Equine rhinitis A virus: molecular pathogenesis and methods for control

Abstract

Equine rhinitis A virus (ERAV) is a respiratory pathogen of horses. ERAV is classified in the family Picornaviridae, genus Aphthovirus. This classification is shared with foot-and-mouth disease virus, a systemic pathogen of cloven hoofed animals which is a major constraint for international trade.

To investigate ERAV pathogenesis, the shedding dynamics and correlates with clinical signs of two Australian ERAV isolates were determined in experimentally infected horses. Two ERAV seronegative 8 month old horses were each intranasally infected with one of two ERAV isolates. Following infection, the challenge ERAV isolates were re-isolated from the nasopharynx and disseminated to distal sites, after a transient viraemia. Viraemia and shedding of infectious virus from the nasopharynx ceased with the appearance of virus neutralising serum antibodies. However, despite a robust neutralising antibody response, ERAV established a persistent infection in both animals, with high levels of virus excreted in the urine at the completion of the trial, on day 37 post infection. Although virus shedding was detected in the nasopharynx and plasma, clinical signs of acute febrile respiratory disease were not observed.

To investigate the chronic urinary shedding of ERAV further, 215 urine samples collected from Thoroughbred race horses were tested by RT-qPCR for ERAV. ERAV was detected in 23% of the urine samples tested, and was most prevalent in samples collected from 2 to 4 year old horses. Phylogenetic analysis of the VP1 sequence from five ERAV isolates, indicates that variants of a single virus, closely related to an Australian ERAV isolate cultured in 1990, are circulating in the Victorian Thoroughbred horse population.

The feasibility of a small animal model to investigate ERAV persistence and chronic urinary shedding was assessed. Three inbred mice strains were susceptible to ERAV infection and the dissemination of ERAV following the intranasal infection of CBA mice was characterised. In this model, an ERAV viraemia was detected as early as 1 day post infection, and virus shedding in the urine detected until day 7 post infection. Virus-specific neutralising antibodies were detected as early as day 3 post infection. ERAV replication in the lungs of animals was confirmed using immunohistochemistry and a negative strand-specific RT-qPCR assay. Although, viral RNA was detected in the hearts and bladder of mice, viral antigen positive cells were not detected in sections by immunohistochemistry.

Several studies have shown that the antigenic structure of the ERAV capsid is more complex than FMDV. In contrast to the linear immunodominant epitope of FMDV, the equivalent site of ERAV is conformational and formed within the quaternary structure of the capsid. Whole virus or virus-like particles (VLPs) would retain this conformational site. The immunogenicity of a plasmid-based DNA vaccine, designed to express as VLPs was assessed in mice. The plasmid construct, pcD.P12A.3C, contained the capsid precursor (P1-2A) and the viral protease 3C, under the transcriptional control of a cytomegalovirus (CMV) promoter. Mature viral capsid proteins and VLPs were detected in vitro in transfected COS7 cells. Immunisation of BALB/c mice with pcD.P12A.3C induced virus neutralising antibodies and enhanced the virus neutralising antibody response to an inactivated ERAV trial vaccine. This study further supports the use of DNA vaccines to elicit neutralising antibodies to complex antigenic proteins.