Detection of Australian gill-associated virus (GAV) and lymphoid organ virus (LOV) of Penaeus monodon by RT-nested PCR

A highly sensitive test based on reverse transcription followed by nested p olymerase chain reaction (RT-nPCR) was developed to detect the Australian y ellow-head-like viruses, gill-associated virus (GAV) and lymphoid organ vir us (LOV) of Penaeus monodon. The RT-nPCR detected viral RNA in as little as 10 fg lymphoid organ total RNA isolated from GAV-infected P. monodon. Ampl ification of serial dilutions of a GAV cDNA clone showed that the nested PC R was sufficiently sensitive to detect a single genome equivalent using a D NA template. The specificity and sensitivity of the RT-nPCR was also demons trated using experimentally infected P. (Marsupenaeus) japonicus, where GAV sequences could be amplified from lymphoid organ and haemocyte RNA as earl y as 6 h post infection (p.i.), and from gills by 24 h p.i. In contrast, tr ansmission electron microscopy (TEM) identified nucleocapsids and virions i n lymphoid organ cells and haemocytes from Days 3 and 6 p.i., respectively, while there was no evidence of infection in gill cells at any time. The pr actical application of the RT-nPCR was demonstrated by screening healthy wi ld-caught P. monodon broodstock. The high prevalence (>98%) of broodstock t hat were positive by RT-nPCR suggests that LOV is endemic in northern Queen sland. In addition, results with lymphoid organ, gill and haemocyte RNA sug gest that small gill biopsies may be best suited to the non-sacrificial tes ting of valuable broodstock. The speed and sensitivity of the RT-nPCR make it a useful adjunct to TEM for diagnosing LOV/GAV infection of P. monodon, with the additional benefit that screening of gill biopsies may facilitate selection of LOV-free broodstock.