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目的体外制备SHIVSF162P3中国恒河猴细胞适应株,建立病毒库,为模型制备提供生物学特性稳定的感染用病毒。方法用SHIVSF162P3体外感染中国恒河猴外周血单个核细胞(PBMCs),定期测定培养液中的P24抗原水平。当病毒复制达高峰期时收集培养上清,分装并冻存。测定病毒RNA载量、P24抗原浓度和TCID50。利用病毒RNAenv区(M33262,6846bp～8481bp,共1636bp)序列分析的方法分析毒株在制备过程中的变异情况。静脉途径感染中国恒河猴G0801V,观察血浆病毒载量、外周血CD4+/CD8的变化情况。结果本研究共制备了275mLSHIVSF162P3病毒,病毒载量为4.389×107copies/mL,P24抗原水平为5.64×102pg/mL,TZM-bl细胞滴定TCID50为8.37×104/mL。gp120序列测定分析表明病毒未发生变异,CCR5的嗜性也未发生改变。感染猴G0801V高峰期病毒载量超过1×108copies/mL,急性期CD4+/CD8+倒置。结论此次制备的SHIVSF162P3细胞适应株生物学特性稳定,适合作为毒种库构建SHIVSF162P3/中国恒河猴模型。
OBJECTIVE: To prepare SHIVSF162P3 Chinese rhesus monkey cell-adapted strain in vitro and establish a virus pool for the purpose of providing a virus with stable biological characteristics for model preparation. Methods Peripheral blood mononuclear cells (PBMCs) from Chinese rhesus monkeys were infected with SHIVSF162P3 in vitro and the levels of P24 antigen in the culture medium were measured periodically. Culture supernatants were harvested at the peak of viral replication, aliquoted and frozen. Viral RNA load, P24 antigen concentration and TCID50 were determined. The virus RNAenv region (M33262,6846bp ~ 8481bp, a total of 1636bp) sequence analysis of the strains in the process of variation. Infection of Chinese Rhesus G0801V by intravenous route, the changes of plasma viral load and peripheral blood CD4 + / CD8 were observed. Results 275mL of SHIVSF162P3 virus were prepared in this study. The viral load was 4.389 × 107copies / mL, the level of P24 antigen was 5.64 × 102pg / mL, and the TCID50 of TZM-bl cells was 8.37 × 104 / mL. gp120 sequence analysis showed that the virus did not mutate, CCR5 tropism did not change. Infected monkey G0801V peak viral load exceeds 1 × 108copies / mL, acute CD4 + / CD8 + upside down. Conclusion The SHIVSF162P3 cell line prepared in this study is stable in biological characteristics and suitable for construction of a SHIVSF162P3 / Chinese rhesus monkey model as a venom library.