一株牦牛源G6P[11]型牛轮状病毒的分离鉴定及部分基因的序列分析
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摘要
本研究的目的是分离、鉴定牦牛源牛轮状病毒(bovine rotavirus, BRV)。将经RT-PCR检测BRV阳性的牦牛腹泻粪便样本接种MA-104细胞,进行病毒分离和鉴定,并对其VP4、VP6和VP7完整基因进行测序,分析其分子特征。结果显示:病毒盲传3代后出现细胞病变,至第7代后出现细胞病变的时间稳定,经蚀斑纯化后测得病毒滴度为10~(8.39)TCID_(50)·mL~(-1)。分离株经RT-PCR、间接免疫荧光和电镜观察证实为BRV,命名为HY-1株;扩增HY-1株VP4、VP6和VP7完整基因序列,分析表明HY-1株为G6P[11]I2型;系统发育树显示,HY-1株VP4、VP6和VP7节段分别与中国牛源DQ-75株和印度牛源M-1和RUBV319株遗传演化关系最近,可能为重配毒株。与国内的G6型和P[11]型BRV毒株相比,HY-1株的VP4和VP7蛋白重要氨基酸位点变化较大。成功分离得到一株牦牛源BRV,基因型为G6P[11]型,为我国首次报道。
The aim of this study was to isolate and identify bovine rotavirus(BRV) from the diarrhea stool samples of Yak. BRV-positive samples detected by RT-PCR was used to inoculate into MA-104 cells for virus isolation and identification, and the complete genes of VP4, VP6 and VP7 were sequenced to determine their molecular characteristics. The cytopathic effect occurred after three continuous blind passages, and the stable time that cytopathic effect occurred was found after seven passages. The virus titer was 10~(8.39)TCID_(50)·mL~(-1) after plaque purification. The isolate, named HY-1 strain, was confirmed to be BRV by RT-PCR, indirect immunofluorescence and electron microscopy. The complete VP4, VP6 and VP7 genes of HY-1 strain were sequenced successfully, showing that HY-1 strain belong to G6 P[11]I2 genotype. The phylogenetic analysis showed that the complete VP4, VP6 and VP7 segments of HY-1 strain were closely genetic related to Chinese bovine DQ-75 strain and Indian bovine M-1 and RUBV319 strains, respectively, which indicated that HY-1 strain may be a reassortant strain. Compared with the domestic G6 and P[11] BRV strains, VP4 and VP7 of HY-1 isolate had significant variation in the important amino acid regions. In this study, a BRV strain was successfully isolated from yak with G6 P[11] genotype. To the best of our knowledge, this is the first report on the identification of G6 P[11] BRV in China.
The aim of this study was to isolate and identify bovine rotavirus(BRV) from the diarrhea stool samples of Yak. BRV-positive samples detected by RT-PCR was used to inoculate into MA-104 cells for virus isolation and identification, and the complete genes of VP4, VP6 and VP7 were sequenced to determine their molecular characteristics. The cytopathic effect occurred after three continuous blind passages, and the stable time that cytopathic effect occurred was found after seven passages. The virus titer was 10~(8.39)TCID_(50)·mL~(-1) after plaque purification. The isolate, named HY-1 strain, was confirmed to be BRV by RT-PCR, indirect immunofluorescence and electron microscopy. The complete VP4, VP6 and VP7 genes of HY-1 strain were sequenced successfully, showing that HY-1 strain belong to G6 P[11]I2 genotype. The phylogenetic analysis showed that the complete VP4, VP6 and VP7 segments of HY-1 strain were closely genetic related to Chinese bovine DQ-75 strain and Indian bovine M-1 and RUBV319 strains, respectively, which indicated that HY-1 strain may be a reassortant strain. Compared with the domestic G6 and P[11] BRV strains, VP4 and VP7 of HY-1 isolate had significant variation in the important amino acid regions. In this study, a BRV strain was successfully isolated from yak with G6 P[11] genotype. To the best of our knowledge, this is the first report on the identification of G6 P[11] BRV in China.
引文
[1] CHO Y I, YOON K J. An overview of calf diarrhea-infectious etiology, diagnosis, and intervention[J]. J Vet Sci, 2014, 15(1):1-17.
