安徽省急性胃肠炎疫情相关GⅡ.P17-GⅡ.17基因型诺如病毒分子特征研究
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摘要
诺如病毒(Noroviruses,NoVs)是引起病毒性胃肠炎的重要病原体,本研究旨在对2015～2016年春季安徽部分地区暴发的急性胃肠炎(Acute gastroenteritis,AGE)疫情标本进行病原检测和分子分型,分析病原的基因特征。收集2015年3月至2016年5月9起AGE疫情流行病学信息和采集病例粪便、肛拭子样本。采用Real-time PCR检测NoVs核酸和RT-PCR扩增RdRp与VP1区基因序列,基因测序后应用BLAST比对和NoVs在线分型工具分析结果。7起疫情发生在中、小学校(77.78%,7/9),2起疫情发生在乡镇,发病人数中位数为6人,男女发病比例为1.41∶1,临床表现主要以恶心、呕吐/腹泻、腹痛为主。77份临床病例样本中检出66份NoVs核酸阳性,基因序列测定获得76条序列,39条序列为RdRp区GⅡ.P17基因型,37条序列为VP1区GⅡ.17基因型。基因进化树分析显示:2015～2016年安徽地区39条RdRp区NoVs GⅡ.P17基因型序列Cluster III进化簇III b分支,与2015年广东、海南、中国台湾地区参考毒株序列有较近的亲缘性关系,核苷酸同源性为99%～100%。37条VP1区NoVs GⅡ.17基因型序列都处在Cluster III进化簇上,其中28条序列属于III a进化分支,与山东2015年LX09株、2016年广东GZ2016-L492株、江苏zj019株、中国香港CUHK-NS-942株以及日本2015年AichiF101毒株序列有较近的亲缘性关系。新型GⅡ.P17-GⅡ.17基因型NoVs是引起2015年和2016年春季安徽部分地区AGE暴发的主要病原体,需加强病毒性胃肠炎流行病学调查与病原监测及分子分型鉴定工作。
To study the genotype and phylogeny of the pathogens related to the epidemic outbreaks of acute gastroenteritis(AGE)in Anhui Province,China,during March 2015 to May 2016,epidemiological data of nine AGE outbreaks and anal swab or fecal samples from the patients were collected. Noroviruses(NoVs)nucleic acids in the samples were tested by real-time PCR,and partial genes of the NoVs RdRp and VP1 regions were amplified by conventional RT-PCR. Genotypes of the sequences were assigned by using BLAST and NoVs genotyping tool(http://www.rivm.nl/mpf/norovirus/typingtool). Seven outbreaks occurred in primary and secondary schools(77.78%,7/9);two outbreaks occurred in villages and towns. The median number of cases was six,and the male to female incidence ratio was 1.41∶1. The main clinical manifestations of patients were nausea,vomiting,diarrhea and abdominal pain. Sixty-six NoVs nucleic acid-positive samples were detected from seventy-seven clinical cases. Seventy-six sequences were obtained successfully,thirty-nine of which were RdRp GⅡ.P17 genotype and thirty-seven of which were VP1 GⅡ.17 genotype. Phylogenetic analysis showed that thirty-nine RdRp NoVs GⅡ.P17 genotype sequences belonged to evolutionary cluster IIIb and were closely related to reference strain sequences from Guangdong,Hainan and Taiwan of China in 2015(nucleotide homology 99%～100%). Thirty-seven NoVs GⅡ.17 VP1 sequences belonged to evolutionary cluster III,twenty-eight of which belonged cluster IIIa. They were closely related to strain LX09 from Shandong Province,China in2015,strain GZ2016-L492 from Guangdong Province in 2016,strain zj019 from Jiangsu Province,China,strain CUHK-NS-942 from Hong Kong,China and strain AichiF101 from Japan in 2015. Norovirus genotype GⅡ.P17-GⅡ.17 was the predominant pathogen causing AGE outbreaks in Anhui Province during 2015～2016. It is necessary to strengthen epidemiological investigation,pathogen monitoring and molecular typing for viral gastroenteritis.
