Assessment of the origin of white spot syndrome virus DNA sequences in farmed Penaeus monodon in Australia

Abstract

Until 2016, the Australian shrimp industry was considered free of White Spot Syndrome Virus (WSSV), a destructive pathogen responsible for the huge economic losses in the shrimp culture industry in most shrimp producing countries worldwide. In December 2016, there was an outbreak of WSSV in commercial Penaeus monodon shrimp farms located on the Logan River in Queensland, northeast Australia which resulted in the loss of all shrimp production on the Logan river either directly from the virus itself or indirectly through destruction of the shrimp production as part of quarantine measures.The first objective of this study was to consider the possible origins of the outbreak, whether the WSSV outbreak arose of an exotic introduction or a local endemic strain. The Australian WSSV DNA sequences, from nine farmed samples, amplified using the 146F2R2 primers spanning relatively conserved regions, were identical to each other and had very close identity to overseas WSSV strains which did not refute the working hypothesis that the outbreak was caused by an invasive species from overseas rather than from entry into aquaculture of a long term Australian endemic strain.The second objective was to consider the possible source of the outbreak (i.e. country or region). To do this, highly variable genomic WSSV regions were identified and novel primers designed for this study to amplify variable genomic regions so it would be possible to conduct DNA sequence comparisons, theoretically, from a hundreds of samples in a relatively short period of time and low cost, something not readily possible with full genomic sequencing. In practice we generated DNA sequences using up to five of these novel primers (and the published ORF 125 variable primers) from three Australian farmed shrimp samples, from two farmed Vietnamese samples (from the north and south of the country), one retail product from Vietnam and one retail product from China sold in Australia and compared these sequences to 10 published overseas WSSV genomes. Available data indicated the Australian sequences were most similar to published sequences from China, and less closely related to sequences from other regions, for example sequences we derived from Vietnamese farmed samples.The third objective was to assess possible physical routes of infection into Australia by testing for the presence of WSSV DNA in aquaculture feed samples used at the time of the outbreak and in imported shrimp retail items. No PCR positives were detected from two samples of shrimp feeds used at the time of the outbreak. Nearly every Australian retail item of imported shrimp product (12 out of 13) tested positive for WSSV and DNA sequences could be obtained from the amplified PCR products indicating that at some point in their history, nearly all the imported samples were in contact with WSSV.In conclusion, our data, considered together, are consistent with the following hypotheses: an exotic shrimp virus, WSSV, which is of considerable economic importance, has breached Australia's biosecurity; the Australian WSSV strain is mostly closely related to one reported from Asia; a plausible physical route of entry may have been through the importation of WSSV infected shrimp for retail sale.