Study On Virus-like Particle (VLP) Of Infectious Hypodermal And Hematopoietic Necrosis Virus (IHHNV)

Infectious hypodermal and hematopoietic necrosis virus(IHNNV) is one of the highly pathogenic shrimp viruses causing diseases in both cultured and wild shrimp.It belongs to the Parvoviridae family and most closely related to the genus brevidensovirus.In previous studies,we have found IHHNV capsid protein(CP) was recombinantly expressed in Escherichia coli and could self-assemble into virus-like particles(VLPs) with homogeneous size and shape similar to the native IHHNV particles.VLPs are non-infectious because of the absence of viral genome.But indirect immunofluorescence microscopy analysis showed that IHHNV-VLPs could efficiently enter primary hemocytes of the shrimp Penaeus wannamei.These results indicate that it may be possible to utilize IHHNV-VLPs as vehicles for developing specific exogenous substances.This papaer is mainly concerned about the assembly mechanism and nucleocapsid structure of IHHNV-VLPs and the nature of nucleic acids encapsidated in IHHNV-VLPs,including the following aspects:(1) In order to study the structure of the capsid,we ultracentrifugated the purified VLPs and the supernatant was studied by transmission electron microscope(TEM),blue-native SDS analysis and gel filtration chromatography. These results have proved that there existed two forms in the supernatant: monomer and pentamer,that is,IHHNV nucleocapsid is only constructed by the pentamer capsomer.To further study the forces to form this stable structure,we treated VLPs with EDTA and dithiothreitol(DTT).VLPs could not be disassembled by EDTA,but its structure was completely disrupted by DTT.Non-reducing SDS/PAGE showed that no intermolecular disulfide bond was formed between the CP molecules,suggesting that intra-CP disulfide bond would be essential for maintaining the capsid structural integrity.(2) Fluorescent virus-like particles(fVLPs) have been more and more widely used in the study of the viral structure and infection.We inserted EGFP into the C-terminal of CP and after co-expression with CP,fVLPs was self-assembled.SDS-PAGE analysis has found CP-EGFP contained in fVLPs is only one-fourteenth of CP.Immuno-gold electron microscope technology indicated EGFP is likely to be displayed on the surface of fVLPs.The assay incubating with primary hemocytes of the shrimp Litopenaeus vannamei showed that the fVLPs could efficiently enter primary shrimp cells.(3) IHHNV-CP could be recombinantly expressed in Escherichia coli and found to self-assemble into VLPs.We have extracted RNA and DNA with a predominant size of 0.5 kb from IHHNV-VLPs.The extract was treated with RNase or DNase and analyzed by agarose gel electrophoresis and ethidium bromide staining showed that the extract was mianly RNA.Northern blot analysis showed that the packaging of RNA is non-specific.(4) In order to package some exogenous substances in VLPs specifically, such as siRNA or dsRNA,we screened IHHNV genome by Systematic Evolution of Ligands by Exponential Enrichment(SELEX).IHHNV genome was randomly fragmented to mostly 200 bp using ultrasonic process and the size-selected fragments were end-filled and linked with a joint,incubated with CP on the Ni-NTA column in the renaturation solution.the screened DNA was amplificated by PCR for the next screening.The probe was denatured for the single-stranded screening.After several rounds of screening,purificated DNA was sequenced to find out a conservative sequence of 65 bp.Conclusively,the extraction of natural IHHNV virus is very complicated, so we hope to get a preliminary understanding about the capsid structure and assembly mechanism by studying the structure of IHHNV-VLPs.We also propose that IHHNV-VLPs may be altered to be a promising vehicle for RNA interference by investigating the nature of nucleic acids encapsidated in VLPs.