Mullan B, Sheehy P, Shanahan F, Fanning L
Hepatitis C Unit, Department of Medicine Department of Microbiology, University College Cork, Cork, Ireland.
J Viral Hepat. 2004 Mar;11(2):108-14. doi: 10.1046/j.1365-2893.2003.00455.x.
Since the first report of genetically heterogeneous, or quasispecies, populations of RNA viruses, the genetic heterogeneity of the RNA genomes of major viral pathogens has been extensively studied. These studies aim to provide insights into the evolutionary pressures that act upon viruses, in order to define windows where anti-viral therapies will be most effective, to take prognostic values from viral genetic distributions at a given time, and to aid the development of novel therapeutic compounds that may tilt viral replication towards information loss. Many methodologies are employed to analyse genetic distributions of a virus in a given sample, but all involve the generation, and subsequent analysis, of the sequence information contained in a reverse-transcription-polymerase chain reaction (RT-PCR) product. Despite the fact that the aim of these RT-PCRs is to obtain sequence information from viral genomes, their application to this task is approached without adequate consideration of this end-goal. The establishment of an RT-PCR for a specific viral target genome generally proceeds in the same fashion as one would apply to establishing a PCR to determine the presence or absence of a specific target sequence in a given sample. However, it is becoming increasingly apparent that RT-PCR products generated by amplification with the ubiquitous thermostable DNA polymerase Taq, coupled with standard cloning and sequencing methodologies, has the potential to yield inaccurate and misleading data as pertains to the information content of populations of RNA viral genomes. This review discusses varying approaches employed to analyse heterogeneous populations of hepatitis C virus RNA genomes.
自从首次报道RNA病毒存在基因异质性群体（即准种）以来，主要病毒病原体RNA基因组的基因异质性已得到广泛研究。这些研究旨在深入了解作用于病毒的进化压力，以便确定抗病毒治疗最有效的时机，从特定时间的病毒基因分布中获取预后价值，并助力开发可能使病毒复制倾向于信息丢失的新型治疗化合物。许多方法被用于分析给定样本中病毒的基因分布，但所有方法都涉及逆转录聚合酶链反应（RT-PCR）产物中所含序列信息的生成及后续分析。尽管这些RT-PCR的目的是从病毒基因组中获取序列信息，但在应用于这项任务时，并未充分考虑这一最终目标。针对特定病毒靶基因组建立RT-PCR的过程，通常与用于建立PCR以确定给定样本中特定靶序列是否存在的过程相同。然而，越来越明显的是，使用普遍存在的耐热DNA聚合酶Taq进行扩增，再结合标准克隆和测序方法所产生的RT-PCR产物，就RNA病毒基因组群体的信息含量而言，有可能产生不准确和误导性的数据。本综述讨论了用于分析丙型肝炎病毒RNA基因组异质性群体的不同方法。
