2021—2022年湖南省PRRSV流行病学调查及基因组特征分析
Epidemiological survey and genomic characteristics of PRRSV in Hunan during 2021-2022
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摘要: 【目的】了解当前湖南省猪繁殖与呼吸综合征病毒 (PRRSV) 流行特点及主要流行基因型的占比情况, 并明确其遗传进化关系及重组特征, 为湖南省PRRS的科学防控提供参考依据。【方法】 2021年3月—2022年10月, 从湖南省14个市 (州) 396个疑似暴发PRRS的猪场采集1184份病料样品, 采用荧光定量PCR检测PRRSV阳性率, 统计不同季节和不同地区的感染情况; 利用RT-PCR扩增部分阳性病料样品ORF5序列后进行序列相似性及遗传进化分析; 将部分病料接种Marc-145细胞进行病毒分离, 采用二代测序技术完成全基因组测序后进行遗传进化分析, 并以SimPlot分析是否发生组重组事件。【结果】共有167份病料样品检测结果呈PRRSV阳性(检出率14.11%), 湖南省不同地区和不同季节的PRRSV样品阳性检出率分别为0~46.43%和6.95%~19.67%; 高致病性PRRSV(HP-PRRSV)株、经典株和类NADC30株的占比分别为43.11%、15.57%和41.32%。共扩增获得21株PRRSV的完整ORF5序列 (600~603 bp), 其中,7株与HP-PRRSV参考株的GP5氨基酸序列相似性较高(92.1%~98.9%), 13株与类NADC30株的相似性较高(91.3%~93.4%); 1株与PRRSV经典株的相似性较高(98.6%~99.1%)。不同PRRSV的GP5氨基酸序列变异程度高, 其中2株类NADC30株GP5氨基酸序列高变区存在氨基酸位点缺失, 且部分病毒株氨基酸变异区域位于GP5氨基酸序列的细胞表位区域。共分离获得2株PRRSV(HuN-A和HuN-B), 其中, HuN-A株属于类NADC30株, 且以类NADC30株为主要亲本株, 以HP-PRRSV株为次要亲本株; HuN-B株属于类HP-PRRSV株。【结论】湖南省PRRSV流行情况较严重, 且基因型繁多, 其中类NADC30株和HP-PRRSV株属于主要流行基因型。分离获得的2株PRRSV中, HuN-A株是类NADC30株与HP-PRRSV株 (JXA1) 发生重组变异而来, HuN-B株可能是JXA1弱毒疫苗株出现毒力返祖而产生。Abstract: 【Objective】 To investigate the epidemiological characteristics and genotype proportion of porcine reproductive and respiratory syndrome virus(PRRSV)in Hunan,which would provide reference for the prevention and control of PRRS in Hunan.【Method】 1184 tissue samples of pigs suspected of PRRSV infection were collected from 396 pig farms in Hunan from 2021,March to 2022,October,and the detected positive rates of different genotypes were determined by fluorescence quantitative PCR,and the PRRSV infection rates in different regions and seasons were counted. The ORF5 sequences of some positive samples were amplified by RT-PCR,and their sequence homology and genetic evolution were analyzed. Part of the disease samples were inoculated with Marc-145 cells for virus isolation. After the whole gene sequence was sequenced by second-generation sequencing technology,genetic evolution analysis was performed,and the occurrence of group recombination events was analyzed by SimPlot.【Result】167 tissue samples(detected positive rate 14.11%)were PRRSV-positive,the positive rate of PRRSV in different regions and seasons in Hunan were 0-46.43% and 6.95%-19.67% respectively. Moreover,the proportions of highly pathogenic PRRSV(HP-PRRSV),classical strains and NADC30-like strains were 43.11%,15.57%,and 41.32% respectively. Complete ORF5 gene sequences(600-603 bp)of 21 strains of PRRSV were amplified in this study,the homology analysis showed that 7 strains had the highest similarity (92.1%-98.9%)with the HP-PRRSV reference strains;13 strains showed the highest amino acid sequence similarity (91.3%-93.4%)with NADC30-like strains;only one strain were genetically identified with the PRRSV classical strains, with the sequence similarity of 98.6%-99.1%. The GP5 amino acid sequence of different PRRSV was highly variable,and two NADC30-like strains had deletion of amino acid sites in the GP5 amino acid sequence highly variable region,and the amino acid variation regions of some PRRSV strains were mainly located in the GP5 cell epitope region. Two PRRSV strains(named HuN-A and HuN-B)were isolated. HuN-A strain belonged to the NADC30-like strain,with NADC30 strain as the main parent and HP-PRRSV strain as the secondary parent. However,the HuN-B strain belonged to HPPRRSV strain.【Conclusion】The prevalence of PRRSV in Hunan was serious and there are many genotypes,among which NADC30 strains and HP-PRRSV strains are the main genotypes. Two PRRSV strains are successfully isolated,the HuN-A strain is recombinant from the NADC30-like strain and the HP-PRRSV like strain(JXA1),while HuN-B strain may be produced by the atavism of JXA1 attenuated vaccine strain.
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