腹泻仔猪肠道病毒组学分析

doi:10.11843/j.issn.0366-6964.2019.03.016

摘要/Abstract

摘要：

为了解上海周边地区腹泻仔猪肠道病毒组学特征，特别是冠状病毒流行情况，采用病毒宏基因组学方法对6个猪场的90份疑似病毒感染导致腹泻粪样进行检测，并通过Geneious和MEGA7.0等生物信息学软件对获得的完整的α冠状病毒属中的PEDV ORF3基因序列进行遗传进化分析。数据显示，腹泻仔猪肠道病毒群落组成主要由小RNA病毒科（Picornaviridae）（63.67%）、星状病毒科（Astroviridae）（12.68%）、杯状病毒科（Caliciviridae）（4.07%）、细小病毒科（Parvoviridae）（2.51%）等组成。在唯一一个检测出PEDV阳性的猪场中，腹泻仔猪PEDV阳性率高达43.33%（13/30）。基因序列遗传进化分析显示，在本研究获得的3个PEDV ORF3基因序列中，有1个属于G2基因型，与其他参考PEDV病毒株相似性较高（96.44%~99.70%）；另外2个ORF3基因序列属于G1基因型，相似性相对较低（95.11%~99.41%）。与PEDV传统株CV777蛋白序列比较分析发现，除piglet91株产生F2S突变以外，三株PEDV存在一致性突变，分别如下：V21A、I70M、V79I、F80V、L85I、L92F，这一特性，有可能是PEDV传统株与流行株基因型的鉴别依据。上海周边地区腹泻仔猪肠道病毒组成丰富，在不同猪群中，不同病原感染的情况有所差异，除了PEDV外，星状病毒和杯状病毒也是仔猪腹泻的主要病原。PEDV的ORF3基因与传统分离株相比具有独特的分子特征，新检测到的毒株突变位点与病毒毒力的关系有待于进一步研究。研究结果有助于了解腹泻仔猪肠道的病毒谱，并为仔猪病毒病防控提供一定的基础数据。

Abstract:

To clarify the epidemiological characteristics of diarrhea in pigs around Shanghai, especially the prevalence of coronavirus, we used viral metagenomic method to detect viruses in stools from 90 diarrhea piglets from 6 swine farms in parts of Shanghai. And then a multiple sequence alignment was carried out using Clustal W and Geneious, and the phylogenetic trees were generated by the distance-based maximum likelihood method using MEGA 7.0. The data showed that the intestinal virus community of diarrhea piglets was mainly composed of Picornaviridae (63.67%), Astroviridae (12.68%), Caliciviridae (4.07%), and Parvoviridae (2.51%). We found that the farm C was the only farm where PEDV is the main cause of piglet diarrhea, and the positive rate of PEDV infection in farm C was as high as 43.33%(13/30). The genetic evolution analysis showed that one of the three PEDV ORF3 gene sequences obtained in this study belonged to the G2 genotype, and the homology with other reference PEDV strains was higher (96.44%-99.70%), which was relatively conservative; the other two ORF3 gene sequences belong to the G1 genotype, and the homology was relatively lower (95.11%-99.41%). In the comparison analysis of protein sequence with the traditional strain CV777, we found that in addition to the piglet91 strain showed a mutation of F2S, three ORF3 sequences both showed consistent mutations, namely:V21A, I70M, V79I, F80V, L85I, L92F. This characteristic may be regarded as the basis for identification of PEDV traditional strains and the popular strain genotype. The enterovirus of diarrhea piglets from surrounding areas of Shanghai were rich in composition. The infection status in the different pigs were significantly different. Apart from PEDV, astrovirus and calicivirus are also the main pathogens of diarrhea piglets. As one of the main pathogens of piglet diarrhea in this particular region, the ORF3 gene of PEDV has unique molecular characteristics compared with traditional isolates. The relationship between mutation site and viral virulence of the newly detected virus needs further experimental research.

中图分类号:

S851.21

沈小娟, 李晶娇, 边疆, 赵婷婷, 华修国, 崔立. 腹泻仔猪肠道病毒组学分析[J]. 畜牧兽医学报, 2019, 50(3): 611-619.

SHEN Xiaojuan, LI Jingjiao, BIAN Jiang, ZHAO Tingting, HUA Xiuguo, CUI Li. Metagenomics Analysis of Feces from Diarrhea Piglets Reveals the Viral Composition and Epidemic Characteristics of Coronavirus[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(3): 611-619.

