Sun Z F, Huang F F, Halbur P G, Schommer S K, Pierson F W, Toth T E, Meng X J
Center for Molecular Medicine and Infectious Diseases, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, 1410 Price's Fork Road, Blacksburg, VA 24061-0342, USA.
Vet Microbiol. 2003 Oct 17;96(2):165-76. doi: 10.1016/s0378-1135(03)00215-3.
Hepatitis E virus (HEV), the causative agent of human hepatitis E, is an important public health problem in many developing countries and is also endemic in many industrialized countries including the US. The discoveries of avian and swine HEVs by our group from chickens and pigs, respectively, suggest that hepatitis E may be a zoonosis. Current methods for molecular epidemiological studies of HEV require PCR amplification of field strains of HEV followed by DNA sequencing and sequence analyses, which are laborious and expensive. As novel or variant strains of HEV continue to evolve rapidly both in humans and other animals, it is important to develop a rapid pre-sequencing screening method to select field isolates for further molecular characterization. In this study, we developed two heteroduplex mobility assays (HMA) (one for swine HEV based on the ORF2 region, and the other for avian HEV based on the ORF1 region) to genetically differentiate field strains of avian and swine HEVs from known reference strains. The ORF2 regions of 22 swine HEV isolates and the ORF1 regions of 13 avian HEV isolates were amplified by PCR, sequenced and analyzed by HMA against reference prototype swine HEV strain and reference prototype avian HEV strain, respectively. We showed that, in general, the HMA profiles correlate well with nucleotide sequence identities and with phylogenetic clustering between field strains and the reference swine HEV or avian HEV strains. Field isolates with similar HMA patterns generally showed similar sequence identities with the reference strains and clustered together in the phylogenetic trees. Therefore, by using different HEV isolates as references, the HMA developed in this study can be used as a pre-sequencing screening tool to identify variant HEV isolates for further molecular epidemiological studies.
戊型肝炎病毒（HEV）是人类戊型肝炎的病原体，在许多发展中国家是一个重要的公共卫生问题，在美国等许多工业化国家也呈地方性流行。我们的研究小组分别从鸡和猪中发现了禽源和猪源HEV，这表明戊型肝炎可能是人畜共患病。目前用于HEV分子流行病学研究的方法需要对HEV野外毒株进行PCR扩增，然后进行DNA测序和序列分析，这些方法既费力又昂贵。由于HEV的新毒株或变异毒株在人类和其他动物中都在迅速进化，因此开发一种快速的测序前筛选方法以选择野外分离株进行进一步的分子特征分析非常重要。在本研究中，我们开发了两种异源双链迁移率分析（HMA）方法（一种基于ORF2区域用于猪源HEV，另一种基于ORF1区域用于禽源HEV），以从已知参考毒株中对禽源和猪源HEV的野外毒株进行基因分型。分别通过PCR扩增了22株猪源HEV分离株的ORF2区域和13株禽源HEV分离株的ORF1区域，进行测序，并分别针对参考原型猪源HEV毒株和参考原型禽源HEV毒株进行HMA分析。我们发现，一般来说，HMA图谱与核苷酸序列同一性以及野外毒株与参考猪源HEV或禽源HEV毒株之间的系统发育聚类密切相关。具有相似HMA模式的野外分离株通常与参考毒株具有相似的序列同一性，并在系统发育树中聚集在一起。因此，通过使用不同的HEV分离株作为参考，本研究中开发的HMA可作为一种测序前筛选工具，用于识别变异的HEV分离株，以进行进一步的分子流行病学研究。
