Persistence and chronic urinary shedding of the aphthovirus equine rhinitis A virus

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Abstract

Equine rhinitis A virus (ERAV) is a member of the Aphthovirus genus, and has many physical and structural similarities to the prototype Aphthovirus foot-and-mouth disease virus (FMDV). The pathogenesis of FMDV has been extensively studied, however, the similarities in the pathogenesis of ERAV and FMDV disease has not been well documented. This study describes and compares the pathogenesis of ERAV both in the natural host and a small animal model alternative (CBA mice). Distinct parallels in the pathogenesis of the acute infection of these two viruses are described where infection in the upper respiratory tract precedes shedding of high levels of virus from the nasopharynx and a transient viraemic phase before dissemination to distal sites. The finding that ERAV is maintained at high levels in the urine of infected horses for at least 37 days post infection, however, is a feature unique to ERAV amongst all of the picornaviruses.

Introduction

Equine rhinitis A virus (ERAV) is a respiratory pathogen of horses classified in the family Picornaviridae, genus Aphthovirus, alongside foot-and-mouth disease virus (FMDV) [1], a systemic pathogen of cloven hoofed animals. ERAV shares many pathogenic features with the prototype aphthovirus FMDV such as a respiratory infection, establishment of viraemia and persistent infection [1]. Furthermore, the key FMDV virulence factors, Lpro and 3Cpro share significant functional similarity to the homologue ERAV proteins [2]. Further investigation into the pathogenesis of ERAV may also highlight additional parallels between these two aphthoviruses, which has previously been described ERAV as a surrogate for FMDV, without the requirement for high-level biocontainment [3], [4], [5].

Sporadic outbreaks of acute febrile respiratory disease following ERAV infection have been reported [6], [7], [8], [9], [10], although, given the high seroprevalence of ERAV within the population, sub-clinical infections of ERAV are likely to be common [8], [11], [12]. It is difficult, however, to establish an estimate of the prevalence of disease caused by ERAV, due to difficulties associated with isolation and propagation of this non-cytopathic virus [7]. Furthermore, recent ERAV challenge studies using highly cell culture adapted virus inoculums have resulted in sub-clinical infections [13], in contrast to the acute febrile respiratory disease observed in the early challenge studies [14]. There has been limited investigation of the host, viral and other factors that may explain the disparate outcomes of experimental infection.

Early experimental challenge studies that documented viraemia and urinary shedding in guinea pigs and rabbits [15] suggested the progression of ERAV infection in a small animal model may be similar to that observed in the horse.

This study describes an investigation into the pathogenesis of ERAV both in the natural host and a small animal model alternative (CBA mice). Using these two models the pathogenesis of ERAV is compared to that of the prototype member, FMDV.

Section snippets

Cells

Viruses were propagated in primary equine foetal kidney (EFK) cells and Vero cells. Cell monolayers were grown in a base media of Dulbecco's minimal essential medium (DMEM, Sigma) containing 5 mM sodium bicarbonate, 10 mM HEPES and 50 μg/ml ampicillin. Uninfected cells were cultured in growth media containing 4 (Vero) or 10% (v/v) (EFK) foetal bovine serum (FBS). Virus was propagated in maintenance media containing 0.5% (v/v) FBS. Virus titrations and neutralisation assays were performed in assay

RT-qPCR sensitivity and specificity

A RT-qPCR assay was designed to target the non-structural internal ribosomal entry site (IRES) of ERAV. The assay was linear over 7 orders of magnitude extending 108–102 copies of in vitro transcribed RNA/RT-qPCR reaction and could detect 10–0.8 TCID50, of virus per reaction, which is 1000 times more virus than previously published assays [19]. The ERAV RT-qPCR assay was highly reproducible with a low inter-assay variation (0.40–2.5 cv% median 0.52) and low intra-assay variation (0.5–1.8 cv%

Discussion

ERAV has been classified as an aphthovirus alongside FMDV since 1996, and although several physical and structural features are conserved among these viruses [1], [2], [3], [4], [5], it is largely unknown whether these also translate into consistencies in pathogenesis. Furthermore the significance of ERAV as a pathogen and a cause of poor performance in horses may be underestimated in part, due to a lack of understanding of the dynamics of viral shedding following infection and sensitive

Acknowledgements

Project funding was from the Australian Research Council and Pfizer Animal Health. S.E.L was a recipient of an Australian Postgraduate Award (Industry) scholarship with Pfizer Animal Health as the industry partner. We thank Drs Gary Muscatello (The University of Sydney), Laura Fenell (The University of Melbourne), John Moody (CSL Ltd.) and Bob Geyer (The University of Melbourne) for assistance with animal experiments, and Nino Ficorilli and Cynthia Brown, Department of Veterinary Science, The

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