University of Pennsylvania School of Medicine, Department of Microbiology, 3610 Hamilton Walk, Philadelphia, PA 19104-6076, USA.
J Virol. 2010 Jun;84(12):6218-28. doi: 10.1128/JVI.02271-09. Epub 2010 Apr 7.
Hepatitis C virus (HCV) replication in infected patients produces large and diverse viral populations, which give rise to drug-resistant and immune escape variants. Here, we analyzed HCV populations during transmission and diversification in longitudinal and cross-sectional samples using 454/Roche pyrosequencing, in total analyzing 174,185 sequence reads. To sample diversity, four locations in the HCV genome were analyzed, ranging from high diversity (the envelope hypervariable region 1 [HVR1]) to almost no diversity (the 5' untranslated region [UTR]). For three longitudinal samples for which early time points were available, we found that only 1 to 4 viral variants were present, suggesting that productive infection was initiated by a very small number of HCV particles. Sequence diversity accumulated subsequently, with the 5' UTR showing almost no diversification while the envelope HVR1 showed >100 variants in some subjects. Calculation of the transmission probability for only a single variant, taking into account the measured population structure within patients, confirmed initial infection by one or a few viral particles. These findings provide the most detailed sequence-based analysis of HCV transmission bottlenecks to date. The analytical methods described here are broadly applicable to studies of viral diversity using deep sequencing.
丙型肝炎病毒 (HCV) 在受感染患者体内的复制会产生大量且多样的病毒群体，从而导致耐药性和免疫逃逸变异。在这里，我们使用 454/Roche 焦磷酸测序技术，对纵向和横向样本中的 HCV 群体在传播和多样化过程进行了分析，总共分析了 174,185 个序列读段。为了采样多样性，我们分析了 HCV 基因组的四个位置，从高多样性（包膜高变区 1 [HVR1]）到几乎没有多样性（5'非翻译区 [UTR]）。对于三个有早期时间点的纵向样本，我们发现只有 1 到 4 种病毒变异体存在，这表明有感染性的感染是由极少数 HCV 颗粒引发的。随后序列多样性逐渐积累，UTR 几乎没有多样化，而包膜 HVR1 在一些受试者中显示出超过 100 种变异。仅计算单个变异体的传播概率，并考虑到患者体内测量的群体结构，证实了初始感染是由一个或几个病毒颗粒引起的。这些发现提供了迄今为止对 HCV 传播瓶颈的最详细的基于序列的分析。这里描述的分析方法广泛适用于使用深度测序研究病毒多样性。