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参考文献

[1] ZHANG J Q, GUY J S, SNIJDER E J, et al. Genomic characterization of equine coronavirus[J]. Virology, 2007, 369(1):92-104.
[2] JUNG K, HU H, EYERLY B, et al. Pathogenicity of 2 porcine deltacoronavirus strains in gnotobiotic pigs[J]. Emerg Infect Dis, 2015, 21(4):650-654.
[3] CHENG V C C, LAU S K P, WOO P C Y, et al. Severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection[J]. Clin Microbiol Rev, 2007, 20(4):660-694.
[4] PEIRIS J S, LAI S T, POON L L M, et al. Coronavirus as a possible cause of severe acute respiratory syndrome[J]. Lancet, 2003, 361(9366):1319-1325.
[5] LIM P L, LEE T H, ROWE E K. Middle east respiratory syndrome coronavirus (MERS CoV):update 2013[J]. Curr Infect Dis Rep, 2013, 15(4):295-298.
[6] 凌云,瞿嵘,罗裕锋. 中国首例输入性中东呼吸综合征患者临床救治分析[J]. 中华危重病急救医学, 2015, 27(8):630-634.
LING Y, QU R, LUO Y F. Clinical analysis of the first patient with imported Middle East respiratory syndrome in China[J]. Chinese Critical Care Medicine, 2015, 27(8):630-634. (in Chinese)
[7] SUN R Q, CAI R J, CHEN Y Q, et al. Outbreak of porcine epidemic diarrhea in suckling piglets, China[J]. Emerg Infect Dis, 2012, 18(1):161-163.
[8] STEVENSON G W, HOANG H, SCHWARTS K J, et al. Emergence of Porcine epidemic diarrhea virus in the United States:clinical signs, lesions, and viral genomic sequences[J]. J Vet Diagn Invest, 2013, 25(5):649-654.
[9] HUANG Y W, DICKERMAN A W, PIÑEYRO P, et al. Origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the united states[J]. mBio, 2013, 4(5):00737-13.
[10] KSIAZEK T G, ERDMAN D, GOLDSMITH C S, et al. A novel coronavirus associated with severe acute respiratory syndrome[J]. N Engl J Med, 2003, 348(20):1953-1966.
[11] PEIRISM. Severe acute respiratory syndrome[M]. Malden:Blackwell Pub., 2005:88-97.
[12] ZAKI A M, VAN BOHEEMEN S, BESTEBROER T M, et al. Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia[J]. N Engl J Med, 2012, 367(19):1814-1820.
[13] BELOUZARD S, MILLET J K, LICITRA B N, et al. Mechanisms of coronavirus cell entry mediated by the viral spike protein[J]. Viruses, 2012, 4(6):1011-1033.
[14] MILLET J K, WHITTAKER G R. Host cell proteases:Critical determinants of coronavirus tropism and pathogenesis[J]. Virus Res, 2015, 202:120-134.
[15] DEHAAN C A M, STADLER K, GODEKE G J, et al. Cleavage inhibition of the murine coronavirus spike protein by a furin-like enzyme affects cell-cell but not virus-cell fusion[J]. J Virol, 2004, 78(11):6048-6054.
[16] KAWASE M, SHIRATO K, MATSUYAMA S, et al. Protease-mediated entry via the endosome of human coronavirus 229E[J]. J Virol, 2009, 83(2):712-721.
[17] SIMMONS G, GOSALIA D N, RENNEKAMP A J, et al. Inhibitors of cathepsin L prevent severe acute respiratory syndrome coronavirus entry[J]. Proc Natl Acad Sci U S A, 2005, 102(33):11876-11881.
[18] QIU Z Z, HINGLEY S T, SIMMONS G, et al. Endosomal proteolysis by cathepsins is necessary for murine coronavirus mouse hepatitis virus type 2 spike-mediated entry[J]. J Virol, 2006, 80(12):5768-5776.
[19] WICHT O, LI W T, WILLEMS L, et al. Proteolytic activation of the porcine epidemic diarrhea coronavirus spike fusion protein by trypsin in cell culture[J]. J Virol, 2014, 88(14):7952-7961.
[20] 方肇寅, 谢华萍,吕红霞,等. 1999~2005年我国婴幼儿人杯状病毒腹泻研究[J]. 病毒学报, 2007, 23(1):9-15.
FANG Z Y, XIE H P, LV H X, et al. Investigation of human calicivirus (HuCV) diarrhea among infantile and young children in China, 1999-2005[J]. Chinese Journal of Virology, 2007, 23(1):9-15. (in Chinese)
