The Development Of The Quantitative Detection Method For EV71 And Its Infection On Primary Kidney Cells Of Tree Shrew

Enterovirus 71(EV71) is one of the major pathogens causing hand foot and mouth disease. This virus mainly infectchildren under 5 years old, transmitted by close contact, respiratory and digestive tract and other channels.Its infection may cause fever, rash and ulcersonin hand, foot and mouth.Some patients can cause myocarditis, pulmonary edema, aseptic meningitis and other complications and even death. So far, there is no effective drugs for hand foot and mouth disease in China except for symptomatic and supportive treatment.The lack of the suitable animal models have severely hampered the development of drugs for this disease.Currently, the available animal models for EV71 are primate macaque, neonatal rat and transgenic mice. Due to the insurmountable problems in these models, these animals are still not the suitable models for this disease.It has been documented that tupaia belangeri could be infected with EV71, they have the potential to be the suitable animal models. The investigation on a mature, stable tree shrew in vitro infection model of EV71 may provid an experimental basis for the establishment of the tree shrew models.Firstly, we need to establish the quantitative detection method for EV71. By comparing EV71 VP1 gene, the highly conserved region was selected to design a set of primers and probe.Then the target gene was obtained and cloned to the vector of pcDNA3.1+, and the transcribed RNA in vitro was diluted to get the standard RNA for setting up the standard cure. After the repeated examination, the optimized concentration of the primers and probe were 300nM and 200nM, respectively. When viral load ranged from 1×103 to 1×109 copies, the R2 value was high up to 1. The low detection limit of this PCR reached to 102 copies/μL. The inter-and intra-assay coefficient of variation was less than 0.5%, and no cross-reaction was found to Coxsackievirus (CA16), Coxsackievirus B1 (CB1), Human rotavirus (HRV) and Herpesvirus (HSV). The successful detection on six samples with EV71 was further verified.The primary kidney epithelial cells were isolated from tree shrews by trypsin digestion and mechanical shock interaction, and then cultured with RPMI1640 containing 5ng/mL EGF. The type of kidney epithelial cells was confirmed by specific binding of cytokeratinl8 (CK18) antibody. The growth characteristics of the kidney cells were measured by MTT. It was showed that the cells grew slowly in the first two days, and exponentially grew between 3-4d, and then came into the stationary phase after 10d, and began to apoptosis in the 14th days.By using the laboratory virus culture method, we get EV71 virus with high viral load of 5×107TCID5o/mL. The virus supernants was used to inoculate the cultured tree shrew kidney epithelial cells. Then collected the culture supernatant and infected tree shrew kidney epithelialcells.By using RT-PCR and quantitative PCR, the target gene could be detected and the viral load was up to 106 copies/μL, which had no significant difference with the viral load in the supernatant harvested from Vero cells infected by EV71. All these results indicated that EV71 could replicate and proliferate in kidney epithelial cells of tree shrew. By measuring the changes of the viral load and the supernatant titers, the optimal multiplicity of infection (MOI) was determined which was 5. EV71 was inoculated in kidney epithelial cells with the optimal MOI. It was found that the virus titer in the supernatant could be up to 5x105TCID50/mL in the 4th days, and began to decline with time increased.After using the detection method of western blotting and indirect immunofluorescence, the target protein of VP1 could be detected in kidney epithelial cells of tree shrew infected by EV71.In summary, the real-time TaqMan quantitative PCR was established successfully for EV71 detection. The trypsin digestion and mechanical shock interaction were used to get the tree shrew kidney epithelial cells. The EV71 VP1 protein could be detected in the kidney cells.The tree shrew kidney epithelial cells could be successfully infected with EV71 with obvious virus replication. These findings have definetly provided a research basis for the final establishment of the tree shrewsanimal model for hand, foot and mouth disease.