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

Abstract

Hepatopancreatic parvovirus (HPV) causes disease in several species of penaeid shrimp. Heavy infections may result in poor growth and reduced production for shrimp farmers. From one southern Thai shrimp pond with a high prevalence of HPV infection, 790 shrimp were sampled randomly and the hepatopancreas (HP) removed. Most HP were preserved in liquid nitrogen. However, every 10th HP (79 total) was divided into 2 parts appropriately fixed for examination by transmission electron microscopy (TEM) and light microscopy. Based on light microscopy, the prevalence of HPV infection in the pond was approximately 30% and its presence was confirmed by TEM of parallel samples. The virus was subsequently purified from hepatopancreatic homogenates of the samples preserved in liquid nitrogen. Negative staining of the purified viral preparation revealed unenveloped, icosahedral viral particles 22 to 24 nm in diameter. Agarose gel electrophoresis of nucleic acid extracts revealed the presence of 2 fragments, one very intense (5.8 kb) and the other weak (4.2 kb). The larger fragment was degraded by DNase I and S1 nuclease, indicating single-stranded DNA (ssDNA) characteristic of the viral family Parvoviridae. The smaller fragment was degraded by DNase I but not by S1 nuclease, indicating that it comprised double-stranded DNA. A genomic DNA library of the 5.8 k b ssDNA was constructed in pUC18 and a clone containing a 659 bp fragment specific and sensitive for HPV was selected for sequencing. Based on this sequence, a n HPV-specific primer set was designed to yield a 156 bp amplicon by polymerase chain reaction (PCR) amplification. The expected 156 bp amplicon was obtained only in the presence of HPV DNA template (at as little as 1 fg purified DNA) and not with nucleic acid templates extracted 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 carriers of HPV in the shrimp cultivation system.

Occurrence, histopathology and experimental transmission of hepatopancreatic parvovirus infection in Penaeus monodon postlarvae

“…The economic impact of high HPV infection, such as that recorded in our samples, has not been established. It was initially suggested that the percentage of HPVinfected larvae prior to pond stocking would approximately correspond to the expected minimum mortality for that pond based on the experiences of Thai shrimp farmers (Sukhumsirichart et al 1999). This means that an infection rate of 60% HPV in PL prior to stocking would result in roughly 60% mortality in culture ponds.…”

“…Harvest losses incurred from stunted shrimp can sometimes be considerable, since stunted shrimp are much less valuable than normal shrimp. Therefore, it is necessary to check also for HPV in PL prior to pond-stocking (Flegel et al 1999).…”

“…Infected shrimp exhibit gross signs, such as anorexia and stunting, which are not specific to HPV. However, Flegel et al (1999) reported that the degree of HPV infection was significantly related to the stunting of cultured Penaeus monodon in Thailand. In addition to poor growth, it could cause significant mortality in juvenile stages, particularly in cases of multiple-agent infections .…”

Immunological-based assays for specific detection of shrimp viruses

“…The other type has been identified Chaivisuthangkura P et al . Detection of shrimp viruses by immunoassays in infected P. monodon from Thailand and India [78,81] . The complete genome of PmDNV isolated from infected P. monodon in Thailand consists of 6321 nucleotides, representing three ORFs and two non-coding termini.…”

“…PmDNV is a non-enveloped icosahedral virus that is 22-23 nm in diameter and contains linear ssDNA. It belongs to the family Parvoviridae in the densovirus group [77,78] . Two Asian types of PmDNV have been characterized at the molecular level.…”