猪繁殖与呼吸综合征商品活疫苗中盖他病毒的分离与鉴定
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摘要
在疫苗生产过程中,外源病毒造成疫苗污染的现象屡有发生。本文在对某猪场的猪繁殖与呼吸综合征(Porcine reproductive and respiratory syndrome, PRRS)减毒活疫苗进行外源病毒检测时,发现盖他病毒(Getah virus,GETV)为阳性。经病毒分离、纯化及鉴定,获得一株GETV分离物,将其命名为GETV-V1。GETV-V1与GenBank中GETV全基因序列相似性为97.3%～99.2%。遗传进化分析显示,GETV-V1与日本毒株14-I-605-C1、12IH26及中国毒株HB0234等处于同一进化分支,与本室分离的猪源GETV毒株HNJZ-S1的全基因序列(KY363862)相似性为99.1%,两者E2基因推导氨基酸序列相似性为99.8%。对同一厂家4批次市售同种疫苗和相应批次留样疫苗RT-PCR检测均为GETV阳性,第三方实验室和疫苗生产厂家分别独立进行的RT-PCR检测得出同样结果。测序结果表明,所有被测样品中E2基因与GETV已知相应序列的相似性均在98%以上。本研究首次报道了我国PRRS减毒活疫苗中GETV的污染,不仅表明猪用活疫苗外源病毒监测标准有待改进,而且提示要重视该病毒污染在疫苗生产、保存和使用过程中潜在的公共卫生风险。
Contamination occurs frequently during the development and production of vaccines. We detected exogenous viruses of a live attenuated porcine reproductive and respiratory syndrome virus(PRRSV) vaccine from a pig farm using reverse transcription-polymerase chain reaction(RT-PCR) and found that the vaccine was contaminated by the Getah virus(GETV). The virus was isolated, passaged,purified by plaques and named "GETV-V1". According to complete-genome analysis, the identity of the sequence between GETV-V1 and reference sequence(GenBank) was 97.3%～99.2%, and the identity was the highest with the Korean pig strain AY702913(Accession number: AY702913). Genetic-evolution analysis showed that GETV-V1 was closely related to the Japanese strains 14-I-605-C1, 14-I-605-C2 and 12 IH26, and Chinese strain HB0234, and was in the same evolutionary branch. The identity of the whole gene sequence between the porcine GETV strain HNJZ-S1(Accession number: KY363862, previously isolated by our lab) and GETV-V1 was 99.1%, while E2-gene deduced amino-acid sequence analysis showed that the identity was 99.8%. The E2 gene of the GETV was detected by RT-PCR in four batches of the vaccine derived from the same manufacturer. All specimens of different batches of the same vaccine derived from the manufacturer above were GETV-positive as well when tested by using RT-PCR conducted by a third-party laboratory and vaccine manufacturer's lab. Sequencing of E2 gene amplified by RT-PCR showed that the identity of sequences between the strains detected in present study and published strains of GETV was >98%. In this study, the contamination of a live attenuated PRRS vaccine with GETV was reported for the first time in China and the results indicated that the monitoring method for exogenous viruses of live vaccines for pigs needs to be improved and people should pay more attention to the effect of live vaccine contaminated with GETV on the potential public health risk.
