Research On Tree HCV Infection In Tree Shrews And Its Variability

Hepatitis C (non-A and non-B hepatitis) is caused by the hepatitis C virus which is leading causes of hepatocellular carcinoma and liver cirrhosis. It is estimated that approximately1.7～2hundred million people are infected with hepatitis C virus all over the world. Hepatitis C virus (HCV) is also a major public health issue in China, with43million people is infected with this virus. Pegylated interferon (PEG IFN) and ribavirin is drug of choice but results are affected due to genotype variation. However, due to the lack of suit small animal models, the development of the treatment and vaccine are seriously impeded.Although some HCV infected cell culture models had been established, these modes have obvious limitation, such as only HCVcc for genotype2a is obtained. Moreover, it is very urgent to develop the animal model of hepatitis C. Recently, Chinese Tree shrew (Tupaia belangeri) was reported to have many valuable features indicating tree shrew are potential to be used as hepatitis C experimental animal model. With the closer relation with human than mouse, small body mass, short life period, low production cast and faster growth rate, together, tree shrew is valuable to be developed as animal model.Hepatitis C virus is a RNA virus. Due to lack of RNA polymerase correction function and existence of body immune selection pressure, many mutations is occurring in almost complete viral genome during HCV replication. In HCV genome, there is a27amino acid region in the E2/NS1gene fragment called as the hypervariable region (HVR), which contains the immune epitope. The understanding on the relationship between virus and host is necessary and has great significance in the evaluation of therapeutic effect and immune prevention.In current study, we further developed the domestication of tree shrew as an experimental animal. The perfusion method, optimization of culture condition was used to get primary tupaia hepatocyte (PTH). By using the laboratory virus culture method, we get HCV (J6/JFH1) virus with high viral load of108copies/ml from inoculated huh7.5.1cell. Furthermore, we inoculate primary tupaia hepatocyte (PTH) with the cultured virus, and then collected the culture supernatant and infected naive cells. Then, nested PCR, quantitative PCR and western blot were used to detect viral RNA and viral protein. It was shown that viral loads in supernatant were up to6×104copies/ml and viral NS3protein could be detected in1Id and13d. The results discovered that tree shrew liver cells were also successfully infected with HCV with obvious virus replication. However, the efficiency of infection varied among different HCV batches and host.Wild tree shrew was placed in a separate cage for domestication. After tree shrew adapted to the environment, tree shrew was infected with HCV through tail vein injection and liver multi-point injection. A week later, virus was inoculated again for strengthen infection. Thereafter, about800μL blood was collected every week, and the obtained serum was stored. A half of serum was used for biochemical indexes analysis and the others were used for extraction of RNA. Qualitative and quantitative test were applied to detect viral RNA. ALT and AST were in the normal range of20～50U/ml. Using reverse transcription nested PCR detection, the experimental group B2, B5, B6, B9and H8appeared intermittent time of infection. Sixteen weeks later, all the experimental groups were negative. And H2and H5were positive in the first and second weeks after the detection, and then they became negative. The infection rate was high during first2weeks, however, after16weeks, we did not detected HCV virus. The highest load reached to4×103copies/ml. It could be confirmed that tree shrew was infected with HCV. Because of the individual genetic and immunological differences, the infection efficiency would be varies.Finally, total RNA was extracted from the positive samples following with PCR amplification and HVR1fragment purification and sequencing. It was shown that the locations of1490,1511,1530,1532,1554nucleotide have appearing mutation in HVR1, which lead to384,391,392,397,398,405amino acids mutation in27amino acids. The results showed that there were different variations in different experimental sample which may be due to the immune selection pressure.In summary, we successfully infected tree shrew and primary tupaia hepatocyte with HCV virus. Although the overall infection rate was low, it is further confirmed that tree shrew could be used as a potential animal model for HCV infection.