The Research On Primary Tupaia Hepatocytes As An Infection Model Of Hepatitis C Virus

Hepatitis C virus is a major pathogen of acute or chronic hepatitis, liver cirrhosis, fibrosis and hepatocellular carcinoma. According released report in 1999 statistics from WHO, there were about 170-200 million people are infected with HCV worldwide. China, with HCV infection rate of 3.2%, was considered as high or middle HCV prevalence area. Except for human beings and chimpanzee, there is no other natural animals susceptible to HCV. The lack of small animal models had severely hampered the research on pathogenesis of hepatitis C. Therefore, there are no more effective drug and preventive vaccine. In 2005, a robust HCV cell culture system has been established by Takaji Wakita and Charles Rice. It was possible to generate HCV with a high titers and same background. The reconstructed J6/JFH1 strains have proved to higher infectious to chimpanzees. Due to the high cost of chimpanzees'purchase and feeding, it is urgent to develop a small animal model of hepatitis C. Tree shrews, considered as a kind of primates with smaller body, mainly distributed in Yunnan, China. Previous reports had implicated that tree shrews could be infected with HCV serum. However, the infectivity of hepatitis C virus to tree shrew has not been finally confirmed due to different source of inoculation serum, low viral titer and unstable tree shrews, etc.Presently, primary tupaia hepatocytes (PTH) were isolated from the domesticated and breed tree shrews with two-step perfusion collagenase digestion method, followed by PTH culture in optimized culture medium containing growth factors. The PTH were got into the logarithmic growth phase grows after an adaptation period of 2-3 days.8-10 days later, apoptosis of PTH began to be obvious, and contaminated fibrous cells appeared growing. The viability of cell was detected with MTT assay, cell growth curve was made on accordance with OD value, and cell morphology was observed in inverted microscope. The change of detected ALT level in PTH culture supernatant was significantly decreased after 2 days, and then maintained at a low level. Lower transaminase levels indicated the little damage to hepatocytes during PTH isolation procedure.More than 108IU/ml viral loads or 106ffu/ml viral titers of J6/JFH1 virion were used to innoculate the grow-well PTH, followed by repeated washing with medium. The HCV load in supernatants was determined up to 4×105IU/ml with real-time quantitative PCR methods. HCV RNA was also proved by qualitative RT-Nested PCR. Furthermore, expressed HCV proteins in PTH were detected with indirect immunofluorescence assay. The green fluorescence in PTH could be observed in fluorescence microscope. These results confirmed HCV proteins'existence in infected PTH. Moreover, the infectivity of PTH generated virion was confirmed by mildly detected viral load in supernatants of Huh7.5.1 cells, which was infected by new produced HCV from PTH. Concurrently, PTH produced HCV has been proved to passage in PTH. The viral load in second passage supernatants was equivalent with the first generation. According to HCV RNA sequencing, the nucleotides sequence in 5'NCR-C and NS5B regions were highly homologous with the corresponding genome regions of J6/JFH1 chimer strains. Of note, the adaptive mutation had really occurred in E1-E2,5'NCR-C and NS5B regions respectively.In summary, primary tupaia hepatocytes were isolated from domesticated and newly breed tree shrews. They grew well enough to meet the requirements of subsequent infection experiments. Quantitative and qualitative nucleic acid and protein level detection had revealed the susceptibility of PTH to J6/JFH1 HCV virion. This had been further confirm by newly produce HCV's passage in PTH and Huh7.5.1, and existence of adaptive mutation through passages. These results provided an experimental basis for the HCV tree shrew animal model development, and would promote the study on mechanism of HCV infection and replication.