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

Abstract

A highly sensitive test based on reverse transcription followed by nested polymerase chain reaction (RT-nPCR) was developed to detect the Australian yellow-head-like viruses, gill-associated virus (GAV) and lymphoid organ virus (LOY) of Penaeus rnonodon. The RT-nPCR detected viral RNA in as little as 10 fg lymphoid organ total RNA isolated from GAV-infected P. monodon. Amplification of serial dilutions of a GAV cDNA clone showed that the nested PCR was sufficiently sensitive to detect a single genome equivalent using a DNA template. The specificity and sensitivity of the RTnPCR was also demonstrated using experimentally infected P. (Marsupenaeus) japonicus, where GAV sequences could be amplified from lymphoid organ and haemocyte RNA as early as 6 h post infection (p.i.), and from gills by 24 h p.i. In contrast, transmission electron microscopy (TEM) identified nucleocapsids and virions in 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 practical application of the RT-nPCR was demonstrated by screening healthy wild-caught P monodon broodstock. The high prevalence (>g8 % ) of broodstock that were positive by RT-nPCR suggests that LOV is endemic in northern Queensland. In addition, results with lymphoid organ, gill and haemocyte RNA suggest that small gill biopsies may be best suited to the non-sacrificial testing of valuable broodstock. The speed and sensitivity of the RTnPCR 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.

RT-nested PCR detection of Mourilyan virus in Australian Penaeus monodon and its tissue distribution in healthy and moribund prawns

“…The nested PCR did not extend the detection limit beyond 2 to 6 RNA copies but amplicon yields were significantly greater and clearly detected in agarose gels. The RT-nested PCR sensitivity thus approaches the theoretical limit of 1 RNA copy and compares favourably to a similar test to detect GAV (Cowley et al 2000a, de la Vega et al 2004) and realtime RT-PCR tests for GAV (de la Vega et al 2004) and other RNA viruses including YHV and TSV (Dhar et al 2002). Moreover, as very few (less than 0.2%) nucleotide changes occurred in MoV RT-PCR products amplified from P. monodon from eastern Australia, the test should be quite robust in detecting minor quasispecies variants present in this prawn population.…”

New yellow head virus genotype (YHV7) in giant tiger shrimp Penaeus monodon indigenous to northern Australia

“…While YHV is exotic to Australia, a closely related genotypic variant, gill-associated virus (GAV, synonymous with YHV2), was discovered not long after the discovery of YHV (Spann et al 1997, Cowley et al 1999. GAV is enzootic and can occur at high prevalence in wild and farmed P. monodon in eastern Australia and can cause disease on farms (Cowley et al 2000, Munro et al 2011. GAV has also been detected in shrimp farmed in Vietnam and Thailand (Wijegoonawardane et al 2008a).…”

Differences in the susceptibility of some penaeid prawn species to gill-associated virus (GAV) infection

“…Spheroids are not typically associated with GAV infection in which there is extensive necrosis of lymphoid organ tissue (Spann et al 1997). Nucleotide sequence comparison of regions in the putative polymerase genes of multiple GAV and LOV isolates has indicated that they are genetically indistinguishable populations (Cowley et al 2000b). GAV and LOV can be regarded as the same virus, which causes either overt or covert infections in P. monodon.…”

“…Screening of broodstock collected in northern Queensland has indicated a prevalence of LOV infection that exceeds 96% (Cowley et al 2000b). LOV-infected prawns show no visible symptoms of disease, but lymphoid organs typically contain discrete foci (spheroids) of hypertrophied, infected cells.…”

“…Penaeus esculentus and Fenneropenaeus merguiensis (also called Penaeus merguiensis) have also been cultured in Australia without evidence of GAV infection. Indeed, screening of wild and cultured penaeids using the sensitive RT-nested PCR test (Cowley et al 2000b) has indicated that P. monodon is the only known natural host of GAV in Queensland. In this paper, we ABSTRACT: Four species of penaeid prawn cultured in Australia (Penaeus monodon, Penaeus esculentus, Marsupenaeus japonicus and Fenneropenaeus merguiensis) were injected with a virulent preparation of gill-associated virus (GAV).…”