山东及周边部分地区猪繁殖与呼吸综合征病毒的变异与遗传演化分析
|
摘要
为了解中国山东及周边部分地区自2017年以来猪繁殖与呼吸综合征病毒(PRRSV)流行株的分子流行病学特征、基因组变化规律,作者收集来自山东省、河南省和江苏省的部分猪场采集及送检的疑似PRRS症状猪组织样品70份,利用RT-PCR方法对其进行PRRSV检测。对部分阳性病料中PRRSV的基因重组、决定中国高致病性PRRSV(HP-PRRSV)致病性和复制力的Nsp9第561、586和592位氨基酸等进行分析,以探明该区域PRRSV的遗传演化规律。结果显示,PRRSV检出率为55.7%,从上述样品筛选11株PRRSV分离株。Nsp2序列分析显示,与经典美洲毒株VR-2332相比,7株PRRSV分离株的Nsp2蛋白分别在第481及533-561位发生了30个氨基酸的不连续缺失,这与我国自2006年以来流行的HP-PRRSV的缺失特征相同;2株PRRSV分离株的Nsp2蛋白在481、533-561及595-597位发生了三个部位的共33个不连续氨基酸的缺失;2株PRRSV分离株的Nsp2蛋白在475-518及533-561位出现了两个部位的共73个不连续氨基酸的缺失。该研究中PRRSV分离毒株的Nsp2基因已发生了明显的新型缺失。采用基因重组分析软件RDP4分析显示,以上后4株新型缺失株存在较大的基因重组,且重组部位和重组片段数量不相同;4株病毒均以高致病性毒株JXA1作为重组的主要亲本毒株,多数重组变化集中在Nsp2蛋白区域,在其他非结构蛋白和次要蛋白区域也可见部分重组变化。另外,Nsp9第561、586和592位氨基酸变异分析显示,该4株病毒在上述三个氨基酸位点与中国HP-PRRSV相符。本研究为深入探索PRRSV的遗传变异规律及相关生物学特性研究积累了数据。
In order to investigate the molecular epidemiological characteristics and genomic changes of porcine reproductive and respiratory syndrome virus(PRRSV) epidemic strains from Shandong province and its neighboring areas in China since 2017, in this study, 70 samples of suspected PRRS symptoms were collected from some pig farms in Shandong, Henan and Jiangsu provinces, and PRRSV was detected by RT-PCR. The genetic recombination of virus in PRRSV positive samples, the 561 st, 586 th and 592 nd amino acids of Nsp9 which determine the pathogenicity and replication of Chinese highly pathogenic PRRSV(HP-PRRSV) were analyzed to explore the genetic evolution of PRRSV isolates. The results showed that 11 strains of PRRSV isolates were screened from the above samples, and the detection rate of PRRSV was 55.7%. Nsp2 sequence analysis showed that compared with the classical American strain VR-2332, 30 amino acids were discontinuously deleted at 481 and 533-561 sites in seven isolates, which were identical to the deletion characteristics of HP-PRRSV prevalent in China since 2006. Two PRRSV isolates had total deletions of 33 discontinuous amino acids in three fragments at 481, 533-561 and 595-597, respectively; and two PRRSV isolates showed a total of 73 discontinuous amino acid deletions in two fragments at 475-518 and 533-561, respectively. The Nsp2 genes of PRRSV mutant strains have undergone significant new deletions. The results of gene recombination analysis by the software RDP4 showed that the last four new type of deleted virus strains had large gene recombination, and the number of recombinant sites and recombinant fragments were different. The four strains all used the highly pathogenic strain JXA1 as the main parental strain for the recombinant and most of the recombination changes were concentrated on Nsp2 protein region, and partial recombination changes were also observed in the non-structural and minor protein regions. In addition, the amino acid variation analysis of positions 561, 586 and 592 of Nsp9 showed that the three critical amino acid sites of the four virus isolates were consistent with Chinese HP-PRRSV. This study accumulated data for further exploration of the genetic variation of PRRSV and related biological characteristics.