To study the genotype and phylogeny of the pathogens related to the epidemic outbreaks of acute gastroenteritis(AGE)in Anhui Province,China,during March 2015 to May 2016,epidemiological data of nine AGE outbreaks and anal swab or fecal samples from the patients were collected. Noroviruses(NoVs)nucleic acids in the samples were tested by real-time PCR,and partial genes of the NoVs RdRp and VP1 regions were amplified by conventional RT-PCR. Genotypes of the sequences were assigned by using BLAST and NoVs genotyping tool(http://www.rivm.nl/mpf/norovirus/typingtool). Seven outbreaks occurred in primary and secondary schools(77.78%,7/9);two outbreaks occurred in villages and towns. The median number of cases was six,and the male to female incidence ratio was 1.41∶1. The main clinical manifestations of patients were nausea,vomiting,diarrhea and abdominal pain. Sixty-six NoVs nucleic acid-positive samples were detected from seventy-seven clinical cases. Seventy-six sequences were obtained successfully,thirty-nine of which were RdRp GⅡ.P17 genotype and thirty-seven of which were VP1 GⅡ.17 genotype. Phylogenetic analysis showed that thirty-nine RdRp NoVs GⅡ.P17 genotype sequences belonged to evolutionary cluster IIIb and were closely related to reference strain sequences from Guangdong,Hainan and Taiwan of China in 2015(nucleotide homology 99%～100%). Thirty-seven NoVs GⅡ.17 VP1 sequences belonged to evolutionary cluster III,twenty-eight of which belonged cluster IIIa. They were closely related to strain LX09 from Shandong Province,China in2015,strain GZ2016-L492 from Guangdong Province in 2016,strain zj019 from Jiangsu Province,China,strain CUHK-NS-942 from Hong Kong,China and strain AichiF101 from Japan in 2015. Norovirus genotype GⅡ.P17-GⅡ.17 was the predominant pathogen causing AGE outbreaks in Anhui Province during 2015～2016. It is necessary to strengthen epidemiological investigation,pathogen monitoring and molecular typing for viral gastroenteritis.
引文
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[12]Matsushima Y,Ishikawa M,Shimizu T,Komane A,Kasuo S,Shinohara M,Nagasawa K,Kimura H,Ryo A,Okabe N,Haga K,Doan Y H,Katayama K,Shi-mizu H. Genetic analyses of GII. 17 norovirus strains in diarrheal disease outbreaks from December 2014 to March 2015 in Japan reveal a novel polymerase sequence and amino acid substitutions in the capsid region[J].Euro Surveill,2015,20(26):21173.
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[14]de Graaf M,van Beek J,Vennema H,Podkolzin A T,Hewitt J,Bucardo F,Templeton K,Mans J,Nordgren J,Reuter G,Lynch M,Rasmussen L D,Iritani N,Chan M C,Martella V,Ambert-Balay K,VinjéJ,White P A,Koopmans M P. Emergence of a novel GII.17 norovirus-End of the GII.4 era[J]. Euro Surveill,2015,20(26):21178.
[15]Parra G I,Green K Y. Genome of emerging norovirus GII.17,United States,2014[J].Emerg Infect Dis,2015,21(8):1477-1479.
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[17]史永林,孔翔羽,靳淼,方金才,施旭东,胡万富.安徽省诺如病毒胃肠炎暴发的分子病原学特征研究[J].中华实验和临床病毒学杂志,2015,29(4):310-312.
[18]Yang Y,Xia M,Tan M,Huang P,Zhong W,Pang X L,Lee B E,Meller J,Wang T,Jiang X. Genetic and phenotypic characterization of GII-4 noroviruses that cir-culated during 1987 to 2008[J].J Virol,2010,84(18):9595-607.
