A novel ssDNA Bidnavirus in the giant freshwater prawn Macrobrachium rosenbergii

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A purported parvovirus producing circular, eosinophilic inclusions in the nuclei of tubule epithelial cells of the hepatopancreas (HP) of the giant freshwater prawn Macrobrachium rosenbergii (Mr) was first reported from Malaysia in 1990. In 2009, similar inclusions in Mr from Thailand were confirmed to contain single-stranded DNA (ssDNA). In 2021, Mr samples showing similar HP inclusions were reported to give positive PCR test results for decapod hepanhamaparvovirus (DHPV) using universal DHPV primers designed from many viral isolates of 3 penaeid shrimp species. However, the matching DNA probe revealed positive in situ hybridization (ISH) results with host HP nuclei only, and not with the intranuclear inclusions. At the time, this anomaly could not be explained. Here we describe metagenomic analysis and de novo assembly from 2016 samples of Mr with similar intranuclear inclusions that showed negative PCR test results using the DHPV universal primers. Genomes identified through metagenomics revealed a novel ssDNA genome sequence of 6723 bases that was confirmed by a single PCR amplicon. ISH using specific probes derived from this genome sequence revealed that the ssDNA was present in both grossly normal nuclei and in the intranuclear inclusions. Tests with archived DNA from the samples used in the 2021 publication yielded positive PCR results for both DHPV and the novel sequence, suggesting that the shrimp had dual infections. Phylogenetic analysis of the novel sequence revealed no relationship to any known DHPV type. Instead, the sequence closely matched the polymerase B gene in the V1 fragment from Bombyx mori bidensovirus 2 (BmBDV2) that is characterized by 2 ssDNA genome fragments and has been classified in the family Bidnaviridae. The 2 BmBDV2 fragments V1 and V2 each produce their own independent capsid protein genes and virions. In contrast, the new Mr virus is independent with a single 6723 base ssDNA genome, and we propose the name Macrobrachium hepatopancreatic bidnavirus (MHBV).