate interference of OvHV-2 neutralizing antibodies in the respiratory tract with virus infection and development of SA-MCF in rabbits.
Objective 2: Develop novel and efficacious MCF vaccines for clinically-susceptible species using novel vaccine vector systems and platforms.
Subobjective 2A. Develop a recombinant BoHV-4 expressing OvHV-2 gB and gH/gL.
Subobjective 2B. Evaluate delivery platforms for immunization with the rBoHV-4/OvHV-2-gB-gH/gL in a rabbit model.
Subobjective 2C. Determine whether immunization with a recombinant BoHV-4 expressing OvHV-2 gB and gH/gL prevents MCF in bison.
Approach
This proposed research addresses a fundamental gap regarding control of OvHV-2: the development of an SA-MCF vaccine. The inability to propagate OvHV-2 in vitro has been a roadblock not only to develop a vaccine for SA-MCF, but also to perform in vitro neutralization assays for antibodies that block, for example, viral entry. Our approach is to develop recombinant non-pathogenic gammaherpesviruses expressing key OvHV-2 glycoproteins to be used as a vaccine and as an in vitro tool for analysis of neutralizing antibody responses. Using recombination-mediated genetic engineering techniques, we will generate recombinant AlHV-1 (rAlHV-1) mutants that express OvHV-2 glycoproteins required for virus entry, as chimeric viruses for in vitro neutralization assays. The assays will be critical to evaluate OvHV-2 neutralizing antibody responses in vaccinated animals and also potentially useful for detecting infection with OvHV-2 and closely related MCF viruses. Moreover, we will develop a recombinant bovine herpesvirus 4 (BoHV-4) expressing OvHV-2 antigens that can stimulate neutralizing antibody responses, as a vaccine to protect clinically-susceptible species from SA-MCF. Overall the proposed research will test two hypotheses: 1) a chimeric virus expressing OvHV-2 glycoprotein(s) propagates in vitro and can be blocked by OvHV-2 neutralizing antibodies; 2) immunization with recombinant BoHV-4 expressing OvHV-2 glycoproteins will stimulate neutralizing antibodies in the respiratory tract, which will correlate with reduced initial viral loads in lung and protection against lethal OvHV-2 challenge. These hypotheses will be tested through the following objectives: 1) develop and evaluate neutralizing antibody assays for OvHV-2 using in vitro and rabbit models with the goal of assessing vaccine efficacy and protection from MCF; and 2) develop novel and efficacious MCF vaccines for clinically-susceptible species using novel vaccine vector systems and platforms.
Progress Report
In support of Sub-objective 2A, research has focused on identifying efficacious methods to deliver ovine herpes-2 (OvHV-2) glycoprotein B (gB) as a vaccine candidate. In vitro and in vivo characterization of a mutant bovine herpesvirus-4 (BoHV-4) constructed in our laboratory (BoHV-4/OvHV-2-gB) was completed. After confirming the virus was infectious, a rabbit model was used to test its safety for animals and its ability to induce protective immune responses. Three out of seven rabbits vaccinated with BoHV-4/OvHV-2-gB remained healthy following OvHV-2 challenge. Samples collected throughout the experiment are being tested to evaluate virus dissemination and host immune responses. Although the protection rate observed in the BoHV-4/OvHV-2-gB vaccinated animals (43 %) was not statistically significant, it demonstrates the potential of gB as a vaccine candidate. With that in mind, additional approaches to deliver OvHV-2-gB are currently being tested in rabbits, including a different vector (a mutant alcelaphine herpesvirus-1) and a combination of DNA and virus immunizations. Significant protection rates (71.4%) have been observed with the new vaccine approaches. As well, in support of Sub-objective 2B, samples obtained from the vaccine experiments are being processed to examine the role of neutralizing antibodies in protection as well as the duration of protective immunity. Data obtained from these vaccine trials in rabbits will define further protocols (Sub-objective 2A) to be tested in bison, as proposed in Sub-objective 2C.
Accomplishments
1. Vaccine delivery systems developed for Malignant Catarrhal Fever (MCF). Malignant Catarrhal Fever (MCF), caused by ovine herpesvirus-2 (OvHV-2), is a fatal disease of ruminants that can cause devastating outbreaks involving large numbers of animals. There is no treatment for MCF and controlling the disease, by avoiding contact with the virus, is sometimes impractical or impossible; therefore, a vaccine is urgently needed. ARS researchers at Pullman, Washington, have modified non-pathogenic viruses to make them produce an OvHV-2 protein and have tested these viruses as vaccine candidates. Vaccine trials performed in rabbits, a laboratory animal model for MCF, showed that a significant number of animals vaccinated with some of these mutant viruses did not develop MCF upon challenge with lethal doses of OvHV-2. These promising results make available mutant viruses that are safe in animals and can be used as delivery systems for an MCF vaccine. Development of an MCF vaccine is an ARS long-term goal, since it is a much-desired biological commodity for several livestock producers, including the bison and deer industries.
2. MCF diagnostic method achieved using fixed tissues. In general, the presence of typical lesions in tissues, associated with molecular detection of DNA of the causal virus in affected animals, is enough to confirm the diagnosis of malignant catarrhal fever (MCF). However, a common problem faced by many veterinarians and pathologists is the unavailability of fresh and/or cryopreserved tissues needed for molecular assays, since collection of fresh tissues during necropsy is not always possible or convenient. The usual practice, especially for necropsies performed at field conditions, is to collect tissues in a fixative reagent, resulting in inadequate material for many molecular assays. ARS researchers at Pullman, Washington, in collaboration with researchers at University of California at Davis, and State University of Londrina, Londrina, Paraná, Brazil, developed diagnostic assays capable of detecting DNA or protein from MCF viruses in fixed tissues. Besides the advantage of using readily available materials, these assays are especially important for the diagnosis of MCF in sheep, where correlating presence of the virus with lesion in tissues is the only way to confirm disease causation. In addition to being a resource for pathologists and diagnostic labs, the developed assays also represent valuable tools for research; for instance, by using these novel detection techniques, scientists can study disease mechanisms and more quickly advance the development of effective control measures, such as vaccines, to benefit MCF affected livestock industries.
Review Publications
Headley, S.A., Oliveria, T.E.S., Li, H., Lisbôa, J.A.N., Queiroz, G.R., Fritzen, J.T.T., Flores, E.F., Alfieri, A.A., Cunha, C.W. 2019. Immunohistochemical detection of intralesional antigens of Ovine gammaherpesvirus-2 in cattle with sheep-associated malignant catarrhal fever. Journal of Comparative Pathology. 174:86-98. https://doi.org/10.1016/j.jcpa.2019.11.002.
Cunha, C.W., Slater, O.M., Macbeth, B., Duignan, P.J., Warren, A., Highland, M.A., Li, H. 2019. Domestic sheep and bighorn sheep carry distinct gammaherpesviruses belonging to the genus Macavirus. Virus Research. 272. https://doi.org/10.1016/j.virusres.2019.197729.
Cunha, C.W., O'Toole, D., Taus, N.S., Shringi, S., Knowles, D.P., Li, H. 2019. A rabbit model for sheep-associated malignant catarrhal fever research: from virus infection to pathogenesis studies and vaccine development. Current Clinical Microbiology Reports. 6:148-155. https://doi.org/10.1007/s40588-019-00126-3.