[19]LeBlanc J J,Pettipas J,Gaston D,Taylor R,Hatch-ette T F, Booth T F, Mandes R, McDermid A,Grudeski E. Outbreak of Norovirus GII. P17-GII. 17 in the Canadian Province of Nova Scotia[J]. Can J Infect Dis Med Microbiol,2016:1280247.
[20]吴冰珊,翁育伟,黄枝妙,何文祥.诺如病毒GII.17新变异株引致福建省2014~2015年病毒性胃肠炎暴发[J].病毒学报,2017,33(3):320-324. DOI:10.13242/j.cnki.bingduxuebao.003142
[21]杨芬,孙立梅,李晖,郭丽丽,方苓,谭小华,龙遗芳,柯昌文,何剑锋.广东省2008-2015年诺如病毒感染暴发的危险因素分析[J].中华流行病学杂志,2017,38(7):906-910.
[22]王璇,雍玮,杜雪飞,张钟,何敏,乔梦凯,石利民,王雅倩,靳淼,丁洁.2014~2017年南京地区胃肠炎暴发中诺如病毒分子流行病学特征[J].病毒学报,2018,34(4):483-490. DOI:10.13242/j. cnki. bingduxue-bao.003393
[2] VinjéJ. Advances in laboratory methods for detection and typing of norovirus[J]. J Clin Microbiol,2015,53(2):373-381.
[3] Lun J H,Hewitt J,Sitabkhan A,Eden J S,Enosi Tuip-ulotu D,Netzler N E,Morrell L,Merif J,Jones R,Huang B,Warrilow D,Ressler K A,Ferson M J,Dw-yer D E,Kok J,Rawlinson W D,Deere D,Crosbie N D,White P A. Emerging recombinant noroviruses iden-tified by clinical and waste water screening[J]. Emerg Microbes Infect,2018,7(1):50.
[4] Fu J G,Ai J,Qi X,Zhang J,Tang F Y,Zhu Y F.Emergence of two novel norovirus genotype II.4 variants associated with viral gastroenteritis in China[J]. J Med Virol,2014,86(7):1226-1234.
[5]买欢,高燕,潘孝本,韩进超,丛旭,魏来.中国7个地区诺如病毒GII.4/Sydney2012变异株分析[J].中华流行病学杂志,2014,35(2):157-162.
[6]史永林,张文艳,金晶,周伟,汤仁树,吴金菊,靳淼,胡万富.安徽省诺如病毒G II.4/Sydney2012子特征分析[J].安徽预防医学杂志,2016,22(6):363-366.
[7] Jin M,Zhou Y K,Xie H P,Fu J G,He Y Q,Zhang S,Jing H B,Kong X Y,Sun X M,Li H Y,Zhang Q,Li K,Zhang Y J,Zhou D Q,Xing W J,Liao Q H,Liu N,Yu H J,Jiang X,Tan M,Duan Z J. Characteriza-tion of the new GII.17 norovirus variant that emerged re-cently as the predominant strain in China[J]. J Gen Vi-rol,2016,97(10):2620-2632.
[8] Fu J,Ai J,Jin M,Jiang C,Zhang J,Shi C,Lin Q,Yuan Z,Qi X,Bao C,Tang F,Zhu Y. Emergence of a new GII.17 norovirus variant in patients with acute gas-troenteritis in Jiangsu,China,September 2014 to March2015[J]. Euro Surveill,2015,20(24):21157.
[9] Lu J,Sun L,Fang L,Yang F,Mo Y,Lao J,Zheng H,Tan X,Lin H,Rutherford S,Guo L,Ke C,Hui L.Gastroenteritis outbreaks caused by norovirus GII. 17,Guangdong province,China,2014-2015[J]. Emerg In-fect Dis,2015,21(7):1240-1242.
