Megalocytiviruses of freshwater ornamental fish and pathogenicity in marine and euryhaline species

Megalocytiviruses affect a broad range of fish species and have caused widespread mortality in aquaculture throughout Asia. The first confirmed report involved mass mortality in red sea bream cultured off Shikoku Island, Japan in 1990. Ornamental fish exhibiting pathology consistent with Megalocytivirus infection have been reported from a range of countries, frequently in association with fish recently imported from south east Asia. Although the relationship between megalocytiviruses in ornamental fish and food fish remains unclear, a potential linkage between a Megalocytivirus from a disease outbreak in Murray cod, Maccullochella peelii, in Australia and those in ornamental fish imported from south east Asia had been demonstrated experimentally. However, no data existed on the strains of virus involved in the first reported cases of Megalocytivirus-like pathology in ornamental fish. Furthermore, the potential for spread from ornamental fish to food fish other than Murray cod was also unknown. This study aimed to characterise Megalocytivirus from archival ornamental fish tissues, and to determine the potential for spread of megalocytiviruses from freshwater ornamental fish to other species, including marine fish. In situ hybridisation (ISH) assays were developed to demonstrate megalocytiviral DNA in tissue sections. The presence of megalocytiviral DNA was confirmed in two archival ornamental fish from 1986 and 1988 which had Megalocytivirus-like pathology. Unlike all other reported cases of Megalocytivirus in ornamental fish, these cases did not involve the Infectious spleen and kidney necrosis (ISKNV)-like genotype. The sequence of the major capsid protein (MCP) and the adenosine triphosphatase (ATPase) genes from the viruses in the archival tissues had a high degree of identity to each other (96.7-100%) and to a turbot reddish body iridovirus (TRBIV)-like Megalocytivirus (96.6-99.9%), reported as a cause of mass mortality in barred knifejaw, Oplegnathus fasciatus, fingerlings in Taiwan. By contrast, MCP sequences of Megalocytivirus in ornamental fish samples from 2002-2010 had almost complete identity to each other (99.9-100%) and to ISKNV, and only 91.5-95.0% identity to the archival samples, providing further evidence that these megalocytiviruses were genetically distinct. Megalocytiviruses in ornamental fish cases during 1986-1993 were TRBIV-like whereas all cases from 1996 onwards were ISKNV-like. Evidence also indicated that a single ornamental fish could be concurrently infected with two different megalocytiviruses. The confirmation of Megalocytivirus infection in ornamental fish prior to 1990 resulted in a revision of the index case for confirmed Megalocytivirus in any fish species. The detection of a novel TRBIV-like strain of Megalocytivirus in ornamental fish raised the possibility that current polymerase chain reaction (PCR) assays may not detect this group of megalocytiviruses. Consequently, new conventional and real-time quantitative (q) PCR assays were developed to differentiate between megalocytiviruses from each of the three major Megalocytivirus genotypes. The discovery of a potential link between historical cases of Megalocytivirus infection and a recent disease outbreak in barred knifejaw, provided further impetus for investigation of the potential for spread of megalocytiviruses from freshwater ornamental fish to marine fish species. The susceptibility of Australian bass, Macquaria novemaculeata, an important euryhaline Australian native species, to a Megalocytivirus (DGIV-10) originating from a recently imported dwarf gourami, Trichogaster lalius, was tested. Australian bass were susceptible to lethal infection with this Megalocytivirus both by direct inoculation and by cohabitation, and transmitted Megalocytivirus to naïve Murray cod through cohabitation. Australian bass could therefore be used as a model to study vector-borne transmission of Megalocytivirus between freshwater and marine fish species. Silver sweep, Scorpis lineolata, an Australian native marine fish was shown to be highly susceptible to DGIV-10 both by direct inoculation and cohabitation, and is the first Australian marine fish with demonstrated susceptibility to a Megalocytivirus originating from ornamental fish. Transmission of DGIV-10 from freshwater Murray cod to euryhaline Australian bass and then to marine silver sweep was demonstrated using cohabitation. Transmission of DGIV-10 from marine silver sweep to euryhaline Australian bass and then to Murray cod was also demonstrated. In summary, megalocytiviruses originating from the freshwater ornamental fish trade were demonstrated to be a potential biosecurity risk not only to Australian native freshwater species, but also to euryhaline and marine species and therefore to marine aquaculture, and recommendations for further research are presented