In order to investigate the molecular epidemiological characteristics and genomic changes of porcine reproductive and respiratory syndrome virus(PRRSV) epidemic strains from Shandong province and its neighboring areas in China since 2017, in this study, 70 samples of suspected PRRS symptoms were collected from some pig farms in Shandong, Henan and Jiangsu provinces, and PRRSV was detected by RT-PCR. The genetic recombination of virus in PRRSV positive samples, the 561 st, 586 th and 592 nd amino acids of Nsp9 which determine the pathogenicity and replication of Chinese highly pathogenic PRRSV(HP-PRRSV) were analyzed to explore the genetic evolution of PRRSV isolates. The results showed that 11 strains of PRRSV isolates were screened from the above samples, and the detection rate of PRRSV was 55.7%. Nsp2 sequence analysis showed that compared with the classical American strain VR-2332, 30 amino acids were discontinuously deleted at 481 and 533-561 sites in seven isolates, which were identical to the deletion characteristics of HP-PRRSV prevalent in China since 2006. Two PRRSV isolates had total deletions of 33 discontinuous amino acids in three fragments at 481, 533-561 and 595-597, respectively; and two PRRSV isolates showed a total of 73 discontinuous amino acid deletions in two fragments at 475-518 and 533-561, respectively. The Nsp2 genes of PRRSV mutant strains have undergone significant new deletions. The results of gene recombination analysis by the software RDP4 showed that the last four new type of deleted virus strains had large gene recombination, and the number of recombinant sites and recombinant fragments were different. The four strains all used the highly pathogenic strain JXA1 as the main parental strain for the recombinant and most of the recombination changes were concentrated on Nsp2 protein region, and partial recombination changes were also observed in the non-structural and minor protein regions. In addition, the amino acid variation analysis of positions 561, 586 and 592 of Nsp9 showed that the three critical amino acid sites of the four virus isolates were consistent with Chinese HP-PRRSV. This study accumulated data for further exploration of the genetic variation of PRRSV and related biological characteristics.
引文
[1] 杨汉春,周磊.猪繁殖与呼吸综合征病毒的遗传变异与演化[J].生命科学,2016,28(3):325-336.YANG H C,ZHOU L.Genetic variation and evolution of porcine reproductive and respiratory syndrome virus[J].Chinese Bulletin of Life Science,2016,28(3):325-336.(in Chinese)
[2] SONG J,SHEN D,CUI J,et al.Accelerated evolution of PRRSV during recent outbreaks in China[J].Virus Genes,2010,41(2):241-245.
[3] 刘杏,王凤雪,温永俊.猪繁殖与呼吸综合征病毒感染和复制机制及其毒力研究进展[J].病毒学报,2015,31(5):585-592.LIU X,WANG F X,WEN Y J.Advances in understanding of the infection/replication mechanisms and virulence determinants of the porcine reproductive and respiratory syndrome virus[J].Chinese Journal of Virology,2015,31(5):585-592.(in Chinese)
[4] TONG G Z,ZHOU Y J,HAO X F,et al.Highly pathogenic porcine reproductive and respiratory syndrome,China[J].Emerg Infect Dis,2007,13(9):1434-1436.
[5] XIE J X,CUI T T,CUI J,et al.Epidemiological and evolutionary characteristics of the PRRSV in Southern China from 2010 to 2013[J].Microb Pathog,2014,75:7-15.
[6] LU W H,SUN B L,MO J Y,et al.Attenuation and immunogenicity of a live high pathogenic PRRSV vaccine candidate with a 32-amino acid deletion in the nsp2 Protein[J].J Immunol Res,2014,2014:810523.
[7] ZHOU L,YANG X R,TIAN Y,et al.Genetic diversity analysis of genotype 2 porcine reproductive and respiratory syndrome viruses emerging in recent years in China[J].BioMed Res Int,2014,2014:748068.
[8] LIU J K,ZHOU X,ZHAI J Q,et al.Genetic diversity and evolutionary characteristics of type 2 porcine reproductive and respiratory syndrome virus in southeastern China from 2009 to 2014[J].Arch Virol,2017,162(9):2603-2615.