Contamination occurs frequently during the development and production of vaccines. We detected exogenous viruses of a live attenuated porcine reproductive and respiratory syndrome virus(PRRSV) vaccine from a pig farm using reverse transcription-polymerase chain reaction(RT-PCR) and found that the vaccine was contaminated by the Getah virus(GETV). The virus was isolated, passaged,purified by plaques and named "GETV-V1". According to complete-genome analysis, the identity of the sequence between GETV-V1 and reference sequence(GenBank) was 97.3%～99.2%, and the identity was the highest with the Korean pig strain AY702913(Accession number: AY702913). Genetic-evolution analysis showed that GETV-V1 was closely related to the Japanese strains 14-I-605-C1, 14-I-605-C2 and 12 IH26, and Chinese strain HB0234, and was in the same evolutionary branch. The identity of the whole gene sequence between the porcine GETV strain HNJZ-S1(Accession number: KY363862, previously isolated by our lab) and GETV-V1 was 99.1%, while E2-gene deduced amino-acid sequence analysis showed that the identity was 99.8%. The E2 gene of the GETV was detected by RT-PCR in four batches of the vaccine derived from the same manufacturer. All specimens of different batches of the same vaccine derived from the manufacturer above were GETV-positive as well when tested by using RT-PCR conducted by a third-party laboratory and vaccine manufacturer's lab. Sequencing of E2 gene amplified by RT-PCR showed that the identity of sequences between the strains detected in present study and published strains of GETV was >98%. In this study, the contamination of a live attenuated PRRS vaccine with GETV was reported for the first time in China and the results indicated that the monitoring method for exogenous viruses of live vaccines for pigs needs to be improved and people should pay more attention to the effect of live vaccine contaminated with GETV on the potential public health risk.
引文
[1]Falcone E,Tollis M,Conti G.Bovine viral diarrhea dis-ease associated with a contaminated vaccine[J].Vac-cine,1999,18(5-6):387.
[2]V?gtlin A,Bruckner L,Ottiger H P.Use of polymerase chain reaction(PCR)for the detection of vaccine con-tamination by infectious laryngotracheitis virus[J].Vac-cine,1999,17(20):2501-2506.
[3]Shah K V.SV40 and human cancer:a review of recent data[J].Int J Cancer,2007,120(2):215-223.
[4]Kulcsar G,Farsang A,Soos T.Testing for viral con-taminants of veterinary vaccines in Hungary[J].Biologi-cals,2010,38(3):346-349.
[5]高金源,陈晓春,韩玉玲,吴华伟,邓永,郎洪武.猪瘟活疫苗(脾淋源)污染外源性兔出血症病毒的检测与启示[J].中国兽药杂志,2011,45(12):16-18.
[6]Zhao P,Dong X,Cui Z.Isolation,identification,and gp85 characterization of a subgroup A avian leukosis vi-rus from a contaminated live Newcastle Disease virus vaccine,first report in China[J].Poultry Sci,2014,93(9):2168-2174.
[7]Berge,T.O.International catalogue of arboviruses,in-cluding certain other viruses of vertebrates.In:DHEWPublication No(CDC)78-8301[M].US Department of Health,Education and Welfare,Public Health Service,Washington DC.1975,p 278.
[8]Xue-Dong L,Fu-Xi Q,Yang Huo R Y N,Charles HcALISHER.Isolation of Getah virus from mosquitos col-lected on Hainan Island,China,and results of a serosur-vey[J].Asian Pac J Trop Med,1992,23(4):730-734.
[9]Kurogi H,Inaba Y,Goto Y,Miura Y,Takahashi,H,Sato K,Matumoto,M.Serologic evidence for etiologic role of Akabane virus in epizootic abortion-arthrogryposis-hydranencephaly in cattle in Japan,1972-1974[J].Arch Virol,1975,47(1),71-83.
[10]Doherty R L.Arboviruses of Australia[J].Aust Vet J,1972,48(4),172-180.
[11]Tajima S,Kotaki A,Yagasaki K,Taniwaki T,Moi ML,Nakayama E,Saijo M,Kurane I,Takasaki T.Identi-fication and amplification of Japanese encephalitis virus and Getah virus propagated from a single porcine serum sample:a case of coinfection[J].Arch Virol,2014,159(11):2969-2975.
[12]矢後啓司,萩原茂,川村齊.A fatal case in newborn piglets with Getah virus infection:Isolation of the virus[J].日本獸醫學雜誌(The Japanese Journal of Veteri-nary Science),1987,49(6):989-994.
[13]Fukunaga Y,Kumanomido T,Kamada M.Getah virus as an equine pathogen[J].Vet Clin North Am Equine Pract,2000,16(3):605-617.
