Experimental infections of Pacific oyster Crassostrea gigas using the Australian OsHV-1 µVar strain

Abstract

In Australia the spread of the Ostreid herpesvirus OsHV-1 microvariant (OsHV-1 µVar) threatens the Pacific oyster industry. There was an urgent need to develop an experimental infection model in order to study the pathogenesis of the virus in controlled laboratory conditions. The present study constitutes the first attempt to use archived frozen oysters as a source of inoculum, based on the Australian OsHV-1 µVar strain. Experiments were conducted to test (i) the virus infectivity, (ii) the dose-response relationship for OsHV-1, and (iii) the best storage conditions to keep infective viral inoculum. Intramuscular injection of a viral inoculum consistently led to an onset of mortality 48h post injection and a final cumulative mortality exceeding 90%, in association with high viral loads (1 x 105 – 3 x 107 copies of virus mg-1) in dead individuals. For the first time an infective inoculum was produced from frozen oysters (tissues stored at -80°C for 6 months). Storage of purified viral inoculum at +4°C for 3 months provided similar results to use of fresh inoculum whereas storage at -20°C, -80°C and room temperature was detrimental to infectivity. A dose-response relationship for OsHV-1 was identified but further research is recommended to determine the most appropriate viral concentration for development of infection models that would be used for different purposes. Overall, this work highlights the best practices and potential issues that may occur in the development of a reproducible and transferable infection model for studying the pathogenicity of the Australian OsHV-1 strain in C. gigas under experimental conditions. Keywords: Ostreid herpesvirus 1; Crassostrea gigas; experimental infection model; dose-response; inoculum preparation; storage conditions