[9] 杨小蓉,荫硕焱,潘梦,等.一株高致病性猪繁殖与呼吸综合征病毒变异毒株的基因组特征[J].畜牧兽医学报,2012,43(2):263-269.YANG X R,YIN S Y,PAN M,et al.Genomic characterization of a variant of highly pathogenic porcine reproductive and respiratory syndrome virus[J].Acta Veterinaria et Zootechnica Sinica,2012,43(2):263-269.(in Chinese)
[10] YU L X,WANG X,YU H,et al.The emergence of a highly pathogenic porcine reproductive and respiratory syndrome virus with additional 120aa deletion in Nsp2 region in Jiangxi,China[J].Transbound Emerg Dis,2018,65(6):1740-1748.
[11] XU L,ZHOU L,SUN W F,et al.Nonstructural protein 9 residues 586 and 592 are critical sites in determining the replication efficiency and fatal virulence of the Chinese highly pathogenic porcine reproductive and respiratory syndrome virus[J].Virology,2018,517:135-147.
[12] ZHAO K,GAO J C,XIONG J Y,et al.Two residues in NSP9 contribute to the enhanced replication and pathogenicity of highly pathogenic porcine reproductive and respiratory syndrome virus[J].J Virol,2018,92(7):9-17.
[13] DE LIMA M,PATTNAIK A K,FLORES E F,et al.Serologic marker candidates identified among B-cell linear epitopes of Nsp2 and structural proteins of a North American strain of porcine reproductive and respiratory syndrome virus[J].Virology,2006,353(2):410-421.
[14] YAN Y L,GUO X,GE X N,et al.Monoclonal antibody and porcine antisera recognized B-cell epitopes of Nsp2 protein of a Chinese strain of porcine reproductive and respiratory syndrome virus[J].Virus Res,2007,126(1-2):207-215.
[15] ZHOU L,WANG Z C,DING Y P,et al.NADC30-like strain of porcine reproductive and respiratory syndrome virus,China[J].Emerg Infect Dis,2015,21(12):2256-2257.
[16] ZHOU L,YANG B N,XU L,et al.Efficacy evaluation of three modified-live virus vaccines against a strain of porcine reproductive and respiratory syndrome virus NADC30-like[J].Vet Microbiol,2017,207:108-116.
[17] WANG H M,LIU Y G,TANG Y D,et al.A natural recombinant PRRSV between HP-PRRSV JXA1-like and NADC30-like strains[J].Transbound Emerg Dis,2018,65(4):1078-1086.
[18] SUN Z,WANG J,BAI X F,et al.Pathogenicity comparison between highly pathogenic and NADC30-like porcine reproductive and respiratory syndrome virus[J].Arch Virol,2016,161(8):2257-2261.
[19] GAO G C,XIONG J Y,YE C,et al.Genotypic and geographical distribution of porcine reproductive and respiratory syndrome viruses in mainland China in 1996-2016[J].Vet Microbiol,2017,208:164-172.
[20] SNIJDER E J,VAN TOL H,ROOS N,et al.Non-structural proteins 2 and 3 interact to modify host cell membranes during the formation of the Arterivirus replication complex[J].J Gen Virol,2001,82:985-994.
[21] GRAHAM R L,SIMS A C,BROCKWAY S M,et al.The nsp2 Replicase proteins of murine hepatitis virus and severe acute respiratory syndrome coronavirus are dispensable for viral replication[J].J Virol,2005,79(21):13399-13411.
[22] GAO Z Q,GUO X,YANG H C.Genomic characterization of two Chinese isolates of Porcine respiratory and reproductive syndrome virus[J].Arch Virol,2004,149(7):1341-1351.
[23] LENG C L,TIAN Z J,ZHANG W C,et al.Characterization of two newly emerged isolates of porcine reproductive and respiratory syndrome virus from Northeast China in 2013[J].Vet Microbiol,2014,171(1-2):41-52.
