W. Sukhumsirichart et al., Characterization and PCR detection of hepatopancreatic parvovirus (HPV) from Penaeus monodon in Thailand, DIS AQU ORG, 38(1), 1999, pp. 1-10
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.