[2] 李鑫,常继涛,刘海军,等. 我国A群牛轮状病毒感染的血清流行病学调查[J]. 中国预防兽医学报, 2010, 32(9):664-667.LI X, CHANG J T, LIU H J, et al. Seroepidemiological survey of the group A bovine rotavirus infection in China[J]. Chinese Journal of Preventive Veterinary Medicine, 2010, 32(9):664-667. (in Chinese)
[3] 周芳. 牦牛轮状病毒的RT-PCR检测方法的建立和应用及病毒的分离培养[D]. 成都:西南民族大学, 2016.ZHOU F. The establishment and application of detection method by RT-PCR and the separation of yak rotavirus[D]. Chengdu: Southwest Minzu University, 2016. (in Chinese)
[4] 李凡,岳华,邱莞思,等. 基于恒温隔绝式荧光PCR技术现场检测牛轮状病毒方法的建立及应用[J]. 畜牧兽医学报, 2017, 48(6):1092-1098.LI F, YUE H, QIU W S, et al. Development and application of an insulated isothermal PCR (iiPCR) for on-site detecting bovine rotavirus[J]. Acta Veterinaria et Zootechnica Sinica, 2017, 48(6):1092-1098. (in Chinese)
[5] DESSELBERGER U. Rotaviruses[J]. Virus Res, 2014, 190:75-96.
[6] MEDICI M C, ABELLI L A, MARTINELLI M, et al. Molecular characterization of VP4, VP6 and VP7 genes of a rare G8P[14] rotavirus strain detected in an infant with gastroenteritis in Italy[J]. Virus Res, 2008, 137(1):163-167.
[7] DA SILVA MEDEIROS T N, LORENZETTI E, ALFIERI A F, et al. Phylogenetic analysis of a G6P[5] bovine rotavirus strain isolated in a neonatal diarrhea outbreak in a beef cattle herd vaccinated with G6P[1] and G10P[11] genotypes[J]. Arch Virol, 2015, 160(2):447-451.
[8] ENNIMA I, SEBBAR G, HARIF B, et al. Isolation and identification of group A rotaviruses among neonatal diarrheic calves, Morocco[J]. BMC Res Notes, 2016, 9:261.
[9] IANIRO G, DELOGU R, FIORE L, et al. Genomic characterization and molecular investigation of VP7 epitopes of uncommon G10P[8] group A rotavirus strains detected in Italy in 2009[J]. J Gen Virol, 2015, 96(7):1801-1810.
[10] LAPPALAINEN S, BLAZEVIC V, MALM M, et al. Rotavirus vaccination and infection induce VP6-specific IgA responses[J]. J Med Virol, 2017, 89(2):239-245.
[11] MATTHIJNSSENS J, CIARLET M, HEIMAN E, et al. Full genome-based classification of rotaviruses reveals a common origin between human Wa-like and porcine rotavirus strains and human DS-1-like and bovine rotavirus strains[J]. JoVirol, 2008, 82(7):3204-3219.
[12] VAN DOORN L J, KLETER B, HOEFNAGEL E, et al. Detection and genotyping of human rotavirus VP4 and VP7 genes by reverse transcriptase PCR and reverse hybridization[J]. J Clin Microbiol, 2009, 47(9):2704-2712.
[13] PAPP H, LáSZLó B, JAKAB F, et al. Review of group A rotavirus strains reported in swine and cattle[J]. Vet Microbiol, 2013, 165(3-4):190-199.
