HearNPV在生长对数期与平台期宿主细胞中的复制差异分析

使用实时荧光定量PCR技术对HearNPV在生长对数期和平台期HzAM1细胞的复制差异进行分析。结果表明,HzAM1细胞生长对数期的倍增时间为22 h,生长对数期的细胞以S期细胞为主(48.6%),而平台期细胞中以G2/M期细胞为主(72.6%)。在这两种不同状态的细胞中,病毒的复制主要在感染后60 h内完成,在感染后14~20 h,病毒复制倍增时间分别为1.8 h和1.9 h,几乎没有差别。但是感染生长对数期细胞时,吸附侵入细胞内的BV数量、BV释放的数量、最终的病毒产量以及病毒表达的蛋白产量明显高于被病毒感染的生长平台期细胞。如生长对数期细胞内复制合成的病毒DNA总量的25%装配形成BV病毒粒子出芽释放到细胞外,而对于平台期细胞,病毒DNA仅有13%装配形成BV病毒粒子出芽释放到细胞外。病毒感染两种生长状态的细胞,病毒DNA均从感染后7~8 h开始复制,没有明显差别;而生长对数期细胞从被感染后18~20 h释放子代病毒BV,生长平台期细胞则在感染后22~25 h开始释放病毒BV。在感染后30~60 h,在生长对数期被感染的细胞释放BV的速度约为483 copies/cell/h,而平台期细胞约为100 copies/cell/h。最初吸附侵入到生长对数期细胞内的BV粒子数量明显多于侵入到生长平台期细胞内的BV数量。实验证实,生长对数期与平台期的细胞膜的流动性有很大差别,推测健康细胞表面有活性的病毒受体数量可能决定了侵入细胞内的BV的数量。

Comparative Study of the Replication Difference of HearNPV in Infected Exponential and Stationary Host Cells

Real-time quantitative PCR was used to characterize HearNPV DNA replication in exponential and stationary phases of HzAM1 cells.Results showed that the doubling time of HzAM1 cells was 22 h in exponential phases.Most of the exponential cells were in S phase(48.6%),and most of the stationary cells in G2/M phase (72.6%).The replication of viral DNA was completed within 60 h post infection(h p.i.) in different phases of HzAM1 cells.During 14 to 20 h p.i.,the doubling time of HearNPV replication was 1.8 h in exponential cells and 1.9 h in stationary cells,and no significant difference was found between them.But the amounts of BV entering and releasing,the final progeny virions and viral protein products in the infected exponential phase cells were obviously higher than that in the stationary phase cells.25% of the total synthesized viral DNAs were released from infected exponential phase cells,but only 13% from the infected stationary phase cells.Viral DNA started to be replicated from 7-8 h p.i.both in infected exponential phase and in stationary phase cells.But in infected exponential phase cells,BVs were started to release from 18-20 h p.i.,and BVs were started to release from 22-25 h p.i.from infected stationary phase cells.During 30-60 h p.i.,the BV releasing rate was about 483 copies/cell/h in the exponential phase cells,but was 100 copies/cell/h in the stationary-phase cells.The initial viral DNA entering into exponential phase cells was much more than that entered into the stationary phase cells.The data of cell membrane fluidity at exponential and stationary phases suggested that the fluidity of cell membrane played an important role during virus entry.