宏基因组学技术分析四川省集约化猪场中仔猪腹泻粪便的病毒种群

doi:10.11843/j.issn.0366-6964.2023.06.026

摘要/Abstract

摘要： 旨在了解四川省集约化猪场腹泻仔猪粪便中病毒种群的变化。于2019年从四川省15个猪场采集腹泻仔猪粪便样本共156份,每个猪场挑取3份样本混合成1个pool样,提取其总RNA并进行高通量测序,利用SOAP denovo软件进行序列组装,并对病毒的种类和分子特征进行研究。分析结果显示,腹泻仔猪粪便样本中的病毒种群包括12个科的20种病毒,以冠状病毒科(Coronaviridae)成员为主,与腹泻相关的主要病原有猪流行性腹泻病毒(porcine pidemic diarrhea virus,PEDV)、猪传染性胃肠炎病毒(porcine infectious gastroenteritis virus,TGEV)和猪轮状病毒(porcine rotavirus,PoRV)。获得PEDV N基因的完整基因组和TGEV、猪星状病毒2型(porcine astrovirus,PAstV-2)、posavirus 1和猪细小病毒4型(porcine parvovirus,PPV-4)的部分基因组。系统发育结果显示,PEDV、TGEV、PAstV-2、posavirus 1和PPV-4均与参考毒株有较近的遗传进化关系。研究结果表明,2019年四川地区仔猪腹泻粪便中病毒种类复杂多样,冠状病毒丰度有所上升,为四川省仔猪腹泻的防控提供了一定的数据支撑。

关键词: 仔猪病毒性腹泻, 宏基因组学, 分子特征

Abstract: The purpose of this study is to investigate the changes of virus populations in the diarrheal feces of piglets in intensive pig farms in Sichuan Province. In 2019, 156 pig diarrheal fecal samples were collected from 15 pig farms in Sichuan Province. Three samples from each farm were mixed into a pool sample, and their total RNA was extracted and subjected to high-throughput sequencing. Sequence assembly was performed using SOAP denovo software, and the virus species and molecular characteristics were studied. The results revealed that the virus population in the fecal samples of diarrheal piglets included 20 viruses from 12 families, mainly Coronaviridae. The main pathogens associated with diarrhea are porcine epidemic diarrhea virus (PEDV), porcine infectious gastroenteritis virus (TGEV) and porcine rotavirus (PoRV). By SOAP denovo software, we assembled and obtained the complete sequences of the PEDV N gene and the partial genomes of TGEV, porcine astrovirus (PAstV-2), Posavirus 1 and PPV-4. The phylogenetic analysis results revealed that PEDV, TGEV, PAstV-2, Posavirus 1 and PPV-4 were more closely related to the reference strains. The above results indicated that the virus species in the feces of piglet diarrhea in Sichuan Province in 2019 were complex and diverse, and that the abundance of Coronaviridae increased, which provided a foundation for the prevention and control of piglet diarrhea in Sichuan Province.

Key words: piglet viral diarrhea, metagenomics, molecular characterization

中图分类号:

S852.651

张家祺, 甲呷格西, 周群, 宋鑫, 张斌. 宏基因组学技术分析四川省集约化猪场中仔猪腹泻粪便的病毒种群[J]. 畜牧兽医学报, 2023, 54(6): 2478-2486.

ZHANG Jiaqi, JIA GA Gexi, ZHOU Qun, SONG Xin, ZHANG Bin. Metagenomics Analysis of Virus Populations in Piglet Diarrheal Feces from Intensive Pig Farms in Sichuan Province[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(6): 2478-2486.

