Preliminary Investigation Of The Prevalence Of Hepatopancreatic Parvovirus Disease And Affinity Screening Exploration Between HPV Nucleic Acids And Nucleocapsid Protein

Hepatopancreatic parvovirus (HPV) is one of the major pathogens thatcause diseases in penaeid shrimp, belonging to the Parvoviridae family,Densovirinae subfamiliy. Major cultured penaeid shrimp (Fenneropenaeuschinensis especially) are natural hosts of HPV. HPV infection in culturedshrimp has been linked to chronic mortalities during the early larval orpostlarval stages and it may result in stunted growth during the juvenilestages. Crabs and other crustaceans can also carry and spread the virus.HPV is a non-enveloped icosahedral virus of22-23nm in diameter with alinear ssDNA.The genome is about6kb, with single nucleocapsid proteinpackaged. The HPV genome has three open reading frames (ORFs), two fornon-structural proteins and one for structural protein, nucleocapsid protein(CP). To date, the complete HPV genomes of five strains isolated fromThailand (DQ002873), Australia (DQ458781), India (FJ410797), China(GU371276) and Korea (JN082231), respectively, were sequenced andavailable in database of GenBank. The gene sequences analysis of different HPV isolates shown lots of genetic vatiations.Currently, main detection methods against HPV usually arehistopathological observation, conventional PCR and so on, which areinternational general methods. And the existing real-time PCR methods forHPV are not suitable for HPV China isolate due to sequence differences ofprimers and probe. Therefore, a TaqMan-based real-time PCR assay wasdeveloped for the detection of HPV in China. The recombinant expressionsystem for HPV-CP has been constructed in our laboratory. By usingSystematic Evolution of Ligands by EXponential enrichment (SELEX)method, we are trying to screen high affinity HPV genomic fragmentsagainst HPV-CP.This work mainly focuses on:1. Sample surveys and analysis. Hundreds of samples were collectedfrom main shrimp farming areas in Shandong Peninsula and around BohaiSea from2011/05to2012/10. These samples were detected with on-sitediagnostic techniques, pathogen detection kit, histopathological observation,conventional PCR method and real-time PCR method. It provided a strongscientific basis for epidemiological survey and disease prevention andcontrol.2. The establishment and application of real-time PCR method againstHPV China isolate. A pair of primers, HPV F/R and a TaqMan probe were selected from HPV genomic sequence (GenBank: GU371276). The primersand TaqMan probe used in this assay were shown to be specific for HPVand did not react with infectious hypodermal and hematopoietic necrosisvirus (IHHNV), white spot syndrome virus (WSSV), and SPF shrimp DNA.The assay had a detection limit of four plasmid HPV DNA copies. Thereproducibility of this method is good. The clinical application of testresults show that high HPV positive rate (76%) was found in F. chinensisand the viral copies were relatively high, too. The positive rate ofLitopenaeus vannamei is low with low number of viral copies. HPV can bedetected in other organisms in the aquaculture water of these samples withhigh viral copies number, too, even in seawater.3. Affinity screening exploration between HPV nucleic acids andnucleocapsid protein. Recombinant expression HPV-CP was purified andrefolded with AKTA explorer100and HPV genome random dsDNAlibrary was built by sonicating. Then we tried to screen high affinity DNAfragment to HPV-CP with SELEX method. In this study, the experimentalprocedure of affinity screening was successfully explored and this provideda reference for future in-depth study.