Location: Animal Disease Research
2018 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
This report highlights significant progress obtained in Sub-objectives 1B, 1C and 2A of the project as well as in several research areas related to the main objectives.
Under Sub-objective 1B, a recombinant alcelapine herpesvirus 1 (AlHV-1), with its glycoprotein B (gB) gene replaced by the ovine herpesvirus 2 (OvHV-2) glycoprotein B (gB) gene (rAlHV-1/OvHV-2gB), was evaluated in an in vitro neutralizing assay. While the recombinant virus was infectious, its ability to infect cells in culture was blocked by anti-OvHV-2 antibodies. These preliminary results indicate that the rAlHV-1/OvHV-2gB-based assay can be used to assess specific neutralizing antibodies against OvHV-2 gB. The assay will be critical in measuring OvHV-2 gB neutralizing antibody responses induced by one of the vaccine vectors to be tested in this project, recombinant bovine herpesvirus 4 (rBoHV-4)/OvHV-2 gB (Objective 2).
Sub-objective 1C is a proof of concept that neutralizing antibodies against OvHV-2 gB and gH/gL present in the respiratory tract are sufficient to block the virus and prevent development of malignant catarrhal fever (MCF) in rabbits following OvHV-2 challenge. OvHV-2 gB-, gH- and gL-specific neutralizing antibodies capable of blocking OvHV-2 infectivity in vitro failed to block the virus when passively transferred to rabbits by intrapulmonary delivery five minutes prior to virus challenge via aerosol. The failure was likely due to immediate clearance of antibodies from the respiratory tract mucosa after delivery. This experiment highlighted the importance of mucosal neutralizing antibodies on MCF vaccine development. As an alternative, rabbits will be infected with a previously constructed virus, rAlHV-1/OvHV-2 gB, to induce anti-OvHV-2 gB neutralizing antibodies in the lung mucosa. This experiment will be performed in the next few months.
Under Sub-objective 2A, the construction of a recombinant BoHV-4 expressing OvHV-2 glycoprotein genes was started. Preliminary results indicated that OvHV-2 gB can replace BoHV-4 gB, although the overall virus viability is yet to be confirmed. Additionally, a pilot experiment was performed to evaluate the virus that will be used as a backbone for the MCF vaccine (BoHV-4 TK gene deletion mutant). Because this virus has a defective gene (TK) and is expected to be attenuated in vivo, it is important to evaluate how it infects, replicates and establishes latency in rabbits before it can be included in a MCF vaccine. Results showed that BoHV-4 TK gene deletion mutant can infect rabbits and induce anti-BoHV-4 antibody responses, indicating that both the virus vector and the rabbit model can be used to evaluate host immune responses in BoHV-4-vectored MCF vaccine trials.
Progress was also made in several research areas related to the main objectives, including development of reagents and methods for detection of viruses and antibodies. A panel of monoclonal antibodies specific to OvHV-2 gB were generated and characterized. These monoclonal antibodies are important molecular tools that can be readily used for the development of an immunohistochemistry assay to study how the virus causes disease. An OvHV-2 unique glycoprotein (called Ov8) that can enhance virus entry into cells was further evaluated for its potential to develop serological assay for detection of OvHV-2 specific antibodies. In collaboration with researchers at the University of Wyoming and University of California, Davis, an OvHV-2 specific and sensitive DNA based probing method called in situ hybridization was developed and validated with tissue samples from cattle, bison, pigs and rabbits with naturally occurring or experimentally induced MCF. Results from this work showed that the developed assay can detect OvHV-2 infected cells in tissues of all animals with OvHV-2-induced MCF and indicated that the distribution of virus infected cells correlates well with lesions in the tissues. Because the OvHV-2 in situ hybridization assay can be used to chronicle the progression of OvHV-2 infection in experimental time-course studies and to address specific aspects of the pathogenesis and diagnosis of MCF, having the assay available is of great value to the current project as well as to MCF research in general.
Accomplishments
1. Solved the mystery of a pathological syndrome in sheep. Sheep systemic necrotizing vasculitis is a sporadic vascular disease of unidentified cause that has puzzled veterinary pathologists and diagnosticians for decades. ARS researchers in Pullman, Washington, in collaboration with researchers at the Oregon State University and University of California, Davis, showed that ovine herpesvirus 2 (OvHV-2) is associated with systemic necrotizing vasculitis in sheep. The study evaluated tissue samples from several sheep from the U.S., United Kingdom and Spain, which had been previously diagnosed with systemic necrotizing vasculitis due to unknown causes. Using molecular assays, high levels of OvHV-2 DNA co-localized with vascular lesions were found in all affected animals, indicating that OvHV-2 plays a role in the development of systemic necrotizing vasculitis in sheep. This finding not only broadens the potential range of malignant catarrhal fever-like disease to include the adapted host species but also identifies at least one causal agent behind vascular diseases in sheep.
2. Discovered that domestic and bighorn sheep carry distinct malignant catarrhal fever (MCF) viruses. It has historically been thought that both domestic and bighorn sheep carry the same MCF virus designated as ovine herpesvirus 2 (OvHV-2). In a recent investigation of “sheep-associated MCF”-like skin lesions in a bighorn at the Banff National Park, ARS researchers in Pullman, Washington, in collaboration with researchers at the University of Calgary, Canada, and a wildlife biologist at the Banff National Park, Canada, discovered that the DNA sequence from the virus infecting the affected bighorn sheep was different from the sequences previously obtained from domestic sheep. To confirm the findings, additional DNA samples from bighorn sheep from different flocks in the U.S. and Canada were obtained and a region of the viral genome that has been extensively used to study the relationship in herpesviruses was sequenced. The study showed that all viral sequences from bighorn samples were identical but shared only 86 percent identity to OvHV-2 sequences derived from domestic sheep around the world, confirming that the MCF viruses carried by domestic and bighorn sheep are distinct. The fact that the viral sequences were very conserved within each species, suggests that the MCF viruses infecting domestic and bighorn sheep are not commonly transmitted from one species to another but rather evolved with each of the hosts over long periods of time. This is critical information for management of domestic and wild sheep regarding the risk of MCF and virus transmission.
Review Publications
Alhajri, S.M., Cunha, C.W., Knowles, D.P., Li, H., Taus, N.S. 2018. Evaluation of glycoprotein Ov8 as a potential antigen for an OvHV-2-specific diagnostic assay. PLoS One. 13(7):30200130. https://doi.org/10.1371/journal.pone.0200130.
Phillips, I.L., Cunha, C.W., Galbraith, D., Highland, M.A., Bildfell, R.J., Li, H. 2018. High copy number of ovine gammaherpesvirus 2 DNA associated with malignant catarrhal fever-like syndrome in a lamb. Journal of Veterinary Diagnostic Investigation. https://doi.org/10.1177/1040638718766976.
Ortiz, K., Javaux, J., Simon, M., Petit, T., Clavel, S., Lamglait, B., Blanc, B., Brunet, A., Myster, F., Li, H., Dewals, B. 2018. Seroprevalence of malignant catarrhal fever virus in captive wildebeest (Connochaetes sp.) in France. Transboundary and Emerging Diseases. https://doi.org/10.1111/tbed.12929.
