Investigation of Leporid herpesvirus 4, an Emerging Pathogen of Rabbits: Infection and Prevalence Studies

Leporid herpesvirus 4 (LeHV-4) is a recently identified alphaherpesvirus that causes lethal respiratory disease in rabbits. Diagnosis has been dependent on the observation of distinctive intranuclear inclusion bodies in affected tissues. The objectives of this body of work were to describe the course of infection in laboratory rabbits, develop a serological test for the detection of antibodies to LeHV-4, and survey Ontario commercial meat rabbits and pet rabbits for LeHV-4 antibody prevalence. Based on the results of an initial dose-range finding pilot study, 22 New Zealand white rabbits were inoculated intranasally with LeHV-4 and monitored for 22 days post-infection (dpi). Clinical signs of infection, including dyspnea, serous oculonasal discharge, pyrexia and weight loss, were evident from 2 to 7 dpi. LeHV-4 was isolated from nasal secretions between 2 and 10 dpi. Gross and microscopic pathology was evaluated and suppurative necrohemorrhagic pneumonia and splenic necrosis were the major findings at peak infection (5 to 7 dpi), at which time eosinophilic herpetic inclusions were present in nasal mucosa, skin, spleen, and lung. Virus neutralization (VN) assay demonstrated serum antibodies starting at 11 dpi and persisting until the study end (22 dpi). Polyclonal antibodies generated to inactivated virus in laboratory rabbits were neutralizing with low titres of 1:64 or less. Serum samples were obtained from 225 commercial meat rabbits and 24 pet rabbits and all were negative for LeHV-4 antibodies by VN assay. To increase the safety of the assay while expediting rabbit screening for antibody to LeHV-4, an indirect enzyme-linked immunosorbant assay (ELISA) was developed using glycoprotein G (gG) as target antigen. Recombinant LeHV-4 gG was generated using a baculovirus expression system. The presence of gG in infected Spodoptera frugiperda (Sf-21) cells was confirmed by Western blot analysis. With this infected cell lysate as target antigen, the indirect ELISA had 100% sensitivity and 99% specificity, and can be used to screen rabbits for exposure to LeHV-4. In addition to characterizing the clinicopathological course of the disease in rabbits, this body of work has demonstrated that LeHV-4 is an uncommon disease, and a latent reservoir is unlikely in Ontario domestic rabbits.