新型鸭呼肠孤病毒分离鉴定及其σC基因序列分析

doi:10.16431/j.cnki.1671-7236.2022.09.026

摘要/Abstract

摘要： 【目的】了解并掌握新型鸭呼肠孤病毒（Novel duck reovirus，NDRV）流行特点及生物学特性，为NDRV的防治提供理论基础和技术支持。【方法】采集河北某鸭场疑似发生鸭呼肠孤病毒感染的组织，无菌处理后接种SPF鸡胚分离病毒，收获第3代尿囊液进行血凝试验，通过PCR、透射电镜观察、间接免疫荧光法（IFA）鉴定病毒，对分离得到的病毒进行体外细胞培养和动物回归试验，并采用Mega 7.0对其σC基因进行遗传进化分析。【结果】分离得到的病毒不能凝集鸡红细胞，可致死鸡胚，死亡鸡胚出血、充血严重；经PCR鉴定，病毒呈NDRV阳性，其他病原（禽呼肠孤病毒、鸭病毒性肝炎、鸭坦布苏病毒、鸭瘟病毒、禽腺病毒血清4型）均呈阴性，将分离得到的病毒命名为BD/CHN/2020株；病毒纯化后，经电镜观察可见直径为60~80 nm、无囊膜的球形病毒粒子，该毒株可在BHK和LMH细胞上稳定增殖并产生细胞融合的细胞病变效应（CPE）；IFA结果显示，BD/CHN/2020株接种BHK细胞后在激光共聚焦显微镜下观察可见特异性绿色荧光；BD/CHN/2020株经皮下接种后，发病鸭出现精神沉郁、排白色稀粪等临床症状，剖检可见脾脏出血、肿大、有白色坏死灶等病理变化；序列比对发现，BD/CHN/2020株与NDRV毒株（TH11、091等毒株）在同一分支，与NDRV SY株核苷酸和氨基酸序列相似性最高，均为99.7%，属于NDRV。与灭活疫苗TH11株（KC493571.1）和弱毒疫苗JS01-105P株（V202168）相比，BD/CHN/2020株第93、120、132、158、253、298位氨基酸处发生了位点突变。【结论】成功分离得到1株NDRV BD/CHN/2020株，分离毒株对北京鸭有较强的致病性，与国内疫苗株相比，BD/CHN/2020株的σC蛋白已经发生了氨基酸位点的突变，结果可为新型鸭呼肠孤病毒病的流行病学及疫苗研发奠定基础。

关键词: 新型鸭呼肠孤病毒(NDRV); 分离鉴定; σC基因

Abstract: 【Objective】 The aim of this study was to understand and master the epidemiological characteristics and biological characteristics of Novel duck reovirus (NDRV), and to provide theoretical basis and technical support for the prevention and treatment of NDRV.【Method】 The tissues suspected of Duck reovirus infection was collected from a duck farm in Hebei, and after aseptic treatment, it was inoculated with SPF chicken embryos to isolate the virus, and the third-generation allantoic fluid was harvested for hemagglutination test and in vitro cell culture.The virus was identified by observation and indirect immunofluorescence assay (IFA).The isolated virus was subjected to animal regression test, and its σC gene was analyzed by Mega 7.0.【Result】 The isolated virus couldn't agglutinate chicken erythrocytes, and could kill chicken embryos.The dead chicken embryos were hemorrhagic and congested.The virus was NDRV positive by PCR identification, and other pathogens (Avian reovirus, Duck hepatitis virus, Duck Tembusu virus, Duck plague virus and Fowl adenovirus 4) were all negative, and the isolated virus was named BD/CHN/2020 strain.Spherical virus particles with a diameter of 60-80 nm were observed by electron microscopy and no envelope could be seen after viruses were purificated.The strain could be proliferated stably on BHK cells and LMH cells and produced a cytopathic effect (CPE) of cell fusion.IFA results showed that specific green fluorescence could be observed under a laser confocal microscope after BD/CHN/2020 strain was inoculated in BHK cells.After the BD/CHN/2020 strain was subcutaneously inoculated, the diseased ducks showed clinical symptoms such as depression and white loose stools.The autopsy showed pathological changes such as spleen hemorrhage, enlargement, and white necrotic foci.Sequence alignment showed that the BD/CHN/2020 strain and NDRV (TH11, 091 and other strains) were in the same branch, and had the highest similarity of nucleotide and amino acid sequences to the NDRV SY strain, which were both 99.7%, and belonged to the NDRV.Compared with the inactivated vaccine TH11 strain (KC493571.1) and the attenuated vaccine JS01-105P strain (CCTCC No.:V202168), the amino acid site mutations of BD/CHN/2020 strain had amino acid site mutations at sites 93, 120, 132, 158, 253 and 298.【Conclusion】 One NDRV BD/CHN/2020 strain was successfully isolated.The isolated strain had strong pathogenicity to Peking duck.Compared with the domestic vaccine strain, the σC protein of the BD/CHN/2020 strain had already undergone amino acid changes.The results laid the foundation for the epidemiology and vaccine development of NDRV disease.

