基于S基因序列分析新型猪流行性腹泻病毒的遗传演化

doi:10.11843/j.issn.0366-6964.2020.01.013

摘要/Abstract

摘要： 最近，笔者实验室在青藏高原地区发现两种新亚型藏猪源猪流行性腹泻病毒（PEDV），为进一步调查新型PEDV是否在四川腹泻猪群中存在或流行，对实验室2018-2019年保存的116份猪腹泻粪便或肠组织样本进行PEDV的检测及其纤突蛋白基因（spike）分子特征研究。结果表明：腹泻样本的PEDV检出率为42.2%（49/116，95% CI=33.1%~51.8%），并获得了13条完整的S基因序列，全长为4 149~4 170 bp，序列相似性为94.2%~99.9%，其中SWUN-H3-CH-SCYA-2019的S基因与藏猪源新G1亚群PEDV的序列相似性高达97.0%~98.6%。遗传演化研究结果表明13株PEDV S基因划分为G1和G2大群，其中SWUN-H3-CH-SCYA-2019位于藏猪源新G1亚群；SWUN-19-CH-SCZY-2018、SWUN-4-CH-SCXC-2018、SWUN-1-CH-SCNJ-2019和SWUN-3CH-CH-SCZG-2019位于G2亚群中一个独立的分支，且与藏猪源新G2亚群毒株有着较近的亲缘关系。为了进一步研究13株PEDV的演化过程，以贝叶斯进化分析软件包（BEAST）进行分歧时间估算，结果表明SWUN-H3-CH-SCYA-2019的分歧时间约为2012.3年，早于藏猪源新G1亚群其余毒株的最早分歧时间（2015.7年）；SWUN-4-CH-SCXC-2018、SWUN-19-CH-SCZY-2018和SWUN-3CH-CH-SCZG-2019的分歧时间约为2014.2年，早于G2亚群的藏猪源毒株2014.7年，所有藏猪源PEDV的分歧时间均晚于四川毒株。本研究在四川地区首次发现了藏猪源PEDV，并且从毒株的分歧时间推断青藏高原的藏猪源PEDV来源于四川，为新型PEDV分子遗传进化的监测提供了依据。

关键词: 猪流行性腹泻病毒, S蛋白, 遗传分析, 分子钟

Abstract: Recently, two novel subgroups of porcine epidemic diarrhea virus (PEDV) were identified in Tibetan pigs on the Qinghai-Tibet Plateau. We further investigated if the novel variant PEDV exists or had been prevalent in Sichuan region. One hundred and sixteen fecal and intestinal tissue samples collected in 2018-2019 from diarrheal pigs of Sichuan were detected for PEDV by RT-PCR, and the molecular characteristics of Spike genes (S) of PEDV were further investigated in this study. The results showed that the detection rate of PEDV was 42.2% (49/116, 95%CI=33.1%-51.8%) in diarrhea samples. Thirteen complete S gene sequences were obtained, and they were 4 149-4 170 bp in length, sharing 94.2%-99.9% identities with each other. Interestingly, SWUN-H3-CH-SCYA-2019 shared 97.0%-98.6% nucleotide sequence identities with those of the novel G1 subgroup of Tibetan Pig PEDVs. Phylogenetic and evolutionary analysis showed that the 13 S genes obtained in this study could be divided into G1 and G2 subgroups, one of which (SWUN-H3-CH-SCYA-2019 strain) fell into the novel G1 subgroup of Tibetan Pig PEDVs; the SWUN-19-CH-SCZY-2018, SWUN-4-CH-SCXC-2018, SWUN-1-CH-SCNJ-2019 and SWUN-3CH-CH-SCZG-2019 were clustered into an unique branch of G2 subgroup, and shared high sequence identities with the novel G2 subgroup of Tibetan Pig PEDVs. To further study the evolution process of 13 PEDVs, the BEAST software was used to estimate the divergence time. The results showed that the divergence time of SWUN-H3-CH-SCYA-2019 was about 2012.3 year, earlier than the earliest divergence time of the other novel G1 subgroup strains (2015.7 year), and the divergence time of SWUN-4-CH-SCXC-2018, SWUN-19-CH-SCZY-2018 and SWUN-3CH-CH-SCZG-2019 were about 2014.2 year, earlier than that of the novel G2 subgroup (2014.7 year). The divergence time of Tibetan pig PEDVs was later than that of strains identified in this study. Tibetan Pig PEDVs were first detected in Sichuan region, and we deduced a conclusion from different divergence time that the novel Tibetan Pig PEDVs were originated from Sichuan region. This study will provide the theoretical basis for monitoring the new variation of PEDV.

