A POLYMERASE CHAIN-REACTION (PCR) TO DETECT EPIZOOTIC HEMATOPOIETIC NECROSIS VIRUS AND BOHLE IRIDOVIRUS

The polymerase chain reaction (PCR) was used to amplify a segment of DNA of the dsDNA genome of epizootic haematopoietic necrosis virus (EHNV). PCR primers were synthesised which spanned an open reading frame. After 30 cycles of amplification of EHNV and Bohle iridovirus (BIV) genomic DNA, PCR products from the primers (P505 and P506) were visible on agarose gels stained with ethidium bromide. No PCR products were obtained from diamond python erythrocytic iridovirus (DPEV) or fish lymphocystis disease virus (FLDV) DNA. EHNV isolates from redfin perch Perca fluviatilis L., rainbow trout Oncorhynchus mykiss Walbaum and BTV isolates from the ornate burrowing frog Limnodynastes ornatus Gray and barramundi Lates calcarifer generated PCR products of 235 bp. The tests were used to amplify DNA extracted from EHNV-infected cell cultures and infected tissues from redfin perch, rainbow trout and barramundi. Specificity of the test was assessed by attempting to amplify DNA from uninfected cell cultures and tissues from uninfected rainbow trout, redfin perch and viruses such as DPEV and FLDV which have not been classified within the iridovirus genus Ranavirus but have been isolated from fish and reptiles within Australia. Hybridisation of P-32-labelled EHNV PCR amplified DNA to Southern blots of NcoI restriction endonuclease digested EHNV and BIV DNA enabled the easy differentiation of EHNV and BTV isolates. The PCR assay described in this paper provides a rapid method to detect/differentiate EHNV and BIV and is a valuable addition to the current EHNV diagnostic tests.