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

Diseases of Aquatic Organisms

Christine M. Dimmock¹  Jeff A. Cowley¹  Kirsten M. Spann¹  Peter J. Walker¹ 

关键词: Yellow head virus (YHV);    Lymphoid organ virus (LOV);    Gill-associated virus (GAV);    Penaeus monodon;    RT-PCR;    Penaeid shrimp;    Prawns;   

DOI  :  10.3354/dao039159

学科分类：生物科学（综合）

来源: Inter-Research

【 摘 要 】

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 (LOV) ofPenaeus monodon. 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 sensitiveto detect a single genome equivalent using a DNA template. The specificity and sensitivity of the RT-nPCR was also demonstrated using experimentally infected P. (Marsupenaeus) japonicus, where GAV sequences could be amplified fromlymphoid 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 (>98%) of broodstock thatwere 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. Thespeed 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.

【 授权许可】

Unknown