流行性出血病病毒(EHDV)血清7型毒株在中国的首次分离与鉴定
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摘要
流行性出血病(EHD)是由流行性出血病病毒(EHDV)感染反刍动物引起的一种虫媒病毒病,为了解中国云南EHDV的感染情况和病毒遗传特征,笔者课题组在云南省师宗县以EHDV血清学和核酸阴性的牛、羊为哨兵动物,拟对流行于云南省的EHDV进行分离与鉴定。将采集于哨兵动物的EHDV核酸阳性血液接种BHK-21细胞进行病毒分离;通过血清中和试验与病毒Seg-2、Seg-3 ORF区的序列分析,确定病毒的血清型与遗传特征;采用C-ELISA和qRT-PCR方法对EHDV感染动物血液中的抗体水平与病毒核酸进行监测。结果如下:从2013年8月采集自云南师宗县哨兵牛的血样中分离出一株EHDV(毒株号YNSZ/V269/2013),血清中和试验结果表明分离的病毒为血清型7型(EHDV-7);Seg-2、Seg-3序列分析表明分离的病毒属EHDV-7 Eastern型,与日本毒株和澳大利亚EHDV-7型毒株具有最近的亲缘关系。哨兵动物病毒核酸转阳后,血清抗体迅速上升,3周后达最高点并能在该水平持续较长时间,而病毒核酸含量却迅速下降,7周后已经检测不到。本研究首次报道了EHDV-7型毒株在中国的分离、毒株序列特征以及在动物上的感染特性。研究结果可为进一步开展中国EHDV-7型病毒的全基因组测序、流行病学调查、诊断方法的建立和致病性等相关研究提供基础。
Epizootic haemorrhagic disease(EHD) is an infectious, non-contagious insect transmitted disease of ruminants. To investigate the infection of epizootic hemorrhagic disease virus(EHDV) among ruminants in Yunnan Province of China and study the virus genetic characteristics, monitoring points were set up in Shizong. Virus isolation was carried out through the inoculating BHK-21 cells with EHDV nucleic acid positive blood samples collected from the sentinel herds. Serotype of the isolated virus was identified by serum neutralization test; ORF of Seg-2 and Seg-3 of the virus were amplified with specific primers, then cloned and sequenced; antibody levels and viral nucleic acid in the EHDV infected animals throughout the year were tested by C-ELISA and qRT-PCR. Results were as follows: In August 2013, an EHDV strain(YNSZ/V269/2013) was isolated from Sentinel Cattle in Shizong, Yunnan. The serum neutralization test showed that the YNSZ/V269/2013 was an EHDV-7 virus. Sequence analysis of Seg-2 and Seg-3 showed that the isolated virus belonged to EHDV-7 Eastern type and had the closest genetic relationship with the Serotype 7 strain in Japan or Australia. The animal serum antibody increased rapidly after being infected by EHDV-7 and the antibody reached the highest point after 3 weeks and lasted for a longer time; meanwhile the nucleic acid content of the virus declined rapidly, 7 weeks later could not be detected in blood samples. This study reported the first isolation of EHDV serotype 7 in China and the virus sequence characterization of Seg-2 and Seg-3. The research results provide the basis for further development of the whole genome sequencing of Chinese EHDV-7 virus, epidemiological investigation, establishment of diagnostic methods and pathogenicity research.
Epizootic haemorrhagic disease(EHD) is an infectious, non-contagious insect transmitted disease of ruminants. To investigate the infection of epizootic hemorrhagic disease virus(EHDV) among ruminants in Yunnan Province of China and study the virus genetic characteristics, monitoring points were set up in Shizong. Virus isolation was carried out through the inoculating BHK-21 cells with EHDV nucleic acid positive blood samples collected from the sentinel herds. Serotype of the isolated virus was identified by serum neutralization test; ORF of Seg-2 and Seg-3 of the virus were amplified with specific primers, then cloned and sequenced; antibody levels and viral nucleic acid in the EHDV infected animals throughout the year were tested by C-ELISA and qRT-PCR. Results were as follows: In August 2013, an EHDV strain(YNSZ/V269/2013) was isolated from Sentinel Cattle in Shizong, Yunnan. The serum neutralization test showed that the YNSZ/V269/2013 was an EHDV-7 virus. Sequence analysis of Seg-2 and Seg-3 showed that the isolated virus belonged to EHDV-7 Eastern type and had the closest genetic relationship with the Serotype 7 strain in Japan or Australia. The animal serum antibody increased rapidly after being infected by EHDV-7 and the antibody reached the highest point after 3 weeks and lasted for a longer time; meanwhile the nucleic acid content of the virus declined rapidly, 7 weeks later could not be detected in blood samples. This study reported the first isolation of EHDV serotype 7 in China and the virus sequence characterization of Seg-2 and Seg-3. The research results provide the basis for further development of the whole genome sequencing of Chinese EHDV-7 virus, epidemiological investigation, establishment of diagnostic methods and pathogenicity research.