[14]张雨,司炳银,常国辉,刘伯华,杨银辉,祝庆余.WJBC株盖塔病毒基因组序列测定及与其他甲病毒的系统进化关系[J].军事医学,2011,35(12):892-895.
[15]中华人民共和国卫生部.卫生部关于印发《人间传染的病原微生物名录》的通知[N/OL].2006-01-27[2018-10-19],http://www.nhfpc.gov.cn/qjjys/s3589/200804/f0840e5958ea40c68ba0cfda9cc138ac.sh-tml
[16]杨火,饶颐年,陈日光,胡定金,李雪东,崔五全,曲小肃,陈德慧,刘景兰,杨佩英,丁建华.海南岛一株甲组虫媒病毒的分离,鉴定和血清抗体调查[J].中华微生物学和免疫学杂志,1984,4(2):107.
[17]冯学平,译.盖他病毒简介及日本盖他病毒病的发生情况[J].中国动物检疫,1990(6):38-39.
[18]Shortridge K F,Mason D K,Watkins K L,Aaskov JG.Serological evidence for the transmission of Getah vi-rus in Hong Kong[J].Vet Rec,1994,134(20):527-528.
[19]白志军,彭翼飞,林立辉,田小东,陈翠华,方美玉,徐春华,蒋廉华,陈唯军.广东、海南两省人血清12种虫媒病毒抗体调查[J].中国人兽共患病杂志,2000,16(1):83-84.
[20]梁勇,张连山,刘永为,董运强,王志强,王峻巍,梁国栋.河北省涉县正常人群盖塔病毒感染状况.河北医药,2010,21(32):3079-3080.
[21]Izumida A,Takuma H,Inagaki S,Kubota M,Hirahara T,Kodama K,Sasaki N.Experimental infection of Ge-tah virus in swine[J].Nihon Juigaku Zasshi,1988,50(3):679-684.
[22]Brown C M,Timoney P J.Getah virus infection of Indi-an horses[J].Trop Anim Health Prot,1998,30(4):241-252.
[23]出水田昭弘,安原寿雄,久保田道雄,吉木研一,平原正,儿玉和夫,佐佐木文存.Establishment and biologi-cal properties of an attenuated strain of getah virus[J].JJapan Vet Med Association(Japan),1991,44:197-201.
[24]周峰,崔丹丹,王傲杰,王新港,常洪涛,陈陆,李永涛,王川庆.我国首株猪盖他病毒的分离与鉴定[J].病毒学报,2018,34(1):59-66.DOI:10.13242/j.cnki.bing-duxuebao.003299
[25]Weaver S C,Reisen W K.Present and future arboviral threats[J].Antiviral Res,2010,85(2):328-345.
[26]梁国栋.虫媒病毒是我国亟待加强的研究领域[J].中华实验和临床病毒学杂志,2005,19(4):305-306.
[27]王仁宏,曹昆.习近平对吉林长春长生生物疫苗案件作出重要指示强调要一查到底严肃问责始终把人民群众的身体健康放在首位坚决守住安全底线[N/OL].人民网,2018-07-23[2018-10-19],http://politics.peo-ple.com.cn/n1/2018/0723/c1024-30165057.html.
[28]贺斌.退市新规出台后,长生生物或将成为第一个被强制退市的上市公司[N/OL].中国新闻周刊,2018-12-2[2018-10-19],http://www.inewsweek.cn/finance/2018-12-02/4483.shtml
[29]Gerber P.An infectious deoxyribonucleic acid derived from vacuolating virus(SV40)[J].Virology,1962,16(1):96-98.
[30]Joseph G.Victoria,Chunlin Wang,Morris S.Jones,Crystal Jaing,Kevin McLoughlin,Shea Gardner,Eric L.Delwart.Viral nucleic acids in live-attenuated vac-cines:detection of minority variants and an adventitious virus[J].J Virol,2010,84(12):6033-6040.
