Location: Animal Disease Research2019 Annual Report
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.
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.
Objective 2 focuses on the development of a novel and efficacious vaccine for malignant catarrhal fever (MCF) using a non-pathogenic virus (bovine herpesvirus 4, BoHV-4) as a delivery platform. Progress in this objective relies in the construction of a mutant BoHV-4 capable of producing the vaccine candidates, the ovine herpesvirus 2 (OvHV-2) proteins known as gB, gH and gH. Under Sub-objective 2A, significant progress was made in developing several versions of a BoHV-4 containing the OvHV-2 gB gene in different regions of its genome. In vitro tests have identified mutant viruses that can infect and grow in mammalian cells and produce the OvHV-2 gB protein. Large scale preparations of selected mutant viruses are currently being processed for animal experiments (Sub-objective 2B). A pilot rabbit experiment testing different routes for inoculation of BoHV-4 (wild type and recombinant viruses) have indicated that the animals can be successfully infected when the virus is administered intravenously, while intranasal inoculations do not result in infection. This is important information regarding the development of vaccines protocols based on BoHV-4 (Sub-objective 2B). Another significant progress was obtained on the development of monoclonal antibodies specific to the OvHV-2 proteins of interest, OvHV-2 gB, gH and gL. These reagents are critical for evaluating expression of these proteins by mutant viruses (Sub-objective 2A); as controls in diagnostic assays, such as the one developed in Sub-objective 1B; and for several other MCF projects, where detection of these OvHV-2 proteins is necessary. Progress has been made in establishing a new MCF diagnostic test. The test is an OvHV-2 in situ hybridization, which has been recently developed for detection of OvHV-2 DNA in tissues. The OvHV-2 in situ hybridization is especially important for the diagnosis of MCF in sheep, where correlating presence of the virus in lesioned tissues is the only way to confirm disease causation. In addition to the OvHV-2 in situ hybridization we are also developing a similar assay for detection of OvHV-2 protein in tissues. Combination of viral DNA (in situ hybridization) and protein (immunohistochemistry) detection in tissues of MCF affected animals will provide important tools to studies aiming at better understanding how the virus cause disease. Significant progress has been also obtained in the development of specific molecular assays for detection of the MCF viruses carried by domestic sheep, OvHV-2, and bighorn sheep, OvHV-3. Contrary to the assumption that both domestic and wild sheep carry the same MCF virus, we have recently identified a new MCF virus infecting bighorn sheep in both the U.S. and in Canada, which has been tentatively identified as OvHV-3. Both OvHV-2 and OvHV-3 can be detected by the polymerase chain reaction (PCR) assay considered as the gold-standard test for MCF viruses in sheep. Therefore, specific assays for each of the viruses were necessary. DNA sequences unique for each virus have been identified and quantitative PCR assays developed. The assays are currently being validated using field samples. The new assays will be of special value to differentiate the viruses in clinical animals that may be susceptible to both viruses.
1. Development of a novel diagnostic assay for detection of OvHV-2-specific antibodies. Currently there is no serological tests for specific detection of antibodies to ovine herpesvirus 2 (OvHV-2), the virus causing sheep-associated malignant catarrhal fever (MCF). All available assays for MCF serological diagnosis are based on alcelaphine herpes virus 1 and broadly detect antibodies to all MCF viruses. Using a newly defined OvHV-2 unique glycoprotein (Ov8) as antigen, ARS researchers in Pullman, Washington, in collaboration with researchers at Washington State University, developed a specific assay for detecting OvHV-2 antibodies. The Ov8-based enzyme linked immunosorbent assay (ELISA) performed highly (100 percent sensitivity and 98.97 percent specificity) in the detection of OvHV-2-specific antibodies. An important feature of the Ov8 ELISA is that it uses recombinant protein, which makes it easy to be adopted for most diagnostic laboratories. The assay is especially valuable for multiple species-operations where detection of OvHV-2 infected animals is necessary.
Seeley, K., Junge, R., Jennings, R., Cunha, C.W., Li, H. 2018. Moose (alces alces) mortality associated with caprine herpesvirus 2 (CPHV-2) in a zoological collection. Journal of Zoo and Wildlife Medicine. 49(3):774-778. https://doi.org/10.1638/2016-0207.1.
Pesavento, P., Cunha, C.W., Li, H., Jackson, K., O'Toole, D. 2018. In situ hybridization for localization of ovine herpesvirus 2, the agent of sheep-associated malignant catarrhal fever, in formalin-fixed tissues. Veterinary Pathology. 56(1):78-86. https://doi.org/10.1177/0300985818798085.
Pesavento, P., Dange, R., Estrada, M., Dasjerdi, A., Pérez, V., Laroca, A., Silván, J., Diab, S., Jackson, K., Phillips, I., Li, H., Cunha, C.W., Wessels, M. 2018. Systemic necrotizing vasculitis in sheep is associated with ovine herpesvirus 2. Veterinary Pathology. 56:(1)87-92. https://doi.org/10.1177/0300985818795166.
O'Toole, D., Li, H. 2018. Malignant catarrhal fever. In: Coetzer, J.A.W., Penrith, M.L., Maclachlan, N.J., Thomson, G.R., editors. Infectious Diseases of Livestock. Anipedia. Available: https://anipedia.org/resources/malignant-catarrahl-fever/1199.