Characterization and PCR detection of hepatopancreatic parvovirus (HPV) from Penaeus monodon in Thailand

Hepatopancreatic parvovirus (HPV) causes disease in several species of pena eid shrimp. Heavy infections may result in poor growth and reduced producti on for shrimp farmers. From one southern Thai shrimp pond with a high preva lence of HPV infection, 790 shrimp were sampled randomly and the hepatopanc reas (HP) removed. Most HP were preserved in liquid nitrogen. However, ever y 10th HP (79 total) was divided into 2 parts appropriately fixed for exami nation by transmission electron microscopy (TEM) and light microscopy. Base d on Light microscopy, the prevalence of HPV infection in the pond was appr oximately 30 % and its presence was confirmed by TEM of parallel samples. T he Virus was subsequently purified from hepatopancreatic homogenates of the samples preserved in Liquid nitrogen. Negative staining of the purified vi ral preparation revealed unenveloped, icosahedral viral particles 22 to 24 nm in diameter. Agarose gel electrophoresis of nucleic acid extracts reveal ed the presence of 2 fragments, one very intense (5.8 kb) and the other wea k (4.2 kb). The larger fragment was degraded by DNase I and S-1 nuclease, i ndicating single-stranded DNA (ssDNA) characteristic of the viral family Pa rvoviridae. The smaller fragment was degraded by DNase I but not by S1 nucl ease, indicating that it comprised double-stranded DNA. A genomic DNA Libra ry of the 5.8 kb ssDNA was constructed in pUC18 and a clone containing a 65 9 bp fragment specific and sensitive for HPV was selected for sequencing. B ased on this sequence, an HPV-specific primer set was designed to yield a 1 56 bp amplicon by polymerase chain reaction (PCR) amplification. The expect ed 156 bp amplicon was obtained only in the presence of HPV DNA template (a t as little as 1 fg purified DNA) and not with nucleic acid templates extra cted from healthy shrimp tissue or other shrimp pathogens. It is hoped that this PCR assay will be useful to shrimp aquaculturists for early detection and screening of shrimp larvae, parental broodstock or other possible carr iers of HPV in the shrimp cultivation system.