引文
[1] MACLACHLAN N J, ZIENTARA S, SAVINI G, et al. Epizootic haemorrhagic disease[J]. Rev Sci Tech, 2015, 34(2):341-351.
[2] ANBALAGAN S, HAUSE B M. Characterization of epizootic hemorrhagic disease virus from a bovine with clinical disease with high nucleotide sequence identity to white-tailed deer isolates[J]. Arch Virol, 2014, 159(10):2737-2740.
[3] SPEDICATO M, CARMINE I, TEODORI L, et al. Innocuity of a commercial live attenuated vaccine for epizootic hemorrhagic disease virus serotype 2 in late-term pregnant cows[J]. Vaccine, 2016, 34(12):1430-1435.
[4] SAVINI G, AFONSO A, MELLOR P, et al. Epizootic haemorrhagic disease[J]. Res Vet Sci, 2011, 91(1):1-17.
[5] ALBAYRAK H, OZAN E, GUR S. A serologic investigation of epizootic hemorrhagic disease virus (EHDV) in cattle and Gazella subgutturosa subgutturosa in Turkey[J]. Trop Anim Health Prod, 2010, 42(8):1589-1591.
[6] NOON T H, WESCHE S L, CAGLE D, et al. Hemorrhagic disease in bighorn sheep in Arizona[J]. J Wildl Dis, 2002, 38(1):172-176.
[7] OIE. Manual of diagnostic tests and vaccines for terres. OIE terrestrial manual[S]. 2014, Chapter 2:1-11.
[8] IWATA H, CHUMA T, ROY P. Characterization of the genes encoding two of the major capsid proteins of epizootic haemorrhagic disease virus indicates a close genetic relationship to bluetongue virus[J]. J Gen Virol, 1992, 73(4):915-924.
[9] ANBALAGAN S, COOPER E, KLUMPER P, et al. Whole genome analysis of epizootic hemorrhagic disease virus identified limited genome constellations and preferential reassortment[J]. J Gen Virol, 2014, 95(2):434-441.
[10] MAAN N S, MAAN S, POTGIETER A C, et al. Development of real-time RT-PCR assays for detection and typing of epizootic haemorrhagic disease virus[J]. Transbound Emerg Dis, 2017, 64(4):1120-1132.
[11] 曹颖颖, 钟华,吴健敏. 鹿流行性出血热病毒研究进展[J]. 中国兽医学报, 2017, 37(3):571-576.CAO Y Y, ZHONG H, WU J M.Research progress of deer epidemic hemorrhagic fever virus[J]. Chinese Journal of Veterinary Science, 2017, 37(3):571-576.(in Chinese)
[12] 张怡轩, 林俊,曹颖颖,等. 广西鹿流行性出血热病毒血清型调查及分布影响分析[J]. 上海畜牧兽医通讯, 2016(4):19-21.ZHANG Y X, LIN J, CAO Y Y,et al. Serological investigation and distribution impact analysis of epizootic hemorrhagic disease virus in Guangxi[J]. Shanghai Journal of Animal Husbandry and Veterinary Medicine, 2016(4):19-21.(in Chinese)
[13] 和东华, 杨振兴,高林,等. 云南省边境地区牛流行性出血病病毒血清学调查[J]. 云南畜牧兽医, 2016(5):7-10.HE D H, YANG Z X, GAO L, et al.Serological investigation of epizootic hemorrhagic disease virus in border areas of Yunnan Province[J]. Yunnan Journal of Animal Science and Veterinary Medicine, 2016(5):7-10.(in Chinese)
[14] 曹颖颖, 吴健敏,林俊,等. 广西首例牛源鹿流行性出血热病毒的分离鉴定[J]. 中国预防兽医学报, 2015, 37(10):746-750.CAO Y Y, WU J M, LIN J, et al.Isolation and identification of the first case of epizootic hemorrhagic disease virus from cattle in Guangxi[J]. Chinese Journal of Preventive Veterinary Medicine, 2015, 37(10):746-750.(in Chinese)
[15] 吕敏娜, 朱建波,李娟,等. 广东一株牛源流行性出血病病毒的分离鉴定[J]. 中国预防兽医学报, 2017, 39(1):67-70.LV M N, ZHU J B, LI J, et al. Isolation and identification of the epizootic hemorrhagic disease virus from cattle in Guangdong[J]. Chinese Journal of Preventive Veterinary Medicine, 2017, 39(1):67-70.(in Chinese)