[24] LAGER K M,SCHLINK S N,BROCKMEIER S L,et al.Efficacy of type 2 PRRSV vaccine against Chinese and Vietnamese HP-PRRSV challenge in pigs[J].Vaccine,2014,32(48):6457-6462.
[25] LI B,XIAO S B,WANG Y W,et al.Immunogenicity of the highly pathogenic porcine reproductive and respiratory syndrome virus GP5 protein encoded by a synthetic ORF5 gene[J].Vaccine,2009,27(13):1957-1963.
[26] 刘建奎,魏春华,杨小燕,等.福建地区PRRSV分离株Nsp2基因新变异序列鉴定[J].中国预防兽医学报,2013,35(4):271-275.LIU J K,WEI C H,YANG X Y,et al.Genetic variation of Nsp2 genes of porcine reproductive and respiratory syndrome virus isolated in Fujian province[J].Chinese Journal of Preventive Veterinary Medicine,2013,35(4):271-275.(in Chinese)
[27] WANG L J,WAN B,GUO Z H,et al.Genomic analysis of a recombinant NADC30-like porcine reproductive and respiratory syndrome virus in China[J].Virus Genes,2018,54(1):86-97.
[2] SONG J,SHEN D,CUI J,et al.Accelerated evolution of PRRSV during recent outbreaks in China[J].Virus Genes,2010,41(2):241-245.
[3] 刘杏,王凤雪,温永俊.猪繁殖与呼吸综合征病毒感染和复制机制及其毒力研究进展[J].病毒学报,2015,31(5):585-592.LIU X,WANG F X,WEN Y J.Advances in understanding of the infection/replication mechanisms and virulence determinants of the porcine reproductive and respiratory syndrome virus[J].Chinese Journal of Virology,2015,31(5):585-592.(in Chinese)
[4] TONG G Z,ZHOU Y J,HAO X F,et al.Highly pathogenic porcine reproductive and respiratory syndrome,China[J].Emerg Infect Dis,2007,13(9):1434-1436.
[5] XIE J X,CUI T T,CUI J,et al.Epidemiological and evolutionary characteristics of the PRRSV in Southern China from 2010 to 2013[J].Microb Pathog,2014,75:7-15.
[6] LU W H,SUN B L,MO J Y,et al.Attenuation and immunogenicity of a live high pathogenic PRRSV vaccine candidate with a 32-amino acid deletion in the nsp2 Protein[J].J Immunol Res,2014,2014:810523.
[7] ZHOU L,YANG X R,TIAN Y,et al.Genetic diversity analysis of genotype 2 porcine reproductive and respiratory syndrome viruses emerging in recent years in China[J].BioMed Res Int,2014,2014:748068.
[8] LIU J K,ZHOU X,ZHAI J Q,et al.Genetic diversity and evolutionary characteristics of type 2 porcine reproductive and respiratory syndrome virus in southeastern China from 2009 to 2014[J].Arch Virol,2017,162(9):2603-2615.
[9] 杨小蓉,荫硕焱,潘梦,等.一株高致病性猪繁殖与呼吸综合征病毒变异毒株的基因组特征[J].畜牧兽医学报,2012,43(2):263-269.YANG X R,YIN S Y,PAN M,et al.Genomic characterization of a variant of highly pathogenic porcine reproductive and respiratory syndrome virus[J].Acta Veterinaria et Zootechnica Sinica,2012,43(2):263-269.(in Chinese)
[10] YU L X,WANG X,YU H,et al.The emergence of a highly pathogenic porcine reproductive and respiratory syndrome virus with additional 120aa deletion in Nsp2 region in Jiangxi,China[J].Transbound Emerg Dis,2018,65(6):1740-1748.
[11] XU L,ZHOU L,SUN W F,et al.Nonstructural protein 9 residues 586 and 592 are critical sites in determining the replication efficiency and fatal virulence of the Chinese highly pathogenic porcine reproductive and respiratory syndrome virus[J].Virology,2018,517:135-147.
