一株G5P[13]型猪轮状病毒的全基因组序列分析

Whole genome sequence analysis of porcine rotavirus strain G5P13

为了解引发贵州省某猪场腹泻疫情的猪轮状病毒(PoRV)基因组特征和遗传变异规律,本研究将鉴定为PoRV阳性的腹泻病死仔猪的肠内容物样品处理后接种Vero细胞,传代后经RT-PCR鉴定并测序,结果显示PoRV属于G5P[13]型。通过透射电镜和间接免疫荧光试验鉴定,结果显示:能观察到直径约70 nm的无囊膜的病毒粒子,接种病毒的Vero细胞有特异性的绿色荧光,表明分离获得一株PoRV,命名为GZAS2020株。根据PoRV国内外参考株序列设计11对兼并引物,经RT-PCR扩增该病毒11个节段并测序后拼接,结果显示:该病毒基因组全长18 514 bp。对GZAS2020株全基因组序列同源性分析,对其VP4、VP7氨基酸差异性及基因重组情况分析。结果显示:GZAS2020株11个节段分别与猪、人、熊猫源轮状病毒的同源性最高且遗传进化树处于同一分支,且VP7、VP4、VP6、VP1~VP3、NSP1~NSP5基因与国内外各基因型轮状病毒的同源性分别高达97.2%、95.6%、98.7%、96.3%、94.5%、96.1%、97.6%、96.5%、96.6%、98.3%、98.7%,该株病毒全基因组基因型为G5-P[13]-I5-R1-C1-M1-A8-N1-T1-E1-H1。GZAS2020株VP4存在23个氨基酸位点的点突变(S^(28)L、H^(42)Q、T^(133)A、A^(185)V、N^(194)S、S^(197)N、E^(200)K、I^(209)V、S^(237)P、H^(248)Q、R^(282)Q、T^(294)A、S^(361)T、Q^(362)P、I^(381)V、M^(393)V、N^(447)S、V^(455)A、T^(460)A、M^(466)I、V^(601)A、S^(655)A、S^(681)N),VP7存在20个氨基酸位点的点突变(C^(44)R、Y^(68)H、F^(86)L、I^(105)V、S^(110)P、D^(116)Y、V^(137)I、R^(154)G、T^(158)I、M^(168)V、F^(175)V、C^(203)Y、S^(221)G、R^(232)C、P^(238)S、C^(262)S、S^(266)R、S^(278)P、T^(282)A、M^(295)V)。重组分析结果显示,GZAS2020株VP4基因和NSP4基因存在重组事件,是由KT727244.1株和MK227934.1株的VP4基因、MK228074.1株和AF144797.1株的NSP4基因重组形成的重组病毒。综上所述,GZAS2020株是由猪源病毒株重组而来,存在跨种间传播,且有传播给人的风险,应引起高度重视。本研究在贵州首次分离到G5P[13]型PoRV,对其遗传演化特征分析为PoRV感染的防控提供参考依据。

To understand the genomic characteristics and genetic variation of porcine rotavirus(PoRV) that caused diarrhea in a pig farm in Guizhou province. In this study, the intestinal contents of piglets identified as PoRV-positive with diarrhea were inoculated into Vero cells. The PoRV strain was identified and sequenced by RT-PCR, and the results showed that the isolated PoRV belonged to the G5P[13] type. Through transmission electron microscopy and indirect immunofluorescence identification,the results showed that the non-envelope virus particles with a shape of about 70nm can be observed, and the Vero cells inoculated with the virus have specific green fluorescence, indicating that a strain of Po RV was isolated and named as GZAS2020 strain.Eleven pairs of primers were designed according to the sequence of Po RV reference strains at home and abroad, and 11 segments of the virus were amplified by RT-PCR and sequenced. The results showed that the full length of the virus genome was 18514 bp. In this study, DNAStar and MEGA 5.0 were used to analyze the nucleotide homology and amino acid difference of VP4 and VP7 in the whole genome of GZAS2020 strain;REDP4 and Simplot were used for gene recombination analysis. The results showed that the 11 segments of GZAS2020 had the highest homology to rotaviruses of pig, human and panda origin and were in the same branch of the genetic evolutionary tree. The homology of domestic and foreign strains is as high as 97.2%, 95.6%, 98.7%, 96.3%,94.5%, 96.1%, 97.6%, 96.5%, 96.6%, 98.3%, 98.7%, and the whole genome genotype is G5-P[13]-I5-R1-C1-M1-A8-N1-T1-E1-H1. There are 23 amino acid point mutations in VP4 of GZAS2020 strain(S^(28)L,H^(42)Q,T^(133)A,A^(185)V,N^(194)S,S^(197)N,E^(200)K,I^(209)V,S^(237)P,H^(248)Q,R^(282)Q,T^(294)A,S^(361)T,Q^(362)P,I^(381)V,M^(393)V,N^(447)S,V^(455)A,T^(460)A,M^(466)I,V^(601)A,S^(655)A,S^(681)N), there are 20 amino acid point mutations in VP7(C^(44)R,Y^(68)H,F^(86)L,I^(105)V,S^(110)P,D^(116)Y,V^(137)I,R^(154)G,T^(158)I,M^(168)V,F^(175)V,C^(203)Y,S^(221)G,R^(232)C,P^(238)S,C^(262)S,S^(266)R,S^(278)P,T^(282)A,M^(295)V). The recombination analysis showed that there was a recombination event between the VP4 gene and the NSP4 gene of the GZAS2020strain, which was a recombinant virus formed by the VP4 gene of the KT727244.1 strain, the VP4 gene of the MK227934.1 strain,the NSP4 gene of the MK228074.1 strain and the NSP4 gene of the AF144797.1 strain. To sum up, the GZAS2020 strain is recombined from the pig-derived parental strain, and there is potential cross-species transmission, and it holds a risk of transmission to humans. This study deeply analyzed the genetic evolution characteristics of the G5P[13] type Po RV that was isolated for the first time in Guizhou province, and provided a reference for Po RV to prevent and control infection.