Key words: porcine epidemic diarrhea virus, S protein, phylogenetic analysis, molecular clock

中图分类号:

S852.659.6

周昱行, 胡承哲, 周群, 王何欣, 董沙沙, 刘娟, 张丹丹, 任玉鹏, 张斌. 基于S基因序列分析新型猪流行性腹泻病毒的遗传演化[J]. 畜牧兽医学报, 2020, 51(1): 109-119.

ZHOU Yuxing, HU Chengzhe, ZHOU Qun, WANG Hexin, DONG Shasha, LIU Juan, ZHANG Dandan, REN Yupeng, ZHANG Bin. Phylogenetic and Evolutionary Analysis of Novel Porcine Epidemic Diarrhea Virus Based on S Gene[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(1): 109-119.

参考文献 26

[1]	陈溥言,王川庆,姜平,等. 兽医传染病学[M]. 6版. 北京:中国农业出版社, 2015:234-237.CHEN P Y, WANG C Q, JIANG P, et al. Veterinary Lemology[M]. 6th ed. Beijing:China Agriculture Press, 2015:234-237. (in Chinese)
[2]	LADINIG A.猪流行性腹泻病毒:各大洲的最新流行状况[J]. 张振东,译. 猪业科学, 2016, 33(4):26-27.LADINIG A. PEDV:an intercontinental update[J]. ZHANG Z D, trans. Swine Industry Science, 2016, 33(4):26-27. (in Chinese)
[3]	翁善钢. 猪流行性腹泻的流行与防控[J]. 养猪, 2015(1):121-122.WENG S G. Epidemiology and prevention of porcine epidemic diarrhea[J]. Swine Production, 2015(1):121-122. (in Chinese)
[4]	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.
[5]	董彦鹏. 2011-2012年我国猪流行性腹泻病毒ORF3基因分子流行病学调查与仔猪致病性研究[D]. 南京:南京农业大学, 2012.DONG Y P. During the period of 2011-2012 molecular epidemiology of Porcine Epidemic Diarrhea Virus based on ORF3 gene and pathogenicity of the virus in piglets[D]. Nanjing:Nanjing Agricultural University, 2012. (in Chinese)
[6]	王隆柏, 林裕胜,车勇良,等. 猪流行性腹泻病毒S、N和ORF3基因的遗传变异分析[J]. 畜牧兽医学报, 2014, 45(11):1830-1836.WANG L B, LIN Y S, CHE Y L, et al. Genetic variation analysis of S, N and ORF3 genes of porcine epidemic diarrhea virus[J]. Acta Veterinaria et Zootechnica Sinica, 2014, 45(11):1830-1836. (in Chinese)
[7]	陆承平. 兽医微生物学[M]. 5版. 北京:中国农业出版社, 2013:449.LU C P. Veterinary microbiology[M]. 5th ed. Beijing:China Agriculture Press, 2013:449. (in Chinese)
[8]	SONG D, PARK B. Porcine epidemic diarrhoea virus:a comprehensive review of molecular epidemiology, diagnosis, and vaccines[J]. Virus Genes, 2012, 44(2):167-175.
[9]	WANG D, FANG L R, XIAO S B. Porcine epidemic diarrhea in China[J]. Virus Res, 2016, 226:7-13.
[10]	WEN Z, LI J, ZHANG Y, et al. Genetic epidemiology of porcine epidemic diarrhoea virus circulating in China in 2012-2017 based on spike gene[J]. Transbound Emerg Dis, 2018, 65(3):883-889.
[11]	HUANG Y W, DICKERMAN A W, PINEYRO P, et al. Origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the United States[J]. mBio, 2013, 4(5):e00737-13.
[12]	QIN S N, HU C Z, YANG D J, et al. Emergence of porcine epidemic diarrhea viruses with the novel S genes in Tibetan pigs in the Qinghai-Tibetan plateau in China[J]. Virus Res, 2019, 270:197652.