[14] 常继涛, 于力. 牛轮状病毒引起的犊牛腹泻研究进展[J]. 中国奶牛, 2016(2):22-25.CHANG J T, YU L. Research progress on calf diarrhea caused by bovine rotavirus[J]. China Dairy Cattle, 2016(2):22-25. (in Chinese)
[15] 王鑫. 牛轮状病毒HQ09株的分离与鉴定[D]. 哈尔滨:东北农业大学, 2012.WANG X. Isolation and identification of bovine rotavirus HQ09 strain[D]. Harbin:Northeast Agricultural University, 2012. (in Chinese)
[16] 闻晓波, 张玲玲,倪宏波,等. G6P[5]型牛轮状病毒的分离鉴定[J]. 黑龙江八一农垦大学学报, 2016, 28(4):36-40.WEN X B, ZHANG L L, NI H B, et al. Isolation and characterization of bovine rotavirus with G6P[5] specificity[J]. Journal of Heilongjiang Bayi Agricultural University, 2016, 28(4):36-40. (in Chinese)
[17] 常继涛, 崔久辉,李建军,等. 我国部分地区牛轮状病毒的病原学调查及一株G10P[11]型牛轮状病毒的分离鉴定[J]. 中国预防兽医学报, 2008, 30(10):755-759.CHANG J T, CUI J H, LI J J, et al. Etiologic investigation of bovine rotavirus in the some regions of China and isolation of a G10P[11] rotavirus[J]. Chinese Journal of Preventive Veterinary Medicine, 2008, 30(10):755-759. (in Chinese)
[18] 谢金鑫. 大庆地区牛轮状病毒的分离及其全序列分析[D]. 大庆:黑龙江八一农垦大学, 2010.XIE J X. Isolated bovine rotavirus in a diary farm in Daqing and analyzed full genomic sequence[D]. Daqing:Heilongjiang Bayi Agricultural University, 2010. (in Chinese)
[19] 栾婧婧, 杨少华,马广强,等. 新分离牛轮状病毒及其血清型的研究[J]. 家畜生态学报, 2006, 27(6):141-144.LUAN J J, YANG S H, MA G Q, et al. Study on the newly isolated rotavirus and its serotype[J]. Acta Ecologiae Animalis Domastici, 2006, 27(6):141-144. (in Chinese)
[20] 姜向阳. 陕西省牛轮状病毒的分离鉴定及其VP7基因的遗传变异分析[D]. 杨凌:西北农林科技大学, 2007.JIANG X Y. Isolation and identification of bovine rotavirus in Shaanxi province and analysis of genetic variation of VP7 gene[D]. Yangling: Northwest A&F University, 2007. (in Chinese)
[21] 张敏,王清江,刘登薇,等. 红原牦牛轮状病毒的研究[J]. 四川畜牧兽医, 1986(3):2-4.ZHANG M, WANG Q J, LIU D W, et al. Study on Hongyuan Yak rotavirus[J]. Sichuan Animal & Veterinary Sciences, 1986(3):2-4. (in Chinese)
[22] 周芳,岳华,张斌,等. 牦牛轮状病毒VP6基因序列分析及RT-PCR检测方法的建立与应用[J]. 畜牧兽医学报, 2016, 47(7): 1465-1473.ZHOU F,YUE H,ZHANG B, et al. Establishment and application of an RT-PCR assay for yak rotavirus based on the sequence analysis of yak rotavirus VP6 gene[J]. Acta Veterinaria et Zootechnica Sinica, 2016, 47(7): 1465-1473. (in Chinese)
[23] MAES P, MATTHIJNSSENS J, RAHMAN M, et al. RotaC:a web-based tool for the complete genome classification of group A rotaviruses[J]. BMC Microbiol, 2009, 9:238.
[24] BADARACCO A, GARAICOECHEA L, RODRíGUEZ D, et al. Bovine rotavirus strains circulating in beef and dairy herds in Argentina from 2004 to 2010[J]. Vet Microbiol, 2012, 158(3-4):394-399.
[25] POURASGARI F, KAPLON J, KARIMI-NAGHLANI S, et al. The molecular epidemiology of bovine rotaviruses circulating in Iran:a two-year study[J]. Arch Virol, 2016, 161(12):3483-3494.
[26] SWIATEK D L, PALOMBO E A, LEE A, et al. Detection and analysis of bovine rotavirus strains circulating in Australian calves during 2004 and 2005[J]. Vet Microbiol, 2010, 140(1-2):56-62.
[27] KHANDOKER N, THONGPRACHUM A, TAKANASHI S, et al. Molecular epidemiology of rotavirus gastroenteritis in Japan during 2014-2015:characterization of re-emerging G2P[4] after rotavirus vaccine introduction[J]. J Med Virol, 2018, 90(6):1040-1046.
[28] STEYER A, NAGLI, et al. Detection and whole-genome analysis of a zoonotic G8P[14] rotavirus strain isolated from a child with diarrhea[J]. Genome Announc, 2017, 5(41):e01053-17.
