RT-SHIV中国恒河猴适应株细胞水平生物学特性分析

In Vitro Characterization of RT-SHIV Adapted in Chinese Rhesus Monkeys

目的体外增值、制备动物感染来源的RT-SHIV病毒中国恒河猴适应株,比较PBMCs和CEMx174两种细胞制备出病毒的差异,同时用TZM-bl、CEMx174、PBMC三种细胞滴定测定病毒TCID50。方法用RT-SHIV病毒静脉感染中国恒河猴,定期采血测定血浆病毒载量,当病毒载量达高峰时采血分离外周血单核淋巴细胞(PBMCs),与正常恒河猴PBMCs或CEMx174细胞共培养,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,滴定病毒的TCID50。结果本研究共制备了78 mL PBMCs来源的RT-SHIV病毒和85 mL CEMx174细胞来源的RT-SHIV病毒。RT基因序列和原始序列的相似度为99%,仅在第254和265位的氨基酸发现突变。RT-SHIV(PBMC)和RT-SHIV(CEMx174)病毒载量分别为1.641×108 copies/mL和8.375×108 copies/mL,P24抗原水平分别为20.745 ng/mL和4.28 ng/mL,TZM-bl、CEMx174、PBMC细胞测定病毒的TCID50分别为3.16×105 TCID50/mL和1×104 TCID50/mL,5×102 TCID50/mL和5×105 TCID50/mL,5×102 TCID50/mL和5×103 TCID50/mL。结论 PBMCs细胞来源制备的病毒较CEMx174制备的病毒具有更高的感染性。

Objective To propagate and titrate the adapted RT-SHIV from Chinese rhesus monkey using TZM-bl,CEMx174 and PBMCs cells,and to compare the possible difference of these viruses which propagated in PBMC and CEMx174,respectively.Methods Rhesus macaques free-living in China were infected with RT-SHIV intravenously.Blood samples were collected regularly and viral loads were monitored.Peripheral blood mononuclear cells（PBMCs） were examined when the viral load reached the peak,and were co-cultured with PBMCs from uninfected rhesus or CEMx174 cells.The P24 antigen level from supernatant was monitored regularly.The co-cultured virus was collected,packed and frozen when the viral replication reaches the peak.The collected viruses as a stock were charactarized with the RNA viral loads,P24 antigen level and titration of TCID50.Results In this study,in tatol of 78 mL of RT-SHIV in monkeys’ PBMCs and 85 mL of RT-SHIV was propagated in CEMx174 cells,respectively.The similarity between RT gene sequence and original sequence was 99%,only two mutation of amino acid at 254 and 265.RT-SHIV（PBMC） and RT-SHIV （CEMx174）viral loads were titrated with TZM-bl,CEMx174 and PBMCs,and their results were 1.641 × 108 copies/mL and 8.375 × 108 copies/mL.respectively,P24 antigenlevel were 20.745 ng/mL and 4.28 ng/mL,respectively,titrations of TCID50 in TZM-bl,CEMx174,PBMC were 3.16 × 105 TCID50/mL and 1 × 104 TCID50/mL;5 × 102 TCID50/mL and 5 ×105 TCID50 /mL;5 × 102 TCID50/mL and 5 × 103 TCID50/mL,respectively.Conclusions RT-SHIV propagated from PBMCs has higher infective ablity than that from the other cells in vitro.