[13]	CHEN Q, LI G W, STASKO J, et al. Isolation and characterization of porcine epidemic diarrhea viruses associated with the 2013 disease outbreak among swine in the United States[J]. J Clin Microbiol, 2014, 52(1):234-243.
[14]	SUN M, MA J L, WANG Y N, et al. Genomic and epidemiological characteristics provide new insights into the phylogeographical and spatiotemporal spread of porcine epidemic diarrhea virus in Asia[J]. J Clin Microbiol, 2015, 53(5):1484-1492.
[15]	董建国, 王瑞,曲哲会,等. 2014-2015年豫南地区猪流行性腹泻病毒S基因变异分析[J]. 畜牧兽医学报, 2018, 49(4):859-864.DONG J G, WANG R, QU Z H, et al. Genetic variations of S gene of porcine epidemic diarrhea virus in Sourthern Henan province from 2014 to 2015[J] Acta Veterinaria et Zootechnica Sinica, 2018, 49(4):859-864. (in Chinese)
[16]	NEFEDEVA M, TITOV I, MALOGOLOVKIN A. Molecular characteristics of a novel recombinant of porcine epidemic diarrhea virus[J]. Arch Virol, 2019, 164(4):1199-1204.
[17]	HANKE D, JENCKEL M, PETROV A, et al. Comparison of porcine epidemic diarrhea viruses from Germany and the United States, 2014[J]. Emerg Infect Dis, 2015, 21(3):493-496.
[18]	LI R F, QIAO S L, YANG Y Y, et al. Genome sequencing and analysis of a novel recombinant porcine epidemic diarrhea virus strain from Henan, China[J]. Virus Genes, 2016, 52(1):91-98.
[19]	张志,董雅琴,刘爽,等. 我国部分省份猪流行性腹泻的流行病学监测[J]. 中国动物检疫, 2014, 31(10):47-51.ZHANG Z, DONG Y Q, LIU S, et al. Survey and surveillance of porcine epidemic diarrhea in some provinces in China[J]. Chinese Journal of Animal Health Inspection, 2014, 31(10):47-51. (in Chinese)
[20]	马凡舒, 张蕾,王洋,等. B细胞抗原表位预测方法的研究进展[J]. 中国畜牧兽医, 2016, 43(1):63-67.MA F S, ZHANG L, WANG Y, et al. Advance on prediction methods of B-cell antigen epitope[J] China Animal Husbandry & Veterinary Medicine, 2016, 43(1):63-67. (in Chinese)
[21]	YANG W H, MARTH J D. Protein glycosylation energizes T cells[J]. Nat Immunol, 2016, 17(6):613-614.
[22]	WRÓBEL B, TORRES-PUENTE M, JIMÉNEZ N, et al. Analysis of the overdispersed clock in the short-term evolution of hepatitis C virus:using the E1/E2 gene sequences to infer infection dates in a single source outbreak[J]. Mol Biol Evol, 2006, 23(6):1242-1253.
[23]	FORSBERG R. Divergence time of porcine reproductive and respiratory syndrome virus subtypes[J]. Mol Biol Evol, 2005, 22(11):2131-2134.
[24]	HE M, DING N Z, HE C Q, et al. Dating the divergence of the infectious hematopoietic necrosis virus[J]. Infect Genet Evol, 2013, 18:145-150.
[25]	CHANG H P, PENG L, CHEN L, et al. Avian influenza viruses (AIVs) H9N2 are in the course of reassorting into novel AIVs[J]. J Zhejiang Univ-Sci B, 2018, 19(5):409-414.
[26]	HOLDER M, LEWIS P O. Phylogeny estimation:traditional and Bayesian approaches[J]. Nat Rev Genet, 2003, 4(4):275-284.