Characterization of a Novel Isolate of Hepatopancreatic Parvovirus From Madagascar

Abstract

Since the late 1980s, farming of finfish, shellfish, and crustaceans has expanded globally. The worldwide growth and expansion of many aquaculture species were possible due to the development of novel technologies and methods such as the development of captive breeding programs, the availability of high-quality formulated diet, and the development of biosecurity protocols to prevent the introduction of diseases in a production system among many other factors. However, after many years of success, the growth and sustainability in the farming of many marine species such as shrimp are threatened by the emergence of infectious diseases, many of which are caused by viruses. More than twenty viruses have been identified in shrimp alone, and some of these diseases have caused more economic losses compared to others and, at times, have changed the trajectory of the industry. For example, the early 1980s IHHNV outbreak in blue shrimp led to the development of a shrimp resistant line and a shift in the captive breeding program from blue shrimp (Penaeus stylirostris) to Pacific white shrimp (P. vannamei). Today, P. vannamei is the most common farmed species. Another virus that has impacted the shrimp aquaculture is the Hepatopancreatic parvovirus (HPV), a small single-stranded DNA virus first reported in farmed marine prawns in Singapore in 1982. Today, the virus is present in all geographic regions where shrimp are farmed. The virus affects post-larval and mid-juvenile stages of penaeid shrimp, increasing mortality and negatively impacting growth, causing economic losses on farms. The viral reservoir and pathogenesis are understudied, and no treatment is available against HPV infection. HPV targets the hepatopancreas, a digestive glad of arthropods and mollusks. The virus is routinely diagnosed by PCR and histology. A key histopathological characteristic of HPV infection is the production of large inclusion bodies in the nuclei of the hepatopancreas tubule cells. Penaeus monodon farmed in Madagascar has been found to carry HPV since the early 1990s. Although at this moment, HPV is not a threat in the hatcheries, recently, a novel strain of the HPV has been discovered in Madagascar where post-larvae occasionally tested positive for HPV by PCR but without any histopathological changes or clinical signs like isolates from other regions. HPV from Madagascar was sequenced by Kathy Tang and colleagues (Tang et al., 2008), but the differences in detecting it by histology and PCR have not been investigated before. The overall goal of this project was to characterize this novel strain of the HPV. The approach was to sequence the full genome of the novel isolate, perform a genomic comparison with homologous isolates, and to compare the histology with conventional and real-time PCR in detecting HPV in P. monodon post-larvae from Madagascar. The analysis showed that the new Madagascan strain was most closely related to the previously sequenced Madagascan strain. The similarity was only 96.9%, and phylogenetic analyses revealed the novel strain constitutes a distinct subclade from the previously sequenced one. The sequence analysis of the HPV genome showed mutations at different sites in the putative capsid promoter region. Further study is needed to determine if those changes may affect the pathogenicity of the strains. The histological analysis of P. monodon post-larvae from Madagascar displayed no inclusion bodies. Total genomic DNA was isolated from tissue samples embedded in the paraffin block, and PCR was performed to detect HPV by conventional and real-time PCR. All samples tested negative by histology and by the PCR using an OIE-recommended method. However, new primers designed to amplify smaller amplicon showed up to 58% samples as positive for HPV. The sequencing and the histopathology data obtained in this study suggest that the novel isolate of HPV from Madagascar may constitute a new species. Further studies need to be carried out to (1) perform bioassay to demonstrate the infectivity of the new isolate, (2) carry out in situ hybridization to localize the infection, (3) conduct a promoter assay to determine the effect of the mutation on the transcription of virally encoded genes, (4) measure the expression of the capsid protein in infected tissue, and (5) screen for secondary diseases in HPV-infected shrimp. This information will help to delineate if the criteria for defining a new species are met by this novel isolate.