摘要
2017年,NADC34-like猪繁殖与呼吸综合征病毒(PRRSV)自北美传入我国后,迄今未见该类病毒与我国本土HP-PRRSV发生重组的报道。为监测该类病毒在四川地区的流行变异情况,本研究从该地区某疑似感染PRRSV发病猪场采集肺脏样品,经ORF5基因分型RT-PCR检测并测序结果显示,该猪场为NADC34-like PRRSV感染。将阳性样品接种至猪肺泡巨噬细胞(PAMs)进行病毒分离,并通过RT-PCR扩增、测序及间接免疫荧光试验(IFA)鉴定结果显示,分离到一株NADC34-like PRRSV,并命名为SCcd2020株。经PCR分段扩增病毒全基因组,测序、拼接后的结果显示,病毒基因组全长15 020 bp。为进一步分析SCcd2020株的遗传演化特征,利用MegAlign分析SCcd2020株全基因组及ORF5基因序列与GenBank中12株PRRSV参考株相应基因的核苷酸及其编码氨基酸序列的相似性;采用MEGA X软件利用NJ法构建SCcd2020株的全基因组及ORF5基因的系统发育树;采用MegAlign分析SCcd2020株Nsp2氨基酸序列的缺失特征;分别利用RDP4、SimPlot软件对SCcd2020株进行重组分析。相似性分析结果显示,SCcd2020株与12株代表PRRSV ORF5基因序列及其编码氨基酸序列的相似性分别为84.4%~97.0%与84.6%~97.0%,其中与IA/2014/NADC34株相似性最高为97%;SCcd2020株与12株PRRSV代表株全基因组序列的相似性为79.8%~89.2%,其中与NADC30株相似性最高为89.2%;ORF5基因进化树结果显示,SCcd2020株与NADC34-like PRRSV聚为一大支;而全基因组进化树结果显示,SCcd2020株与NADC30-like PRRSV聚为一大支。Nsp2氨基酸序列分析结果显示,SCcd2020株该区域存在“111+1+19aa”缺失模式,而NADC34-like PRRSV为“100 aa”缺失模式;上述结果表明,SCcd2020株基因组可能发生了重组。重组分析结果结果显示,SCcd2020株是由NADC30-like PRRSV、HP-PRRSV以及NADC34-like PRRSV之间形成的重组病毒。综上所述,SCcd2020株为国内首次报道与我国本土HP-PRRSV发生复杂重组的NADC34-like PRRSV,表明我国PRRSV遗传多样性进一步增加,应高度重视。本研究结果有助于深入了解我国NADC34-like PRRSV的遗传演化特征,为科学防控该类病毒的感染提供参考依据与数据支持。
In 2017 NADC34-like PRRSV emerged in China from North America. So far, there has been no report on recombination of the NADC34-like strain with high-pathogenic PRRSV(HP-PRRSV) strain. In order to monitor the epidemic variation of NADC34-like virus in Sichuan province, the lung samples collected from a pig farm suspected to be PRRSV-positive were detected by RT-PCR with ORF5 genotyping primers and sequenced. The results showed that a NADC34-like PRRSV strain was identified in this pig farm. The positive samples were inoculated into porcine alveolar macrophages(PAMs) for virus isolation, RT-PCR,sequencing and indirect immunofluorescence assay(IFA) were then carried out. The result showed that a NADC34-like PRRSV strain was successfully isolated, named as SCcd2020. The full length genome of the virus was 15020bp. In order to further analyze the genetic evolution characteristics of SCcd2020 strain, MegAlign software was used to analyze the nucleotide(nt) and amino acid(aa) similarity of both whole genome and ORF5 gene among SCcd2020 strain and 12 representative PRRSV strains in GenBank;MegAlign was also used to analyze the deletion characteristics of Nsp2 amino acid sequence of SCcd2020 strain. RDP4 and SimPlot software were used to recombine and analyze the isolated strain SCcd2020. The results showed that the nucleotide similarity of ORF5 gene among SCcd2020 strain and 12 representative PRRSV was 84.4%-97.0%, and the similarity of deduced amino acid was 84.6%-97.0%, among which the highest similarity was with IA/2014/NADC34 strain(97%);The nucleotide similarity among the whole genome and 12 representative PRRSV strains was 79.8%-89.2%, among which the highest similarity was with IA/2014/NADC34 strain(97%);The results of ORF5 gene phylogenetic tree showed that SCcd2020 strain and NADC34-like PRRSV clustered into one branch;The whole genome evolutionary tree showed that SCcd2020 strain and NADC30-like PRRSV clustered into one branch. The deduced Nsp2 amino acid results showed that there was "111 + 1 + 19aa" deletion mode in Nsp2 region of SCcd2020 strain, while the other NADC34-like PRRSV was "100aa" deletion mode. These results suggest that there may be recombination events in the genome of SCcd2020 strain. The recombination results suggest that SCcd2020 strain may be a recombinant virus emerged from NADC30-like PRRSV, HP-PRRSV and NADC34-like PRRSV strains. SCcd2020 strain is also the first report of NADC34-like PRRSV with complex recombination with HP-PRRSV in China, indicating that the genetic diversity of PRRSV in China has been increased and more attention should be paid to the current situation. This study will contribute to a deeper understanding of the genetic evolution characteristics of NADC34-like PRRSV in China, and provide theoretical basis and data support for scientific prevention and control of such strains infection.
作者
隆耀平
周军
肖璐
于吉峰
张斌
周泷
LONG Yao-ping;ZHOU Jun;XIAO Lu;YU Ji-feng;ZHANG Bin;ZHOU Long(College of Animal&Veterinary Sciences,Southwest Minzu University,Chengdu 610041,China;Sichuan Boce Testing Technology Co.,Ltd.,Chengdu 610023,China;Sichuan Animal Science Academy,Chengdu 610066,China)
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2022年第5期544-549,554,共7页
Chinese Journal of Preventive Veterinary Medicine
基金
西南民族大学中央高校基本科研业务费专项资金项目资助(2021NYYXS87)
四川应用基础研究计划(2020YJ0247)
四川省省级科研院所基本科研业务费(SASA20201)。
作者简介
隆耀平(1998-),男,重庆石柱人,硕士研究生,主要从事动物病原学研究;通信作者:周泷,E-mail:zhoulongscu@163.com。
