Studies On Establishment And Characteristics Of Hepatitis G Virus Transgenic Mouse Models

A novel pathogenic agent, named GB virus C or hepatitis G virus, was isolated by Simons and Linnen of two independent groups in 1995 and 1996. GBV-C and IIGV share high nucleotide and protein sequence homology representative of different isolates of the same virus. Both isolates are hereafter referred to as HGV. HGV is an enveloped virus containing a positive strand RNA genome of approximately 9400 nucleotides, which consists of a large open reading frame encoding a 2900 amino acid polyprotein. The ORF is flanked at the 5'end by a nontranslated region that functions as an internal ribosome entry site and at the 3?end by a sequence essential for genome replication. Due to the similarity to HCV , HGV is presumed to be a flavivirus. And the full-length HGV cDNA clone, HGVqz, was spliced in 1998 in our Lab. (GenBankNo AF081782). Transgenic mouse system has been instrumental in providing new insights into virus and its related medical aspects, especially to the virus whose host range is narrow. The lack of optional culture and replication system in vitro and in vivo hampered the study of mechanism of replication and pathogenesis of nOV. In this study, we established a transgenic mouse model, carrying hepatitis G virus (I-IGV) genes and genomic HGVqz for the studies of characteristics of HGVqz. The transgenic mouse models were explored to study the replication, expression, pathogenssis and hepatotropism of HGV. 1. Establishment of transgenic mouse line HGVcns3 TGM Plasmid pHGVns3 was transfected into Chang liver cell line. RT-PCR and Western blotting of the transfected cells indicated that the HGV genes can be expressed in vitro. The transgenic mice were produced by microinjecting the linearized plasmid pHGVns3 digested by Sail and XmnI with conventional method. The integrated exogenous gene was identified by PCR and Southern blotting with tail tissue DNA from mice. The exogenous HGV gene can be transmitted through germ line. Detection of RT-PCR indicated that IJGV RNA was positive in liver tissue and monocytes of blood in transgenic mouse. HGV antigen was detected in liver cells. HGV gene expression products tend to cause the mild damage in liver of the transgenic mice under the pathological slice observation. However, this transgenics only include the partial genes of I-IGV. It is essential to develop a transgenics carrying HGV whole genome. 2. Transgenic mouse model carrying hepatitis G virus genome The full-length of HGVqz is 9373bp in length, which encodes 2874 AA. The coding region in located from 442nt to 9063nt. The start AA is MGPP, which is different from other HOV isolates. However, it has the conservative splicing site in its protein sequence. The HGVqz was explored to construct two express vector regulated by CMV or CMV and Alb promotors respectively. Then, the HGVqz and two express vectors were expressed in vitro. The precursor of HGVqz 310 KD specific band, was detected by Western blotting, but the precursor of HGVqz can't be spliced in vitro. So the transgenics carrying the whole genome including the 5 'NCR, structural region, non-structural region, and 3' nontranslated region was produced by microinjection.This transgenics line was named HGVFTGM. The exogenous genes can be transmitted stablely from founder mice to F1, F1 to F2. The positive rate of F1 and F2 is 71% and 76%,respective. The HGV genome was integrated into multiple sites in chromosomes. The HGV RNA can be detected in serum, and many tiss...