Gouvea V, Hoke C H, Innis B L
Department of Virus Diseases, Walter Reed Army Institute of Research, Washington, DC 20307-5100, USA.
J Virol Methods. 1998 Jan;70(1):71-8. doi: 10.1016/s0166-0934(97)00172-9.
The genomic variability of hepatitis E virus (HEV) was examined by restriction endonuclease analysis (REA) of four genomic cDNA copies comprising a 499 bp segment of the putative polymerase gene, a 264 bp segment of the helicase gene, and two, 680 bp and 448 bp, segments of the capsid gene. Analysis of the deduced restriction sites of all 27 HEV sequences currently available in the GenBank, and digestion of reverse-transcribed and nested PCR amplified segments obtained from six Nepali isolates were used to devise and test a REA genotyping assay. The assay allowed easy discrimination between the Mexico and Asian genotypes, and the classification of the Asian genotypes into three, or perhaps four subgenotypes. In addition, endonucleases identifiers of individual isolate or clusters of isolates were found. This assay permits rapid identification of a large number of HEV isolates directly from clinical specimens for studies on the molecular epidemiology and evolution of HEV.
通过对四个基因组cDNA拷贝进行限制性内切酶分析（REA）来检测戊型肝炎病毒（HEV）的基因组变异性，这四个基因组cDNA拷贝包括推定的聚合酶基因的499 bp片段、解旋酶基因的264 bp片段以及衣壳基因的两个片段（680 bp和448 bp）。对GenBank中目前可用的所有27个HEV序列推导的限制性位点进行分析，并对从六个尼泊尔分离株获得的逆转录和巢式PCR扩增片段进行酶切，以设计和测试一种REA基因分型检测方法。该检测方法能够轻松区分墨西哥基因型和亚洲基因型，并将亚洲基因型分为三个或可能四个亚基因型。此外，还发现了单个分离株或分离株簇的内切酶标识符。该检测方法可直接从临床标本中快速鉴定大量HEV分离株，用于HEV分子流行病学和进化研究。
