Potential universal PCR method to detect decapod hepanhamaparvovirus (DHPV) in crustaceans

Abstract Parvoviruses that infect the penaeid shrimp previously called hepatopancreatic parvoviruses (HPV) are now called Hepanhamaparvovirus and referred to as decapod hepanhamaparvovirus, designated here as DHPV. However, a virus that causes similar lesions to the hepatopancreas of Macrobrachium rosenbergii has previously been known since 1990 and resemble what is now called hepanhamaparvovirus by microscopy and histochemistry. Unfortunately, no genome information is yet available and PCR detection methods that work for DHPV in P. monodon do not work with M. rosenbergii. For hatchery samples of M. rosenbergii in Thailand with DHPV-like lesions, we hypothesized it might be possible to design primer pairs from 8 full DHPV genome sequences at GenBank for use in PCR detection of DHPV in M. rosenbergii. Using this strategy, we successfully designed a new set of primers and a PCR protocol called the DHPV-U method that produced an amplicon of the expected size with DNA extracts from larvae of M. rosenberigii samples that showed DHPV-like lesions, while extracts from normal larvae gave none. DNA extracts from P. monodon infected with DHPV also gave amplicons of the expected size. At the same time, the conventional PCR method for DHPV in P. monodon gave no amplicon with the M. rosenbergii DNA extracts. The DHPV-U amplicons from P. monodon and M. rosenbergii shared 99% sequence identity, and in situ hybridization (ISH) assays using the DIG-labeled amplicon gave positive histochemical results in the HP tissue of both P. monodon and M. rosenbergii. The DHPV-U method is now being used in Thailand for detection of DHPV in both P. monodon and M. rosenbergii. Overall, the results support the proposal that the HP virus in M. rosenbergii is also a hepanhamaparvovirus. Based on 100% sequence identity of the target region in the currently published DHPV sequences at GenBank, the DHPV-U method may also work for detection of other DHPV isolates.