[29] TACHAROENMUANG R, KOMOTO S, GUNTAPONG R, et al. Characterization of a G10P[14] rotavirus strain from a diarrheic child in Thailand:evidence for bovine-to-human zoonotic transmission[J]. Infect, Genet Evol, 2018, 63:43-57.
[30] BWOGI J, JERE K C, KARAMAGI C, et al. Whole genome analysis of selected human and animal rotaviruses identified in Uganda from 2012 to 2014 reveals complex genome reassortment events between human, bovine, caprine and porcine strains[J]. PLoS One, 2017, 12(6):e0178855.
[31] GHOSH S, VARGHESE V, SAMAJDAR S, et al. Evidence for bovine origin of VP4 and VP7 genes of human group A rotavirus G6P[14] and G10P[14] strains[J]. J Clin Microbiol, 2007, 45(8):2751-2753.
[32] MALIK Y S, KUMAR N, SHARMA K, et al. Multispecies reassortant bovine rotavirus strain carries a novel simian G3-like VP7 genotype[J]. Infect, Genet Evol, 2016, 41:63-72.
[33] DORMITZER P R, NASON E B, PRASAD B V V, et al. Structural rearrangements in the membrane penetration protein of a non-enveloped virus[J]. Nature, 2004, 430(7003):1053-1058.
[34] MOHANTY E, DEHURY B, Satapathy A K, et al. Design and testing of a highly conserved human rotavirus VP8* immunogenic peptide with potential for vaccine development[J]. J Biotechnol, 2018, 281:48-60.
[35] SUN X M, LI D D, QI J X, et al. Glycan binding specificity and mechanism of human and porcine P[6]/P[19] rotavirus VP8*s[J]. J Virol, 2018, 92(14):JVI. 00538-18.
[36] AOKI S T, SETTEMBRE E C, TRASK S D, et al. Structure of rotavirus outer-layer protein VP7 bound with a neutralizing Fab[J]. Science, 2009, 324(5933):1444-1447.
[37] JING Z, ZHANG X, SHI H, et al. A G3P[13] porcine group A rotavirus emerging in China is a reassortant and a natural recombinant in the VP4 gene[J]. Transbound Emerg Dis, 2018, 65(2):e317-e328.
[2] 李鑫,常继涛,刘海军,等. 我国A群牛轮状病毒感染的血清流行病学调查[J]. 中国预防兽医学报, 2010, 32(9):664-667.LI X, CHANG J T, LIU H J, et al. Seroepidemiological survey of the group A bovine rotavirus infection in China[J]. Chinese Journal of Preventive Veterinary Medicine, 2010, 32(9):664-667. (in Chinese)
[3] 周芳. 牦牛轮状病毒的RT-PCR检测方法的建立和应用及病毒的分离培养[D]. 成都:西南民族大学, 2016.ZHOU F. The establishment and application of detection method by RT-PCR and the separation of yak rotavirus[D]. Chengdu: Southwest Minzu University, 2016. (in Chinese)
[4] 李凡,岳华,邱莞思,等. 基于恒温隔绝式荧光PCR技术现场检测牛轮状病毒方法的建立及应用[J]. 畜牧兽医学报, 2017, 48(6):1092-1098.LI F, YUE H, QIU W S, et al. Development and application of an insulated isothermal PCR (iiPCR) for on-site detecting bovine rotavirus[J]. Acta Veterinaria et Zootechnica Sinica, 2017, 48(6):1092-1098. (in Chinese)
[5] DESSELBERGER U. Rotaviruses[J]. Virus Res, 2014, 190:75-96.
[6] MEDICI M C, ABELLI L A, MARTINELLI M, et al. Molecular characterization of VP4, VP6 and VP7 genes of a rare G8P[14] rotavirus strain detected in an infant with gastroenteritis in Italy[J]. Virus Res, 2008, 137(1):163-167.
[7] DA SILVA MEDEIROS T N, LORENZETTI E, ALFIERI A F, et al. Phylogenetic analysis of a G6P[5] bovine rotavirus strain isolated in a neonatal diarrhea outbreak in a beef cattle herd vaccinated with G6P[1] and G10P[11] genotypes[J]. Arch Virol, 2015, 160(2):447-451.
[8] ENNIMA I, SEBBAR G, HARIF B, et al. Isolation and identification of group A rotaviruses among neonatal diarrheic calves, Morocco[J]. BMC Res Notes, 2016, 9:261.
