| 摘要: |
| 【目的】对猪流行性腹泻病毒安徽毒株AH-2018-HF1进行全基因组测序，了解该毒株的分子生物学特征和遗传进化情况，为PEDV的进化和变异研究提供参考。【方法】从PEDV阳性病料（肠道组织）中提取RNA，反转录获得cDNA。将PEDV基因分为9段，设计相应的引物，采用分段重叠法对PEDV基因组进行分段扩增，扩增产物经克隆测序后，采用DNAstar软件拼接，获得全基因组，将此全基因序列与GenBank上公布的国内外25株PEDV毒株进行比对和分析。【结果】PCR扩增获得了4 036，3 724，2 874，3 600，3 950，4 187，3 576，2 953和1 138 bp的9条目标片段，测序拼接后获得了27 937 bp的PEDV全长基因组。AH-2018-HF1株与经典株CV777（疫苗株）全基因序列同源性为97.7%，S基因同源性为96.7%，ORF3基因同源性为90.8%。全基因组序列对比发现，AH-2018-HF1株与SD-M株同源性最高,为99.9%;与PEDV/MEX/QRO/02/2017同源性最低,为96%。S基因序列分析结果显示，AH-2018-HF1株在2 319-2 320位缺失3 bp碱基（GTT）；与CV777、LZC株相比，AH-2018-HF1株在459-460位缺失3 bp碱基（ATA）；和国内外变异毒株相比，AH-2018-HF1株在174-175位缺失12 bp 碱基(CAGGGTGTCAAT)，在461-462位插入6 bp碱基（TGGAAA）。ORF3基因序列分析结果显示，AH-2018-HF1株在245-296位有49个碱基缺失。全基因组系统进化树分析表明，AH-2018-HF1与SD-M亲缘性最近，与CV777亲缘性较近，与国内外变异毒株的亲缘关系较远；S基因系统进化结果与全基因组系统进化结果相似；ORF3基因系统进化分析结果与全基因组系统进化结果不同。【结论】AH-2018-HF1仍属于经典毒株，但存在一定的变异，表明PEDV处于不断的进化中。 |
| 关键词: 猪流行性腹泻病毒 全基因组 序列分析 安徽省 |
| DOI： |
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| 基金项目:合肥市农业行业首席专家工作室（生猪饲养及疫防）项目；现代农业产业技术体系建设专项（CARS-36-生猪）；安徽省生猪产业技术体系项目；农业农村部猪肉质量安全控制重点实验室项目；安徽省自然科学基金项目（1708085MC83） |
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| Whole genome sequencing and analysis of porcine epidemic diarrhea virus AH-2018-HF1 strain |
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SUN Pei,WANG Zhenzhen,LI Liang,et al
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| Abstract: |
| 【Objective】The whole genome sequencing of the Anhui strain AH-2018-HF1 of porcine epidemic diarrhea virus (PEDV) was obtained to understand the molecular biological characteristics and genetic evolution of the strain.【Method】RNA was extracted from PEDV positive disease material (intestinal tissue) and was reversely transcribed to obtain cDNA.PEDV gene was divided into 9 segments,corresponding primers were designed,and the PEDV genome was segmented by segmented overlap method.After cloning and sequencing,the amplified products were spliced using the DNAstar software to obtain the entire genome.The 25 domestic and foreign PEDV strains published on GenBank were then compared and analyzed.【Result】A total of 9 target fragments of 4 036,3 724,2 874,3 600,3 950,4 187,3 576,2 953 and 1 138 bp were obtained by PCR amplification.After sequencing and splicing,a full-length genome of PEDV of 27 937 bp was obtained.The whole gene sequence homology between AH-2018-HF1 strain and CV777 strain was 97.7%,the nucleotide homology of S gene was 96.7%,and the nucleotide homology of ORF3 gene was 90.8%.The highest nucleotide homology between AH-2018-HF1 strain and SD-M strain was 99.9%,and the lowest nucleotide homology with PEDV/MEX/QRO/02/2017 was 96%.S gene sequence analysis results showed that the AH-2018-HF1 strain deleted 3 bp at positions 2 319-2 320 (GTT).Compared with the CV777 and LZC strains,the AH-2018-HF1 strain deleted 3 bp at positions 459-460 (ATA).Compared with the domestic and foreign mutant strains,the AH-2018-HF1 strain deleted 12 bp (CAGGGTGTCAAT) at positions 174-175 and inserted 6 bp (TGGAAA) at positions 461-462.ORF3 gene sequence analysis showed that the AH-2018-HF1 strain had 49 base deletions at positions 245-296.Genome wide phylogenetic tree analysis showed that AH-2018-HF1 was closest to SD-M and closer to CV777,while it was far from domestic and foreign mutant strains.The genome wide system evolution results were similar to the S gene system evolution results,and the ORF3 gene system evolution analysis results were different from the genome wide system evolution results.【Conclusion】AH-2018-HF1 is still a classic strain,but there are certain variations,indicating that PEDV is in continuous evolution. |
| Key words: porcine epidemic diarrhea virus genome sequence analysis Anhui province |