Key words: Novel duck reovirus (NDRV); isolation and identification; σC gene

中图分类号:

S858.32

张薇, 武华, 阴雅洁, 李松励, 侯绍华. 新型鸭呼肠孤病毒分离鉴定及其σC基因序列分析[J]. 中国畜牧兽医, 2022, 49(9): 3520-3529.

ZHANG Wei, WU Hua, YIN Yajie, LI Songli, HOU Shaohua. Isolation and Identification of Novel Duck Reovirus and Its σC Gene Sequence Analysis[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(9): 3520-3529.

参考文献

[1] YANG Y, LI L, LIU X, et al.Quantitative proteomic analysis of duck embryo fibroblasts infected with Novel duck reovirus[J].Frontiers in Veterinary Science, 2020, 7:577370.
[2] LIU Q, ZhANG G, HUANG Y, et al.Isolation and characterization of a Reovirus causing spleen necrosis in Pekin ducklings[J].Veterinary Microbiology, 2011, 148(2-4):200-206.
[3] FARKAS S L, VARGA-KUGLER R, MARTON S, et al.Genomic sequence and phylogenetic analyses of two Novel orthoreovirus strains isolated from Pekin ducks in 2014 in Germany[J].Virus Research, 2018, 257:57-62.
[4] XIAO R, MI X, SUN J, et al.Interaction between translocation-associated membrane protein 1 and σC protein of Novel duck reovirus controls virus infectivity[J].Virus Genes, 2020, 56(3):347-353.
[5] ZHANG H, SONG X, LI T, et al.DDX1 from Cherry valley duck mediates signaling pathways and anti-NDRV activity[J].Veterinary Research, 2021, 52(1):9.
[6] 朱旭.新型鸭呼肠孤病毒基因组序列解析及其对雏鸭致病性差异的病理学研究[D].武汉:华中农业大学, 2018. ZHU X.Genomic characteristics and pathological study on the pathogenicity of Novel duck reovirus in duckings[D].Wuhan:Huazhong Agricultural University, 2018.(in Chinese)
[7] 陈少莺, 陈仕龙, 林锋强, 等.新型鸭呼肠孤病毒的分离与鉴定[J].病毒学报, 2012, 28(3):224-230. CHEN S Y, CHEN S L, LIN F Q, et al.Isolation and identification of a Novel duck reovirus[J].Chinese Journal of Virology, 2012, 28(3):224-230.(in Chinese)
[8] YAN H, XU G, ZHU Y, et al.Isolation and characterization of a naturally attenuated Novel duck reovirus strain as a live vaccine candidate[J]. Veterinary Microbiology, 2021, 261(5):109214.
[9] 王丹.鸭源呼肠孤病毒的表型和分子特征分析及基因2型毒株编码的新蛋白的鉴定[D].北京:中国农业大学, 2016. WANG D.Analysis of phenotypic and molecular characteristics of duck-origin Reoviruses and identification of novel proteins encoded by the genotype 2 strain[D].Beijing:China Agricultural University, 2016.(in Chinese)
[10] 袁远华, 吴志新, 黄兴国, 等.新型鸭呼肠孤病毒病研究进展[J].养禽与禽病防治, 2012, 10:18-20. YUAN Y H, WU Z X, HUANG X G, et al.Research progress of Novel duck reovirus disease[J].Poultry Husbandry and Disease Control, 2012, 10:18-20.(in Chinese)
[11] 谢碧林, 林志敏, 林彬彬, 等.新型鸭呼肠孤病毒病研究进展[J].福建畜牧兽医, 2021, 43(1):23-26. XIE B L, LIN Z M, LIN B B, et al.Research progress of Novel duck reovirus disease[J].Fujian Journal of Animal Husbandry and Veterinary Medicine, 2021, 43(1):23-26.(in Chinese)
[12] 中国兽药典委员会.中国人民共和国兽药典[M].北京:中国农业出版社, 2020. CVPC.Veterinary Pharmacopoeia of the People's Republic of China[M].Beijing:China Agriculture Press, 2020.(in Chinese)
