Wei Hua-Mian, Wang Xue-Feng, Wang Shan-Shan, Du Cheng, Liu Hai-Fang, Liu Qiang, Zhou Jian-Hu
State Key Laboratory of Veterinary Biotechnology, Harbin, China.
Bing Du Xue Bao. 2012 Jun;28(4):431-8.
Our previous studies found that the Chinese attenuated EIAV vaccine was composed of a pool of quasispecies, which showed a complicated diversity called "multi-species". Further determining the viral composition of these species in the vaccine should improve the identification of predominant viruses in the vaccine and facilitate the analysis of in vivo evolution of EIAV and the vaccine. In this study, the comparison of fidelities in amplifying and sequencing the V3 to V5 fragment of EIAV envelope gp90 gene by either a single-genome amplification (SGA) approach or the traditional RT-PCR (bulk PCR) was performed. Results revealed that the diversities were 1.84% and 1.88% for SGA- and bulk PCR-derived sequences, respectively. Futher analysis revealed that beside the sequences highly homologous to those derived by the bulk PCR, nine of 73 sequences derived by SGA contained a deduced amino acid domain that was identical to the corresponding domain in the virulent strain LN40. In addition, sequences with deletion of one predicted amino acid residual was detected by using SGA The presence of these less populated sequences provided additional evidence for the "multi-species" hypothesis for the action mechanism of the EIAV vaccine. Furthermore, based on the analysis of sampling bias, Our results that the difference in copy number of each viral specie in the pool of quasispecies resulted in the inefficiency to amplify viral sequences that were in low population by bulk PCR. Therefore, the sequences amplified by bulk PCR could not correctly represent the composition of quasispecies. As an approach based on the amplification and sequencing single isolated genome, SGA significantly improved the weakness of bulk PCR and appeared its advantage in analysis of EIAV genome composition with high variety.
我们之前的研究发现，中国研制的减毒马传染性贫血病毒（EIAV）疫苗是由一群准种组成，呈现出一种被称为“多物种”的复杂多样性。进一步确定疫苗中这些物种的病毒组成，应有助于识别疫苗中的优势病毒，并促进对EIAV及疫苗体内进化的分析。在本研究中，我们比较了通过单基因组扩增（SGA）方法或传统逆转录聚合酶链反应（批量PCR）扩增和测序EIAV包膜糖蛋白90（gp90）基因V3至V5片段时的保真度。结果显示，SGA法和批量PCR法获得的序列多样性分别为[X]%和[X]%。进一步分析表明，除了与批量PCR法获得的序列高度同源的序列外，SGA法获得的73个序列中有9个包含一个推导氨基酸结构域，该结构域与强毒株LN40中的相应结构域相同。此外，使用SGA法检测到了缺失一个预测氨基酸残基的序列。这些数量较少的序列的存在，为EIAV疫苗作用机制的“多物种”假说提供了额外证据。此外，基于抽样偏差分析，我们的结果表明，准种库中各病毒物种拷贝数的差异导致批量PCR法无法有效扩增低丰度的病毒序列。因此，批量PCR法扩增的序列不能正确代表准种的组成。作为一种基于单分离基因组扩增和测序方法，SGA法显著改善了批量PCR法的不足，并在分析高度多样的EIAV基因组组成方面显示出优势。
