SHIV-KB9病毒中国恒河猴细胞适应株的制备

The Propagation and Evaluation of SHIV-KB9 Adapative Virus Stock in Chinese Original Rhesus Macaques

试验旨在研究体外制备SHIV-KB9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。试验将SHIV-KB9半长质粒连接后转染CEMx174细胞,转染上清与正常恒河猴PBMCs共培养。定期测定培养液中的P27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载量和TCID50。静脉感染中国恒河猴,研究该批次SHIV-KB9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。结果表明,本研究共制备了95mLSHIV-KB9病毒原液,病毒载量为2.678×105拷贝/mL,TZM-bl细胞测定病毒的TCID50为3.16×103/mL,gp120序列分析表明病毒未发生变异。10倍稀释的3个不同浓度的SHIV-KB9均静脉成功感染中国恒河猴,引起外周血CD4+/CD8+大幅下降。因此,此次制备的SHIV-KB9细胞适应株生物学特性稳定,适合作为毒种库建立SHIV-KB9/中国恒河猴模型。

To propagate SHIV-KB9 virus stock via passaged in Chinese-original monkey PBMCs and realize viral replication and immunological trauma in the monkey after inoculation intravenously with 10 fold dilution of SHIV-KB9.Infectious virus was generated by ligating pSHIV-KB9 5′ and pSHIV-KB9 3′,and transfecting CEMx174 cells.The peripheral blood mononuclear cells（PMBCs） from Chinese-original rhesus macaque were cocultured with the supernatant of transfection and the virus stock was collected when the viral replication reached the peak area.The variation of env gene was analized,so did the viral loads and tissue culture infective dose（TCID50）with TZM-bl cells.Three Chinese-origin rhesus monkeys were infected with this batch of SHIV-KB9 intravenously with 10 fold dilution and the viral loads in plasma and the change of CD4＋ and CD8＋ T lymphocytes were analysed during the whole infection.In this study,totally 95 mL virus stock was propagated in monkeys PBMCs and without any variation about the gp120 sequence of env gene.The viral loads was 2.678×105 copies/mL and had 3.16×103 TCID50 in 1 mL of cell-free SHIV-KB9 stock.3 rhesus macaques were infected and presented severe and rapid depletion of CD4＋ cells during acute phase.This panel of SHIV-KB9 adapted in Chinese-original rhesus monkeys＇ PBMCs were highly infectious and suitable for the animal modle.