[9] IANIRO G, DELOGU R, FIORE L, et al. Genomic characterization and molecular investigation of VP7 epitopes of uncommon G10P[8] group A rotavirus strains detected in Italy in 2009[J]. J Gen Virol, 2015, 96(7):1801-1810.
[10] LAPPALAINEN S, BLAZEVIC V, MALM M, et al. Rotavirus vaccination and infection induce VP6-specific IgA responses[J]. J Med Virol, 2017, 89(2):239-245.
[11] MATTHIJNSSENS J, CIARLET M, HEIMAN E, et al. Full genome-based classification of rotaviruses reveals a common origin between human Wa-like and porcine rotavirus strains and human DS-1-like and bovine rotavirus strains[J]. JoVirol, 2008, 82(7):3204-3219.
[12] VAN DOORN L J, KLETER B, HOEFNAGEL E, et al. Detection and genotyping of human rotavirus VP4 and VP7 genes by reverse transcriptase PCR and reverse hybridization[J]. J Clin Microbiol, 2009, 47(9):2704-2712.
[13] PAPP H, LáSZLó B, JAKAB F, et al. Review of group A rotavirus strains reported in swine and cattle[J]. Vet Microbiol, 2013, 165(3-4):190-199.
[14] 常继涛, 于力. 牛轮状病毒引起的犊牛腹泻研究进展[J]. 中国奶牛, 2016(2):22-25.CHANG J T, YU L. Research progress on calf diarrhea caused by bovine rotavirus[J]. China Dairy Cattle, 2016(2):22-25. (in Chinese)
[15] 王鑫. 牛轮状病毒HQ09株的分离与鉴定[D]. 哈尔滨:东北农业大学, 2012.WANG X. Isolation and identification of bovine rotavirus HQ09 strain[D]. Harbin:Northeast Agricultural University, 2012. (in Chinese)
[16] 闻晓波, 张玲玲,倪宏波,等. G6P[5]型牛轮状病毒的分离鉴定[J]. 黑龙江八一农垦大学学报, 2016, 28(4):36-40.WEN X B, ZHANG L L, NI H B, et al. Isolation and characterization of bovine rotavirus with G6P[5] specificity[J]. Journal of Heilongjiang Bayi Agricultural University, 2016, 28(4):36-40. (in Chinese)
[17] 常继涛, 崔久辉,李建军,等. 我国部分地区牛轮状病毒的病原学调查及一株G10P[11]型牛轮状病毒的分离鉴定[J]. 中国预防兽医学报, 2008, 30(10):755-759.CHANG J T, CUI J H, LI J J, et al. Etiologic investigation of bovine rotavirus in the some regions of China and isolation of a G10P[11] rotavirus[J]. Chinese Journal of Preventive Veterinary Medicine, 2008, 30(10):755-759. (in Chinese)
[18] 谢金鑫. 大庆地区牛轮状病毒的分离及其全序列分析[D]. 大庆:黑龙江八一农垦大学, 2010.XIE J X. Isolated bovine rotavirus in a diary farm in Daqing and analyzed full genomic sequence[D]. Daqing:Heilongjiang Bayi Agricultural University, 2010. (in Chinese)
[19] 栾婧婧, 杨少华,马广强,等. 新分离牛轮状病毒及其血清型的研究[J]. 家畜生态学报, 2006, 27(6):141-144.LUAN J J, YANG S H, MA G Q, et al. Study on the newly isolated rotavirus and its serotype[J]. Acta Ecologiae Animalis Domastici, 2006, 27(6):141-144. (in Chinese)
[20] 姜向阳. 陕西省牛轮状病毒的分离鉴定及其VP7基因的遗传变异分析[D]. 杨凌:西北农林科技大学, 2007.JIANG X Y. Isolation and identification of bovine rotavirus in Shaanxi province and analysis of genetic variation of VP7 gene[D]. Yangling: Northwest A&F University, 2007. (in Chinese)
[21] 张敏,王清江,刘登薇,等. 红原牦牛轮状病毒的研究[J]. 四川畜牧兽医, 1986(3):2-4.ZHANG M, WANG Q J, LIU D W, et al. Study on Hongyuan Yak rotavirus[J]. Sichuan Animal & Veterinary Sciences, 1986(3):2-4. (in Chinese)
[22] 周芳,岳华,张斌,等. 牦牛轮状病毒VP6基因序列分析及RT-PCR检测方法的建立与应用[J]. 畜牧兽医学报, 2016, 47(7): 1465-1473.ZHOU F,YUE H,ZHANG B, et al. Establishment and application of an RT-PCR assay for yak rotavirus based on the sequence analysis of yak rotavirus VP6 gene[J]. Acta Veterinaria et Zootechnica Sinica, 2016, 47(7): 1465-1473. (in Chinese)
[23] MAES P, MATTHIJNSSENS J, RAHMAN M, et al. RotaC:a web-based tool for the complete genome classification of group A rotaviruses[J]. BMC Microbiol, 2009, 9:238.
