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 polymerase chain reaction (RT-nPCR) was developed to detect the Australian yellow-head-like viruses, gill-associated virus (GAV) and lymphoid organ virus (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. 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 RT-nPCR 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 (>98%) 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 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.
Cowley
J A
JA
Cooperative Research Centre for Aquaculture, CSIRO Tropical Agriculture, Indooroopilly, Australia. jeff.cowley@tag.csiro.au
Dimmock
C M
CM
Spann
K M
KM
Walker
P J
PJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001706:Biopsy / Q000662:veterinary; D003001:Cloning, Molecular; D003332:Coronaviridae / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D017931:DNA Primers / Q000737:chemistry; D004279:DNA, Viral / Q000737:chemistry; D005260:Female; D005880:Gills / Q000473:pathology / Q000821:virology; D006434:Hemocytes / Q000473:pathology / Q000821:virology; D008221:Lymphoid Tissue / Q000473:pathology / Q000821:virology; D008297:Male; D008854:Microscopy, Electron / Q000662:veterinary; D008969:Molecular Sequence Data; D033561:Penaeidae / Q000821:virology*; D011793:Queensland; D012367:RNA, Viral / Q000737:chemistry / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
D017931:DNA Primers; D004279:DNA, Viral; D012367:RNA, Viral