不同种属细胞系感染HCoV-OC43 VR1558株的病变效应与复制动力学

Cytopathic effects and replication kinetics of HCoV-OC43 VR1558 strain in cell lines derived from different species

摘要:

目的 探讨HCoV-OC43 VR1558株在不同种属细胞系中的致细胞病变效应与病毒复制增殖特征。方法 选择来自人源(MRC-5、HRT-18、Huh7、Huh7.5、RD、HeLa)、非人灵长动物(LLC-MK2、Vero)和啮齿类动物(17Cl-1、Mv.1Lu、BHK-21、BHK-21-APN、Neuro 2a)的13种细胞系，将HCoV-OC43 VR1558株感染上述细胞系，分别于感染后连续观察细胞病变效应(cytopathic effects, CPE)，同时每天收集病毒感染的细胞与上清，通过实时定量逆转录聚合酶链式反应(reverse transcription quantitative real-time PCR, RT-qPCR)监测病毒RNA核酸拷贝数变化，通过病毒半数组织培养感染剂量(tissue culture infectious dose 50%, TCID₅₀)测定具有感染能力的活病毒载量，绘制病毒复制动力学曲线。结果 HCoV-OC43 VR1558株感染上述不同种属来源的13种细胞系后均能观察到CPE，主要表现为细胞聚集、皱缩、变圆与脱落等特征；其中，MRC-5、Mv.1Lu、HeLa及17Cl-1 4株细胞CPE出现较快，感染后72 h内即可观察到明显的细胞病变，其他细胞直至感染后120 h才能观察到明显的CPE，且HRT-18与Huh7.5的CPE最轻。RT-qPCR结果表明，病毒感染后的24 h内核酸拷贝数上升最为明显，但达到高峰期的时间与核酸拷贝数不同；其中，感染Huh7.5细胞后24 h达到高峰(10⁷ copies/mL)；感染17Cl-1、BHK-21-APN、Mv.1Lu、BHK-21细胞后48 h到达高峰(10⁷-10⁹ copies/mL)；感染MRC-5、LLC-MK2、Neuro 2a、Vero 细胞后72 h达到复制高峰(10⁶-10⁹ copies/mL)；而感染HRT-18、HeLa、Huh7以及RD细胞后的病毒拷贝数在96 h后才达到高峰(10⁸-10⁹ copies/mL)。TCID₅₀检测结果显示，病毒感染24 h内快速增殖，但达到高峰期的时间与滴度不同；感染BHK-21-APN细胞后48 h达高峰(2.68×10⁷ TCID₅₀/mL)；BHK-21、Neuro 2a在感染后72 h达峰值(10⁶-10⁷ TCID₅₀/mL)，而MRC-5、17Cl-1、HeLa、Huh7、Huh7.5、Mv.1Lu、LLC-MK2及RD细胞于感染后96 h达到滴度峰值(10⁶-10⁸ TCID₅₀/mL)，Vero细胞至感染后120 h到达峰值(10⁵ TCID₅₀/mL)，而HRT-18细胞则在感染后144 h达到峰值(10⁸ TCID₅₀/mL)。结论 HCoV-OC43 VR1558株具有较广泛的细胞感染谱，病毒感染不同种属来源13株细胞系后均能在24 h内快速复制增殖，但达到高峰期的时间不同。最高感染滴度可达到10⁸ TCID₅₀/mL (在MRC-5和HRT-18 细胞)。该研究为深入开展人冠状病毒OC43的复制特征、感染致病机制研究及抗病毒药物筛选评价提供参考。

Abstract:

Objective To study the cytopathic effects (CPE) and replication characteristics of HCoV-OC43 VR1558 strain in cell lines derived from different species.Methods Thirteen cell lines from humans (MRC-5, HRT-18, Huh7, Huh7.5, RD, and HeLa), non-human primates (LLC-MK2 and Vero), and rodents (17Cl-1, Mv.1Lu, BHK-21, BHK-21-APN, and Neuro 2a) were selected and infected with HCoV-OC43 VR1558 strain. CPE were observed for several consecutive days. Virus-infected cells and supernatants were collected daily. RT-qPCR was conducted to monitor the changes in viral RNA copy number. The load of viruses with infectious ability was determined based on the tissue culture infectious dose 50% (TCID₅₀), and the kinetic curves of viral replication in different cell lines were established.Results Following infection with HCoV-OC43 VR1558 strain, CPE were observed in all the 13 cell lines. CPE primarily manifested as cell aggregation, shrinkage, rounding, and detachment. CPE appeared early in MRC-5, Mv.1Lu, HeLa, and 17Cl-1 cells, being noticeable within 72 h post-infection (hpi). The virus induced CPE in other cell lines after 120 hpi, and CPE were the mildest in HRT-18 and Huh7.5 cells. RT-qPCR results indicated that the viral RNA copy number increased most significantly within 24 hpi, although the time to reach the peak and the peak copy number varied among cell lines. Specifically, the RNA copy number in Huh7.5 cells reached the peak (10⁷ copies/mL) at 24 hpi, and that in 17Cl-1, BHK-21-APN, Mv.1Lu, and BHK-21 cells reached the peaks (10⁷ to 10⁹ copies/mL) at 48 hpi. In MRC-5, LLC-MK2, Neuro 2a, and Vero cells, the replication peaks (10⁶ to 10⁹ copies/mL) occurred at 72 hpi. In HRT-18, HeLa, Huh7, and RD cells, the viral RNA copy number peaked after 96 hpi, reaching 10⁸ to 10⁹ copies/mL. TCID₅₀ assay results demonstrated rapid viral proliferation within 24 hpi, while the time to reach the peak titer and the peak titers varied. The peak titer (2.68× 10⁷ TCID₅₀/mL) in BHK-21-APN cells was observed at 48 hpi. In BHK-21 and Neuro 2a cells, the peak titers (10⁶ to 10⁷ TCID₅₀/mL) were observed at 72 hpi. In MRC-5, 17Cl-1, HeLa, Huh7, Huh7.5, Mv.1Lu, LLC-MK2, and RD cells, the peak titers (10⁶ to 10⁸ TCID₅₀/mL) were observed at 96 hpi. In Vero cells, the virus strain reached the peak titer (10⁵ TCID₅₀/mL) at 120 hpi, while the strain reached the peak titer of 10⁸ TCID₅₀/mL in HRT-18 cells at 144 hpi.Conclusion HCoV-OC43 VR1558 strain exhibits a wide spectrum of cell tropism, demonstrating rapid replication and proliferation within 24 hpi across 13 cell lines derived from various species. However, the time to reach the replication peak varied among different cell lines. The highest viral titer achieved was 10⁸ TCID₅₀/mL, observed in MRC-5 and HRT-18 cells. This study provides experimental reference for further investigation of the replication characteristics, infection mechanism, and pathogenicity of HCoV-OC43, as well as for the screening and evaluation of antiviral drugs.