[16] 朱建波, 杨振兴,肖雷,等. 流行性出血病多克隆抗体C-ELISA检测方法的建立[J]. 畜牧兽医学报, 2018, 49(7):1440-1450.ZHU J B, YANG Z X, XIAO L, et al.Establishment of a rapid competitive ELISA for detecting antibodies against epizootic haemorrhagic disease virus[J]. Acta Veterinaria et Zootechnica Sinica, 2018, 49(7):1440-1450.(in Chinese)
[17] 中华人民共和国国家质量监督检验检疫总局, 中国国家标准化管理委员会. GB/T 18089-2008 蓝舌病病毒分离、鉴定及血清中和抗体检测技术[S]. 北京:中国标准出版社, 2008.People's Republic of China State Administration of Quality Supervision, Inspection and Quarantine, China National Standardization Management Committee. GB/T 18089-2008 Isolation, identification and serum neutralization antibody test in Bluetongue virus[S]. Beijing: China Standard Press, 2008.(in Chinese)
[18] TAMURA K, STECHER G, PETERSON D, et al. MEGA6:Molecular evolutionary genetics analysis version 6. 0[J]. Mol Biol Evol, 2013, 30(12):2725-2729.
[19] ANTHONY S J, MAAN N, MAAN S, et al. Genetic and phylogenetic analysis of the non-structural proteins NS1, NS2 and NS3 of epizootic haemorrhagic disease virus (EHDV)[J]. Virus Res, 2009, 145(2):211-219.
[20] SHIRAFUJI H, KATO T, YAMAKAWA M, et al. Characterization of genome segments 2, 3 and 6 of epizootic hemorrhagic disease virus strains isolated in Japan in 1985-2013:Identification of their serotypes and geographical genetic types[J]. Infect Genet Evol, 2017, 53:38-46.
[21] FORZAN M, PIZZURRO F, ZACCARIA G, et al. Competitive enzyme-linked immunosorbent assay using baculovirus-expressed VP7 for detection of epizootic haemorrhagic disease virus (EHDV) antibodies[J]. J Virol Methods, 2017, 284:212-216.
[22] LEI W W, GUO X F, FU S H, et al. Isolation of Tibet orbivirus, TIBOV, from Culicoides collected in Yunnan, China[J]. PLoS One, 2015, 10(8):e0136257.
[23] WANG J L, LI H C, HE Y W, et al. Isolation and genetic characterization of Mangshi Virus:a newly discovered seadornavirus of the reoviridae family found in Yunnan Province, China[J]. PLoS One, 2015, 10(12):e0143601.
[2] ANBALAGAN S, HAUSE B M. Characterization of epizootic hemorrhagic disease virus from a bovine with clinical disease with high nucleotide sequence identity to white-tailed deer isolates[J]. Arch Virol, 2014, 159(10):2737-2740.
[3] SPEDICATO M, CARMINE I, TEODORI L, et al. Innocuity of a commercial live attenuated vaccine for epizootic hemorrhagic disease virus serotype 2 in late-term pregnant cows[J]. Vaccine, 2016, 34(12):1430-1435.
[4] SAVINI G, AFONSO A, MELLOR P, et al. Epizootic haemorrhagic disease[J]. Res Vet Sci, 2011, 91(1):1-17.
[5] ALBAYRAK H, OZAN E, GUR S. A serologic investigation of epizootic hemorrhagic disease virus (EHDV) in cattle and Gazella subgutturosa subgutturosa in Turkey[J]. Trop Anim Health Prod, 2010, 42(8):1589-1591.
[6] NOON T H, WESCHE S L, CAGLE D, et al. Hemorrhagic disease in bighorn sheep in Arizona[J]. J Wildl Dis, 2002, 38(1):172-176.
[7] OIE. Manual of diagnostic tests and vaccines for terres. OIE terrestrial manual[S]. 2014, Chapter 2:1-11.
[8] IWATA H, CHUMA T, ROY P. Characterization of the genes encoding two of the major capsid proteins of epizootic haemorrhagic disease virus indicates a close genetic relationship to bluetongue virus[J]. J Gen Virol, 1992, 73(4):915-924.
[9] ANBALAGAN S, COOPER E, KLUMPER P, et al. Whole genome analysis of epizootic hemorrhagic disease virus identified limited genome constellations and preferential reassortment[J]. J Gen Virol, 2014, 95(2):434-441.