[13] 李静.新型鸭呼肠孤病毒的分离鉴定及油乳剂灭活疫苗的制备[D].泰安:山东农业大学, 2020. LI J.Isolation and identification of a Novel duck reovirus and screening of oil emulsion inactivated vaccine[D].Tai'an:Shandong Agricultural University, 2020.(in Chinese)
[14] 李海琴, 傅光华, 康昭风, 等.新型鸭呼肠孤病毒、新型鹅细小病毒和鸭坦布苏病毒多重PCR检测方法的建立及初步应用[J].福建农业学报, 2022, 37(1):1-6. LI H Q, FU G H, KANG Z F, et al.Establishment and preliminary application of a multiple PCR assay for detection of NDRV, NGPV and DTMUV[J].Fujian Journal of Agricultural Sciences, 2022, 37(1):1-6.(in Chinese)
[15] 于可响, 路晓, 刘存霞, 等.山东地区新型鸭呼肠孤病毒致病特点与变异分析[J].中国动物传染病学报, 2018, 26(4):24-28. YU K X, LU X, LIU C X, et al.Pathogenic characteristics and variation analysis of Novel duck reovirus isolated from Shandong province[J].Chinese Journal of Animal Infectious Diseases, 2018, 26(4):24-28.(in Chinese)
[16] 陆婧, 李敏, 王艳, 等.鸭呼肠孤病毒TaqMan与SYBR Green荧光定量检测方法的建立及比较[J].中国兽医学报, 2022, 42(2):237-243. LU J, LI M, WANG Y, et al.Establishment and comparison of TaqMan and SYBR Green Real-time fluorescence quantitative PCR detection methods for methods for Novel duck reovirus[J].Chinese Journal of Veterinary Science, 2022, 42(2):237-243.(in Chinese)
[17] 刘晓丽, 刘婷, 刘波, 等.鸭呼肠孤病毒人工感染SPF鸡胚的病理学研究[J].中国农业科学, 2016, 49(14):2844-2849. LIU X L, LIU T, LIU B, et al.The pathogenicity of Duck reovirus on SPF chicken embryo[J].Scientia Agricultura Sinica, 2016(14):2844-2849.(in Chinese)
[18] 武鸿, 康亚男, 唐荣宏, 等.3株樱桃谷肉鸭源和2株番鸭源新型鸭呼肠孤病毒的分离与鉴定[J].南京农业大学学报, 2019, 42(2):328-335. WU H, KANG Y N, TANG R H, et al.The isolation and identification of Novel duck reovirus from Cherry valley ducks(3 strains) and Muscovy ducks(2 strains)[J].Journal of Nanjing Agricultural University, 2019, 42(2):328-335.(in Chinese)
[19] 马芹, 许明清, 汪建华, 等.新型鸭呼肠孤病毒山东株的分离鉴定及致病性分析[J].中国兽医杂志, 2021, 57(12):44-47. MA Q, XU M Q, WANG J H, et al.Isolation, identification and pathogenic analysis of a Novel duck reovirus isolated from Shandong province[J].Chinese Journal of Veterinary Medicine, 2021, 57(12):44-47.(in Chinese)
[20] 晁锦, 卢凤英, 潘群兴, 等.新型鸭呼肠孤病毒SY株的分离鉴定及培养特性[J].中国动物传染病学报, 2020, 5:11-18. CHAO J, LU F Y, PAN Q X, et al.Isolation, identification and culture characteristics of Novel duck reovirus strain SY[J]. Chinese Journal of Animal Infectious Diseases, 2020, 5:11-18.(in Chinese)
[21] BI Z, ZHU Y, CHEN Z, et al.Induction of a robust immunity response against Novel duck reovirus in ducklings using a subunit vaccine of sigma C protein[J].Scientific Reports, 2016, 6:39092.
[22] 田昂.新型鸭呼肠孤病毒间接ELISA检测方法的建立及应用[D].泰安:山东农业大学, 2021. TIAN A.Establishment and application of indirect EL1SA detection method for Novel duck reovirus[D].Tai'an:Shandong Agricultural University, 2021.(in Chinese)
[23] LUO D, LIU R, WENG L, et al.Genomic sequences and pathogenic characteristics of two variant Duck reoviruses associated with spleen necrosis[J]. Infection Genetics and Evolution, 2021, 92:104847.