[21] 王新珍. 东北稻田水体蓝藻病毒基因多样性与水体病毒宏基因组学研究[D]. 长春:中国科学院大学(中国科学院东北地理与农业生态研究所), 2017.
WANG X Z. Analysis of cyanophage genetic diversity and viral metagenomics in paddy waters in Northeast China[D]. Changchun:Chinese Academy of Sciences (Northeast Institute of Geography and Agroecology), 2017. (in Chinese)
[22] 刘文彬, 张翠媛,虞结梅,等. 中国广西龙虎山野生猕猴粪便的病毒宏基因组学分析[J]. 病毒学报, 2016, 32(3):273-282.
LIU W B, ZHANG C Y, YU J M, et al. Metagenomic analysis of wild rhesus monkey virome in Longhu Mountain in Guangxi Area, China[J]. Chinese Journal of Virology, 2016, 32(3):273-282. (in Chinese)
[23] 舒鹏. 宏基因组测序技术在肠道菌群研究和临床样本检测中的应用[D]. 合肥:安徽医科大学, 2016.
SHU P. The application of metagenomics sequencing technology in the research of intestinal floraand the detection of clinical samples[D]. Hefei:Anhui Medical University, 2016. (in Chinese)
[24] 张若曦, 张志,顾文源,等. 河北省猪腹泻相关病毒的检测及猪流行性腹泻病毒S基因遗传变异分析[J]. 畜牧兽医学报, 2018, 49(3):597-605.
ZHANG R X, ZHANG Z, GU W Y, et al. Detection of porcine diarrhea associated virus and genetic variation analysis of S gene of porcine epidemic diarrhea virus in Hebei province[J]. Acta Veterinaria et Zootechnica Sinica, 2018, 49(3):597-605. (in Chinese)
[25] 施标,董世娟,朱于敏,等. 中国猪流行性腹泻病毒分子流行病学研究进展[J]. 中国农业科学, 2013, 46(20):4362-4369.
SHI B, DONG S J, ZHU Y M, et al. Advances in study of molecular epidemiology of porcine epidemic diarrhea virus in China[J]. Scientia Agricultura Sinica, 2013, 46(20):4362-4369. (in Chinese)
[26] SUN R Q, LENG Z M, ZHAI S L, et al. Genetic variability and phylogeny of current Chinese porcine epidemic diarrhea virus strains based on Spike, ORF3, and Membrane genes[J]. Sci World J, 2014, 2014:208439.
[27] GE F F, YANG D Q, JU H B, et al. Epidemiological survey of porcine epidemic diarrhea virus in swine farms in Shanghai, China[J]. Arch Virol, 2013, 158(11):2227-2231.
[28] 韩文,罗玉子,赵碧波,等. 基于宏基因组学的猪群样本病毒探测方法的建立[J]. 微生物学报, 2013, 53(2):197-203.
HAN W, LUO Y Z, ZHAO B B, et al. Metagenomics-based detection of swine viruses[J]. Acta Microbiologica Sinica, 2013, 53(2):197-203. (in Chinese)
[29] BREITBART M, HEWSON I, FELTS B, et al. Metagenomic analyses of an uncultured viral community from human feces[J]. J Bacteriol, 2003, 185(20):6220-6223.
[30] LI L L, VICTORIA J G, WANG C L, et al. Bat guano virome:predominance of dietary viruses from insects and plants plus novel mammalian viruses[J]. J Virol, 2010, 84(14):6955-6965.
[31] WILLNER D, FURLAN M, HAYNES M, et al. Metagenomic analysis of respiratory tract DNA viral communities in cystic fibrosis and non-cystic fibrosis individuals[J]. PLoS One, 2009, 4(10):e7370.
[32] ALLANDER T, EMERSON S U, ENGLE R E, et al. A virus discovery method incorporating DNase treatment and its application to the identification of two bovine parvovirus species[J]. Proc Natl Acad Sci U S A, 2001, 98(20):11609-11614.
[33] RONDON M R, AUGUST P R, BETTERMANN A D, et al. Cloning the soil metagenome:a strategy for accessing the genetic and functional diversity of uncultured microorganisms[J]. Appl Environ Microbiol, 2000, 66(6):2541-2547.
[34] HENNE A, SCHMITZ R A, BOMEKE M, et al. Screening of environmental DNA libraries for the presence of genes conferring lipolytic activity on Escherichia coli[J]. Appl Environ Microbiol, 2000, 66(7):3113-3116.
[35] 单同领. 病毒宏基因组学分析儿童和猪肠道病毒群落及23株病毒的初步研究[D]. 上海:上海交通大学, 2011.
SHAN T L. Viral metegenomic analysis of viromes of stool samples in children and swine and preliminary study of 23 strains of viruses[D]. Shanghai:Shanghai Jiaotong University, 2011. (in Chinese)