[31]Gregersen J P,Schmitt H J,Trusheim H,Michael Br?ker.Safety of MDCK cell culture-based influenza vaccines[J].Future Microbiol,2011,6(2):143-152.
[32]Palomares R A,Marley S M,Givens M D,Gallardo RA,Brock K V.Bovine viral diarrhea virus fetal persis-tent infection after immunization with a contaminated modified-live virus vaccine[J].Theriogenology,2013,79(8):1184-1195.
[33]崔丹丹.不同PRRSV毒株的分离鉴定,序列及致病性分析[D].河南农业大学,2017.
[2]V?gtlin A,Bruckner L,Ottiger H P.Use of polymerase chain reaction(PCR)for the detection of vaccine con-tamination by infectious laryngotracheitis virus[J].Vac-cine,1999,17(20):2501-2506.
[3]Shah K V.SV40 and human cancer:a review of recent data[J].Int J Cancer,2007,120(2):215-223.
[4]Kulcsar G,Farsang A,Soos T.Testing for viral con-taminants of veterinary vaccines in Hungary[J].Biologi-cals,2010,38(3):346-349.
[5]高金源,陈晓春,韩玉玲,吴华伟,邓永,郎洪武.猪瘟活疫苗(脾淋源)污染外源性兔出血症病毒的检测与启示[J].中国兽药杂志,2011,45(12):16-18.
[6]Zhao P,Dong X,Cui Z.Isolation,identification,and gp85 characterization of a subgroup A avian leukosis vi-rus from a contaminated live Newcastle Disease virus vaccine,first report in China[J].Poultry Sci,2014,93(9):2168-2174.
[7]Berge,T.O.International catalogue of arboviruses,in-cluding certain other viruses of vertebrates.In:DHEWPublication No(CDC)78-8301[M].US Department of Health,Education and Welfare,Public Health Service,Washington DC.1975,p 278.
[8]Xue-Dong L,Fu-Xi Q,Yang Huo R Y N,Charles HcALISHER.Isolation of Getah virus from mosquitos col-lected on Hainan Island,China,and results of a serosur-vey[J].Asian Pac J Trop Med,1992,23(4):730-734.
[9]Kurogi H,Inaba Y,Goto Y,Miura Y,Takahashi,H,Sato K,Matumoto,M.Serologic evidence for etiologic role of Akabane virus in epizootic abortion-arthrogryposis-hydranencephaly in cattle in Japan,1972-1974[J].Arch Virol,1975,47(1),71-83.
[10]Doherty R L.Arboviruses of Australia[J].Aust Vet J,1972,48(4),172-180.
[11]Tajima S,Kotaki A,Yagasaki K,Taniwaki T,Moi ML,Nakayama E,Saijo M,Kurane I,Takasaki T.Identi-fication and amplification of Japanese encephalitis virus and Getah virus propagated from a single porcine serum sample:a case of coinfection[J].Arch Virol,2014,159(11):2969-2975.
[12]矢後啓司,萩原茂,川村齊.A fatal case in newborn piglets with Getah virus infection:Isolation of the virus[J].日本獸醫學雜誌(The Japanese Journal of Veteri-nary Science),1987,49(6):989-994.
[13]Fukunaga Y,Kumanomido T,Kamada M.Getah virus as an equine pathogen[J].Vet Clin North Am Equine Pract,2000,16(3):605-617.
[14]张雨,司炳银,常国辉,刘伯华,杨银辉,祝庆余.WJBC株盖塔病毒基因组序列测定及与其他甲病毒的系统进化关系[J].军事医学,2011,35(12):892-895.
[15]中华人民共和国卫生部.卫生部关于印发《人间传染的病原微生物名录》的通知[N/OL].2006-01-27[2018-10-19],http://www.nhfpc.gov.cn/qjjys/s3589/200804/f0840e5958ea40c68ba0cfda9cc138ac.sh-tml
[16]杨火,饶颐年,陈日光,胡定金,李雪东,崔五全,曲小肃,陈德慧,刘景兰,杨佩英,丁建华.海南岛一株甲组虫媒病毒的分离,鉴定和血清抗体调查[J].中华微生物学和免疫学杂志,1984,4(2):107.