参考文献 28

[1]	王红宁. 仔猪腹泻成因及综合防治技术措施[J]. 中国畜牧杂志, 2006, 42(6):58-60.WANG H N. The cause and integrated control of piglet diarrhea[J]. Chinese Journal of Animal Science, 2006, 42(6):58-60. (in Chinese)
[2]	LI G, ZHAI S L, ZHOU X, et al. Phylogeography and evolutionary dynamics analysis of porcine delta-coronavirus with host expansion to humans[J]. Transbound Emerg Dis, 2022, 69(5):e1670-e1681.
[3]	PÉREZ-RIVERA C, RAMÍREZ-MENDOZA H, MENDOZA-ELVIRA S, et al. First report and phylogenetic analysis of porcine deltacoronavirus in Mexico[J]. Transbound Emerg Dis, 2019, 66(4):1436-1441.
[4]	ZHOU P, FAN H, LAN T, et al. Fatal swine acute diarrhoea syndrome caused by an HKU2-related coronavirus of bat origin[J]. Nature, 2018, 556(7700):255-258.
[5]	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)
[6]	胡承哲, 周群, 李玉, 等. 利用宏基因组学鉴定藏猪腹泻粪便病毒种群[J]. 畜牧兽医学报, 2019, 50(12):2479-2487.HU C Z, ZHOU Q, LI Y, et al. Identification of viral community in diarrheal feces of Tibetan pig by metagenomics[J]. Acta Veterinaria et Zootechnica Sinica, 2019, 50(12):2479-2487. (in Chinese)
[7]	LU R J, ZHAO X, LI J, et al. Genomic characterisation and epidemiology of 2019 novel coronavirus:implications for virus origins and receptor binding[J]. Lancet, 2020, 395(10224):565-574.
[8]	ZHANG B, TANG C, YUE H, et al. Viral metagenomics analysis demonstrates the diversity of viral flora in piglet diarrhoeic faeces in China[J]. J General Virol, 2014, 95(7):1603-1611.
[9]	CHENG W X, LI J S, HUANG C P, et al. Identification and nearly full-length genome characterization of novel porcine bocaviruses[J]. PLoS One, 2010, 5(10):e13583.
[10]	DELWART E L. Viral metagenomics[J]. Rev Med Virol, 2007, 17(2):115-131.
[11]	何彪, 涂长春. 病毒宏基因组学的研究现状及应用[J]. 畜牧兽医学报, 2012, 43(12):1865-1870.HE B, TU C C. The advances and applications of viral metagenomics[J]. Acta Veterinaria et Zootechnica Sinica, 2012, 43(12):1865-1870. (in Chinese)
[12]	CHEN X, ZHANG B, YUE H, et al. A novel astrovirus species in the gut of yaks with diarrhoea in the Qinghai-Tibetan Plateau, 2013[J]. J General Virol, 2015, 96(12):3672-3680.
[13]	FAHSBENDER E, ALTAN E, SEGUIN M A, et al. Chapparvovirus DNA found in 4% of dogs with diarrhea[J]. Viruses, 2019, 11(5):398.
[14]	ABI K M, YU Z H, JING Z Z, et al. Identification of a novel Aichivirus D in sheep[J]. Infect Genet Evolut, 2021, 91:104810.
[15]	PAN Y F, TIAN X Y, QIN P, et al. Discovery of a novel swine enteric alphacoronavirus (SeACoV) in southern China[J]. Vet Microbiol, 2017, 211:15-21.
[16]	GONG L, LI J, ZHOU Q F, et al. A New Bat-HKU2-like coronavirus in Swine, China, 2017[J]. Emerg Infect Dis, 2017, 23(9):1607-1609.
[17]	FAN K Z, WANG R. Complete genome of a porcine calicivirus strain in Anhui province, China, is significantly shorter than that of the other Chinese strain[J]. J Virol, 2012, 86(24):13823.
[18]	OBA M, NAOI Y, ITO M, et al. Metagenomic identification and sequence analysis of a Teschovirus A-related virus in porcine feces in Japan, 2014-2016[J]. Infect Genet Evolut, 2018, 66:210-216.
[19]	ZHOU L, HU C Z, ZHOU Q, et al. Viral communities associated with porcine diarrhoeal disease and genetic characterization of a bufavirus in Tibetan pigs in China[J]. Arch Virol, 2021, 166(2):613-617.
[20]	徐囡, 吴长德, 张英俊, 等. 猪血凝性脑脊髓炎研究进展[J]. 动物医学进展, 2016, 37(11):94-97.XU N, WU C D, ZHANG Y J, et al. Progress on porcine hemagglutinating encephalomyelitis[J]. Progress in Veterinary Medicine, 2016, 37(11):94-97. (in Chinese)
[21]	沈小娟, 李晶娇, 边疆, 等. 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 X J, LI J J, BIAN J, et al. 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. (in Chinese)
[22]	ZHANG X M, HERBST W, KOUSOULAS K G, et al. Biological and genetic characterization of a hemagglutinating coronavirus isolated from a diarrhoeic child[J]. J Med Virol, 1994, 44(2):152-161.
[23]	WOO P C Y, LAU S K P, LAM C S F, et al. Discovery of seven novel Mammalian and avian coronaviruses in the genus Deltacoronavirus supports bat coronaviruses as the gene source of Alphacoronavirus and Betacoronavirus and avian coronaviruses as the gene source of Gammacoronavirus and Deltacoronavirus[J]. J Virol, 2012, 86(7):3995-4008.
[24]	SHEN H G, ZHANG J F, GAUGER P C, et al. Genetic characterization of porcine sapoviruses identified from pigs during a diarrhoea outbreak in Iowa, 2019[J]. Transbound Emerg Dis, 2022, 69(3):1246-1255.
[25]	YANG F, LIU X W, ZHOU Y C, et al. Histopathology of Porcine kobuvirus in Chinese piglets[J]. Virol Sin, 2015, 30(5):396-399.
[26]	SAUVAGE V, AR GOUILH M, CHEVAL J, et al. A member of a new Picornaviridae genus is shed in pig feces[J]. J Virol, 2012, 86(18):10036-10046.
[27]	ZAULET M, PETROVAN V, BIRLADEANU A M, et al. Identification and prevalence of swine pasivirus 1 in eastern Romanian pig farms[J]. J Vet Diagnost Investigat, 2017, 29(3):305-311.
[28]	CHEN Q, WANG L Y, ZHENG Y, et al. Metagenomic analysis of the RNA fraction of the fecal virome indicates high diversity in pigs infected by porcine endemic diarrhea virus in the United States[J]. Virol J, 2018, 15(1):95.