SHIV1157ipd3N4细胞适应株的制备及其生物特性

Propagation and Characterization of SHIV1157ipd3N4 Adaptive Virus Stock in Chinese-Origin Rhesus Macaques

目的体外制备SHIV1157ipd3N4病毒中国恒河猴细胞适应株,在细胞水平和中国恒河猴体内评价其生物学特性。方法用SHIV1157ipd3N4病毒阴道感染中国恒河猴,在血浆病毒载量高峰期采血分离外周血单核淋巴细胞(PBMCs),与正常中国恒河猴PBMCs共培养。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。静脉感染中国恒河猴,研究该批次SHIV1157ipd3N4在体内的病毒学、免疫学指标变化及变异情况,分析其基本的生物学特性。结果本研究共制备了243 mL SHIV1157ipd3N4病毒原液,gp120序列分析表明病毒未发生变异,CCR5的嗜性也未发生改变。病毒载量为1.586×108 copies/mL,P24抗原水平为1.16×103 pg/mL,TZM-bl细胞测定病毒的TCID50为3.16×103/mL。1 mL SHIV1157ipd3N4静脉成功感染中国恒河猴G1004V,高峰期病毒载量达到1.0×106 copies/mL以上。结论此次制备的SHIV1157ipd3N4细胞适应株生物学特性稳定,适合作为毒种库构建SHIV1157ipd3N4/中国恒河猴模型。

Objective To propagate SHIV1157ipd3N4 virus stock via passages in Chinese-origin monkey peripheral blood mononuclear cells（PMBCs）.Methods The PBMCs from Chinese-origin rhesus macaque were cocultured with SHIV1157ipd3N4,a CCR5-tropic chimeric simian-human immunodeficiency virus（SHIV） strain.The level of P24 antigen in the supernatant was tested continuously.The virus stock was collected when the viral duplication reached a peak.The env gene of SHIV1157ipd3N4 was analyzed,and the viral load,P24 antigen level and TCID50were determined.The rhesus monkey G1004V was infected with this batch of SHIV1157ipd3N4 intravenously.The viral load in plasma and the changes of CD4 ＋/ CD8 ＋ ratio were analyzed by real-time RT-PCR and flow cytometry.Results Totally 243 mL virus stock was propagated in monkeys PBMCs.The viral load was 1.586 × 108 copies/mL,the level of P24 antigen was 1.16 × 103 pg/mL and had 3.16 × 103 50% tissue culture infective dose（TCID50） in 1 mL of cell-free SHIV1157ipd3N4.There was no variation in the gp120 sequence of env gene and this SHIV1157ipd3N4 stock was still exclusively CCR5-tropic.The monkey G1004V was infected and had a high level of viral loads in plasma.Conclusion This panel of pathogenic R5-tropic SHIV1-157ipd3N4 was adapted in Chinese-origin rhesus monkeys PBMCs,and is stable and suitable to serve as an animal model.