HCV Infection And Passage In Primary Tupaia Hepatocytes And Transfected Receptors Strengthen Virus Infection

As the cause of Hepatitis C, hepatitis C virus is prevalent worldwide. According to the released data, it was estimated that about1.7-2.0hundred million population are infected with HCV. China is the mid-high HCV prevalence area with HCV infection rate of3.2%among general population. So far, the effective treatment and protective vaccine is still poor due to the lack of effective culture system and suit animal model. In2005, the robust HCV cell culture system had been established. It made the seeking and development of the suit small animal model become more urgent. However, there is no evidence showing any other animals was infected with HCV except for human being and chimpanzee. Tree shrews (Tupaia belangeri chinensis) are small animals, closely related to primates, mainly spreaded in Yunnan province, which adapt easily to a laboratory environment. Although it was reported that Tupaia could be infected by HCV derived from hepatitis C patient's serum, the infection of HCV to Tupaia has not been confirmed yet because of uncertain origin of HCV, low HCV viral load, and unclear background of host animal.In this study, we further determine the suspection of tree shrew as hepatitis C animal model. The two-step perfusion, collagenase digestion technology was used to separate and get primary tupaia hepatocyte (PTH). The used perfusion fluid, centrifugal speed and the culture medium and other factors of PTH culture were optimized. Finally the goted number of PTH are determined with Trypan Blue method and the PTH vatality is evaluated with MTT method. It was showed that D-Hank's is the best perfusion fluid, centrifugal speed of800rpm for3min,600rpm for3min,400rpm for2min are more suit to PTH operation. The best culture medium contains1x ITS formula,1%DMSO,200uM glutamine,0.15%cow serum albumin,100U/ml penicillin,100ug/ml Streptomycin,0.1uM dexamethasone and WEM medium. PTH could grow for longer period with better state under this optimizated circumstance. It is qualified for plsmid transfection and HCV infection.To further increase the PTH's suspectivity for HCV, the constructed plasmid which expressing CD81receptor gene successfully, was introdueced into PTH with the lipidosome. The expressed RNA was qualitvely and quantitavitly determined with PCR and real time PCR method. The results showed transfected PTH could express the human CD81with234times than control cells which transfected with mimic plasmid. The transfected PTH was inoculated with HCV supernants with107copies/ml viral load, which from J6/JFH1-Huh7.5.1HCV culture system. It was found that the viral load of PTH infection is between104and105IU/ml both in transfected cells and non-transfected cells, and the highest the viral load in transfected cells is106IU/ml on the seventh culture day.In summary, Tupaia belangeri chinensis is conducted with the two step perfusion technique, to achieve the primary Tupaia hepatocytes. The culture factors was optimized to optimized to get qualified hepatocytes. Moreover, the human CD81was transfected into PTH and highly expressed. The tranfected PTH could support the infection and replication of HCV derived from J6/JFH1-Huh7.5.1HCV culture system. Our results further confirmed the possibility of Tupaia as a HCV small animal model, and may provide a useful technique for the research on HCV infection and replication.