[17]冯学平,译.盖他病毒简介及日本盖他病毒病的发生情况[J].中国动物检疫,1990(6):38-39.
[18]Shortridge K F,Mason D K,Watkins K L,Aaskov JG.Serological evidence for the transmission of Getah vi-rus in Hong Kong[J].Vet Rec,1994,134(20):527-528.
[19]白志军,彭翼飞,林立辉,田小东,陈翠华,方美玉,徐春华,蒋廉华,陈唯军.广东、海南两省人血清12种虫媒病毒抗体调查[J].中国人兽共患病杂志,2000,16(1):83-84.
[20]梁勇,张连山,刘永为,董运强,王志强,王峻巍,梁国栋.河北省涉县正常人群盖塔病毒感染状况.河北医药,2010,21(32):3079-3080.
[21]Izumida A,Takuma H,Inagaki S,Kubota M,Hirahara T,Kodama K,Sasaki N.Experimental infection of Ge-tah virus in swine[J].Nihon Juigaku Zasshi,1988,50(3):679-684.
[22]Brown C M,Timoney P J.Getah virus infection of Indi-an horses[J].Trop Anim Health Prot,1998,30(4):241-252.
[23]出水田昭弘,安原寿雄,久保田道雄,吉木研一,平原正,儿玉和夫,佐佐木文存.Establishment and biologi-cal properties of an attenuated strain of getah virus[J].JJapan Vet Med Association(Japan),1991,44:197-201.
[24]周峰,崔丹丹,王傲杰,王新港,常洪涛,陈陆,李永涛,王川庆.我国首株猪盖他病毒的分离与鉴定[J].病毒学报,2018,34(1):59-66.DOI:10.13242/j.cnki.bing-duxuebao.003299
[25]Weaver S C,Reisen W K.Present and future arboviral threats[J].Antiviral Res,2010,85(2):328-345.
[26]梁国栋.虫媒病毒是我国亟待加强的研究领域[J].中华实验和临床病毒学杂志,2005,19(4):305-306.
[27]王仁宏,曹昆.习近平对吉林长春长生生物疫苗案件作出重要指示强调要一查到底严肃问责始终把人民群众的身体健康放在首位坚决守住安全底线[N/OL].人民网,2018-07-23[2018-10-19],http://politics.peo-ple.com.cn/n1/2018/0723/c1024-30165057.html.
[28]贺斌.退市新规出台后,长生生物或将成为第一个被强制退市的上市公司[N/OL].中国新闻周刊,2018-12-2[2018-10-19],http://www.inewsweek.cn/finance/2018-12-02/4483.shtml
[29]Gerber P.An infectious deoxyribonucleic acid derived from vacuolating virus(SV40)[J].Virology,1962,16(1):96-98.
[30]Joseph G.Victoria,Chunlin Wang,Morris S.Jones,Crystal Jaing,Kevin McLoughlin,Shea Gardner,Eric L.Delwart.Viral nucleic acids in live-attenuated vac-cines:detection of minority variants and an adventitious virus[J].J Virol,2010,84(12):6033-6040.
[31]Gregersen J P,Schmitt H J,Trusheim H,Michael Br?ker.Safety of MDCK cell culture-based influenza vaccines[J].Future Microbiol,2011,6(2):143-152.
[32]Palomares R A,Marley S M,Givens M D,Gallardo RA,Brock K V.Bovine viral diarrhea virus fetal persis-tent infection after immunization with a contaminated modified-live virus vaccine[J].Theriogenology,2013,79(8):1184-1195.
[33]崔丹丹.不同PRRSV毒株的分离鉴定,序列及致病性分析[D].河南农业大学,2017.