[12] ZHAO K,GAO J C,XIONG J Y,et al.Two residues in NSP9 contribute to the enhanced replication and pathogenicity of highly pathogenic porcine reproductive and respiratory syndrome virus[J].J Virol,2018,92(7):9-17.
[13] DE LIMA M,PATTNAIK A K,FLORES E F,et al.Serologic marker candidates identified among B-cell linear epitopes of Nsp2 and structural proteins of a North American strain of porcine reproductive and respiratory syndrome virus[J].Virology,2006,353(2):410-421.
[14] YAN Y L,GUO X,GE X N,et al.Monoclonal antibody and porcine antisera recognized B-cell epitopes of Nsp2 protein of a Chinese strain of porcine reproductive and respiratory syndrome virus[J].Virus Res,2007,126(1-2):207-215.
[15] ZHOU L,WANG Z C,DING Y P,et al.NADC30-like strain of porcine reproductive and respiratory syndrome virus,China[J].Emerg Infect Dis,2015,21(12):2256-2257.
[16] ZHOU L,YANG B N,XU L,et al.Efficacy evaluation of three modified-live virus vaccines against a strain of porcine reproductive and respiratory syndrome virus NADC30-like[J].Vet Microbiol,2017,207:108-116.
[17] WANG H M,LIU Y G,TANG Y D,et al.A natural recombinant PRRSV between HP-PRRSV JXA1-like and NADC30-like strains[J].Transbound Emerg Dis,2018,65(4):1078-1086.
[18] SUN Z,WANG J,BAI X F,et al.Pathogenicity comparison between highly pathogenic and NADC30-like porcine reproductive and respiratory syndrome virus[J].Arch Virol,2016,161(8):2257-2261.
[19] GAO G C,XIONG J Y,YE C,et al.Genotypic and geographical distribution of porcine reproductive and respiratory syndrome viruses in mainland China in 1996-2016[J].Vet Microbiol,2017,208:164-172.
[20] SNIJDER E J,VAN TOL H,ROOS N,et al.Non-structural proteins 2 and 3 interact to modify host cell membranes during the formation of the Arterivirus replication complex[J].J Gen Virol,2001,82:985-994.
[21] GRAHAM R L,SIMS A C,BROCKWAY S M,et al.The nsp2 Replicase proteins of murine hepatitis virus and severe acute respiratory syndrome coronavirus are dispensable for viral replication[J].J Virol,2005,79(21):13399-13411.
[22] GAO Z Q,GUO X,YANG H C.Genomic characterization of two Chinese isolates of Porcine respiratory and reproductive syndrome virus[J].Arch Virol,2004,149(7):1341-1351.
[23] LENG C L,TIAN Z J,ZHANG W C,et al.Characterization of two newly emerged isolates of porcine reproductive and respiratory syndrome virus from Northeast China in 2013[J].Vet Microbiol,2014,171(1-2):41-52.
[24] LAGER K M,SCHLINK S N,BROCKMEIER S L,et al.Efficacy of type 2 PRRSV vaccine against Chinese and Vietnamese HP-PRRSV challenge in pigs[J].Vaccine,2014,32(48):6457-6462.
[25] LI B,XIAO S B,WANG Y W,et al.Immunogenicity of the highly pathogenic porcine reproductive and respiratory syndrome virus GP5 protein encoded by a synthetic ORF5 gene[J].Vaccine,2009,27(13):1957-1963.
[26] 刘建奎,魏春华,杨小燕,等.福建地区PRRSV分离株Nsp2基因新变异序列鉴定[J].中国预防兽医学报,2013,35(4):271-275.LIU J K,WEI C H,YANG X Y,et al.Genetic variation of Nsp2 genes of porcine reproductive and respiratory syndrome virus isolated in Fujian province[J].Chinese Journal of Preventive Veterinary Medicine,2013,35(4):271-275.(in Chinese)
[27] WANG L J,WAN B,GUO Z H,et al.Genomic analysis of a recombinant NADC30-like porcine reproductive and respiratory syndrome virus in China[J].Virus Genes,2018,54(1):86-97.