庚肝病毒全其因组RNA猴肝接种及其致病性研究

Hepatitis G virus (HGV), identified in 1995, has been considered a member of the Flaviviridae. The early researches suggest that HGV might be associated with post-transfusion hepatitis as well as acute and chronic hepatitis. The pathogenicity, pathogenisis and the replication and expression of HGV is not very clear since lacking of appropriate animal and cell models. In order to clarify the above questions, we constructed a full-length HGV cDNA clone pHGVqz, and prepared HGV RNA transcripts in vitro. Experiments were carried out to study the replication, expression and pathogenicity of HGV by transfecting the Rhesus monkeys and producing transgenic mice. In addition, a phage-displayed HGV library was constructed and antigen epitopes were screened. (1) In vitro transcription of HGVqz and transfection of Rhesus monkeys with HGV RNA transcripts: HGV genomic RNA transcripts were prepared in vitro with the template of HGV full-length clone HGVqz, which encodes 2873 amino acids with the genome of 9373 base pairs. HGV RNA transcripts were prepared in vitro and transfected into HepG2 cells with lipofectamin. HGV plus and minus RNA strands and E2 protein were detectable in the transfected cells. HGV could replicate for generations in the cells. HGV RNA could also be detected in the culture supernatants and the frozen-thawed HepG2 cells. The findings suggest that HepG2 cell model for HGV replication should be preliminary established.Five Rhesus monkeys were used in this experiment, sera were collected weekly/bi-weekly from infected macaques to detect the ALT level, HGV RNA and anti-HGV. All of the 5 Rhesus monkeys have been detectable with HGV RNA and anti-HGV. All of the macaques showed slightly inflammatory changes in liver. The results of immunohistochemical staining with monoclonal antibody (MAb) showed that HGV E2 protein was mainly expressed in the cytoplasm of hepatocytes, and also in heart and spleen cells. The electron microscopy of liver from one Rhesus monkey indicated that HGV could produce two types of virus-like particles in the dissae of hepatcocyte. HGV specific probe tagged with fluorescence was used to detect the dynamic changes of HGV in sera and tissues of the experimental Rhesus monkeys. HGV replicates not only in the liver but also in other tissues such as heart and spleen. All results showes that Rhesus monkey is susceptible to the inoculation of HGV RNA transcripts, and could be used as an experimental animal model. HGV could be transmitted in the macaques for at least three generations and cause mild hepatitis. HGV could replicate in the liver of macaques, and might replicate in the other tissues such as heart and spleen.(2) Screening HGV antigen epitope from phage-displayed library: Phage-displayed random library of HGV genome was constructed, and showed good randomness and integrality verified by restriction enzyme analysis, neucleic acid hybridization and DNA sequencing. HGV epitopes from a phage-displayed random peptide library were sreened out using HGV Mab and sera from patients. The 8 epitope positive clones were proved to have the core sequence WA(W/Y)WXH, which has high homology with HGV. These data suggests that the core sequence seems to be the mimic epitope of HGV. The other 9 clones were selected from the othere phage-displayed random peptide library, and 4 of them shared the same amino acid sequence of VXSPL, which is homologous with aa 218-222 of HGV E2 protein. So, VXSPL was supposed to be a linear antigen epitope. In addition, 4 HGV synthetic peptides were tested with competitive inhibition experiments and proved to be epitope of HGV E2. (3) Transgenic mouse model of subgnome and genome HGV were constructed. The results generated from the animals suggest HGV is not a completely hepatropism virus, lymphocyte and liver are also the sites for HGV replication and expression. HGV could cause some pathological changes.

本室最近成功地获得了庚肝病毒（HGV）全长基因组的单个完整克隆。该课题拟利用这一特械幕虿牧现苯又趾锔文冢缓罅鄄焓笛槎锾迥诘牟《咎宓男纬杉捌涓腥拘浴⒅虏⌒约疤迥谕饷庖哂Υ鸱从χ副辍１鞠钅渴褂肏GV全基因组而非混合血清标本进行动物接种，目的是更客观明确地回答HGV是否致病这一有争议的热点问题。