[24] BADARACCO A, GARAICOECHEA L, RODRíGUEZ D, et al. Bovine rotavirus strains circulating in beef and dairy herds in Argentina from 2004 to 2010[J]. Vet Microbiol, 2012, 158(3-4):394-399.
[25] POURASGARI F, KAPLON J, KARIMI-NAGHLANI S, et al. The molecular epidemiology of bovine rotaviruses circulating in Iran:a two-year study[J]. Arch Virol, 2016, 161(12):3483-3494.
[26] SWIATEK D L, PALOMBO E A, LEE A, et al. Detection and analysis of bovine rotavirus strains circulating in Australian calves during 2004 and 2005[J]. Vet Microbiol, 2010, 140(1-2):56-62.
[27] KHANDOKER N, THONGPRACHUM A, TAKANASHI S, et al. Molecular epidemiology of rotavirus gastroenteritis in Japan during 2014-2015:characterization of re-emerging G2P[4] after rotavirus vaccine introduction[J]. J Med Virol, 2018, 90(6):1040-1046.
[28] STEYER A, NAGLI, et al. Detection and whole-genome analysis of a zoonotic G8P[14] rotavirus strain isolated from a child with diarrhea[J]. Genome Announc, 2017, 5(41):e01053-17.
[29] TACHAROENMUANG R, KOMOTO S, GUNTAPONG R, et al. Characterization of a G10P[14] rotavirus strain from a diarrheic child in Thailand:evidence for bovine-to-human zoonotic transmission[J]. Infect, Genet Evol, 2018, 63:43-57.
[30] BWOGI J, JERE K C, KARAMAGI C, et al. Whole genome analysis of selected human and animal rotaviruses identified in Uganda from 2012 to 2014 reveals complex genome reassortment events between human, bovine, caprine and porcine strains[J]. PLoS One, 2017, 12(6):e0178855.
[31] GHOSH S, VARGHESE V, SAMAJDAR S, et al. Evidence for bovine origin of VP4 and VP7 genes of human group A rotavirus G6P[14] and G10P[14] strains[J]. J Clin Microbiol, 2007, 45(8):2751-2753.
[32] MALIK Y S, KUMAR N, SHARMA K, et al. Multispecies reassortant bovine rotavirus strain carries a novel simian G3-like VP7 genotype[J]. Infect, Genet Evol, 2016, 41:63-72.
[33] DORMITZER P R, NASON E B, PRASAD B V V, et al. Structural rearrangements in the membrane penetration protein of a non-enveloped virus[J]. Nature, 2004, 430(7003):1053-1058.
[34] MOHANTY E, DEHURY B, Satapathy A K, et al. Design and testing of a highly conserved human rotavirus VP8* immunogenic peptide with potential for vaccine development[J]. J Biotechnol, 2018, 281:48-60.
[35] SUN X M, LI D D, QI J X, et al. Glycan binding specificity and mechanism of human and porcine P[6]/P[19] rotavirus VP8*s[J]. J Virol, 2018, 92(14):JVI. 00538-18.
[36] AOKI S T, SETTEMBRE E C, TRASK S D, et al. Structure of rotavirus outer-layer protein VP7 bound with a neutralizing Fab[J]. Science, 2009, 324(5933):1444-1447.
[37] JING Z, ZHANG X, SHI H, et al. A G3P[13] porcine group A rotavirus emerging in China is a reassortant and a natural recombinant in the VP4 gene[J]. Transbound Emerg Dis, 2018, 65(2):e317-e328.