[10]Chan M C,Lee N,Hung T N,Kwok K,Cheung K,Tin E K,Lai R W,Nelson E A,Leung T F,Chan P K. Rapid emergence and predominance of a broadly rec-ognizing and fast-evolving norovirus GII. 17 variant in late 2014[J].Nat Commun,2015,6:10061.
[11]Lee C C,Feng Y,Chen S Y,Tsai C N,Lai M W,Chiu C H. Emerging norovirus GII. 17 in Taiwan[J].Clin Infect Dis,2015,61(11):1762-1764.
[12]Matsushima Y,Ishikawa M,Shimizu T,Komane A,Kasuo S,Shinohara M,Nagasawa K,Kimura H,Ryo A,Okabe N,Haga K,Doan Y H,Katayama K,Shi-mizu H. Genetic analyses of GII. 17 norovirus strains in diarrheal disease outbreaks from December 2014 to March 2015 in Japan reveal a novel polymerase sequence and amino acid substitutions in the capsid region[J].Euro Surveill,2015,20(26):21173.
[13]Dang Thanh H,Than V T,Nguyen T H,Lim I,Kim W. Emergence of norovirus GII.17 variants among chil-dren with acute gastroenteritis in South Korea[J/OL].PLoS One,2016,11(5):e0154284.
[14]de Graaf M,van Beek J,Vennema H,Podkolzin A T,Hewitt J,Bucardo F,Templeton K,Mans J,Nordgren J,Reuter G,Lynch M,Rasmussen L D,Iritani N,Chan M C,Martella V,Ambert-Balay K,VinjéJ,White P A,Koopmans M P. Emergence of a novel GII.17 norovirus-End of the GII.4 era[J]. Euro Surveill,2015,20(26):21178.
[15]Parra G I,Green K Y. Genome of emerging norovirus GII.17,United States,2014[J].Emerg Infect Dis,2015,21(8):1477-1479.
[16]Gao Z,Li X,Yan H,Li W,Jia L,Hu L,Hu H,Liu B,Li J,Wang Q. Human calicivirus occurrence among outpatients with diarrhea in Beijing, China, between April 2011 and March 2013[J]. J Med Virol,2015,87(12):2040-2047
[17]史永林,孔翔羽,靳淼,方金才,施旭东,胡万富.安徽省诺如病毒胃肠炎暴发的分子病原学特征研究[J].中华实验和临床病毒学杂志,2015,29(4):310-312.
[18]Yang Y,Xia M,Tan M,Huang P,Zhong W,Pang X L,Lee B E,Meller J,Wang T,Jiang X. Genetic and phenotypic characterization of GII-4 noroviruses that cir-culated during 1987 to 2008[J].J Virol,2010,84(18):9595-607.
[19]LeBlanc J J,Pettipas J,Gaston D,Taylor R,Hatch-ette T F, Booth T F, Mandes R, McDermid A,Grudeski E. Outbreak of Norovirus GII. P17-GII. 17 in the Canadian Province of Nova Scotia[J]. Can J Infect Dis Med Microbiol,2016:1280247.
[20]吴冰珊,翁育伟,黄枝妙,何文祥.诺如病毒GII.17新变异株引致福建省2014~2015年病毒性胃肠炎暴发[J].病毒学报,2017,33(3):320-324. DOI:10.13242/j.cnki.bingduxuebao.003142
[21]杨芬,孙立梅,李晖,郭丽丽,方苓,谭小华,龙遗芳,柯昌文,何剑锋.广东省2008-2015年诺如病毒感染暴发的危险因素分析[J].中华流行病学杂志,2017,38(7):906-910.
[22]王璇,雍玮,杜雪飞,张钟,何敏,乔梦凯,石利民,王雅倩,靳淼,丁洁.2014~2017年南京地区胃肠炎暴发中诺如病毒分子流行病学特征[J].病毒学报,2018,34(4):483-490. DOI:10.13242/j. cnki. bingduxue-bao.003393