[10] MAAN N S, MAAN S, POTGIETER A C, et al. Development of real-time RT-PCR assays for detection and typing of epizootic haemorrhagic disease virus[J]. Transbound Emerg Dis, 2017, 64(4):1120-1132.
[11] 曹颖颖, 钟华,吴健敏. 鹿流行性出血热病毒研究进展[J]. 中国兽医学报, 2017, 37(3):571-576.CAO Y Y, ZHONG H, WU J M.Research progress of deer epidemic hemorrhagic fever virus[J]. Chinese Journal of Veterinary Science, 2017, 37(3):571-576.(in Chinese)
[12] 张怡轩, 林俊,曹颖颖,等. 广西鹿流行性出血热病毒血清型调查及分布影响分析[J]. 上海畜牧兽医通讯, 2016(4):19-21.ZHANG Y X, LIN J, CAO Y Y,et al. Serological investigation and distribution impact analysis of epizootic hemorrhagic disease virus in Guangxi[J]. Shanghai Journal of Animal Husbandry and Veterinary Medicine, 2016(4):19-21.(in Chinese)
[13] 和东华, 杨振兴,高林,等. 云南省边境地区牛流行性出血病病毒血清学调查[J]. 云南畜牧兽医, 2016(5):7-10.HE D H, YANG Z X, GAO L, et al.Serological investigation of epizootic hemorrhagic disease virus in border areas of Yunnan Province[J]. Yunnan Journal of Animal Science and Veterinary Medicine, 2016(5):7-10.(in Chinese)
[14] 曹颖颖, 吴健敏,林俊,等. 广西首例牛源鹿流行性出血热病毒的分离鉴定[J]. 中国预防兽医学报, 2015, 37(10):746-750.CAO Y Y, WU J M, LIN J, et al.Isolation and identification of the first case of epizootic hemorrhagic disease virus from cattle in Guangxi[J]. Chinese Journal of Preventive Veterinary Medicine, 2015, 37(10):746-750.(in Chinese)
[15] 吕敏娜, 朱建波,李娟,等. 广东一株牛源流行性出血病病毒的分离鉴定[J]. 中国预防兽医学报, 2017, 39(1):67-70.LV M N, ZHU J B, LI J, et al. Isolation and identification of the epizootic hemorrhagic disease virus from cattle in Guangdong[J]. Chinese Journal of Preventive Veterinary Medicine, 2017, 39(1):67-70.(in Chinese)
[16] 朱建波, 杨振兴,肖雷,等. 流行性出血病多克隆抗体C-ELISA检测方法的建立[J]. 畜牧兽医学报, 2018, 49(7):1440-1450.ZHU J B, YANG Z X, XIAO L, et al.Establishment of a rapid competitive ELISA for detecting antibodies against epizootic haemorrhagic disease virus[J]. Acta Veterinaria et Zootechnica Sinica, 2018, 49(7):1440-1450.(in Chinese)
[17] 中华人民共和国国家质量监督检验检疫总局, 中国国家标准化管理委员会. GB/T 18089-2008 蓝舌病病毒分离、鉴定及血清中和抗体检测技术[S]. 北京:中国标准出版社, 2008.People's Republic of China State Administration of Quality Supervision, Inspection and Quarantine, China National Standardization Management Committee. GB/T 18089-2008 Isolation, identification and serum neutralization antibody test in Bluetongue virus[S]. Beijing: China Standard Press, 2008.(in Chinese)
[18] TAMURA K, STECHER G, PETERSON D, et al. MEGA6:Molecular evolutionary genetics analysis version 6. 0[J]. Mol Biol Evol, 2013, 30(12):2725-2729.
[19] ANTHONY S J, MAAN N, MAAN S, et al. Genetic and phylogenetic analysis of the non-structural proteins NS1, NS2 and NS3 of epizootic haemorrhagic disease virus (EHDV)[J]. Virus Res, 2009, 145(2):211-219.
[20] SHIRAFUJI H, KATO T, YAMAKAWA M, et al. Characterization of genome segments 2, 3 and 6 of epizootic hemorrhagic disease virus strains isolated in Japan in 1985-2013:Identification of their serotypes and geographical genetic types[J]. Infect Genet Evol, 2017, 53:38-46.
[21] FORZAN M, PIZZURRO F, ZACCARIA G, et al. Competitive enzyme-linked immunosorbent assay using baculovirus-expressed VP7 for detection of epizootic haemorrhagic disease virus (EHDV) antibodies[J]. J Virol Methods, 2017, 284:212-216.
[22] LEI W W, GUO X F, FU S H, et al. Isolation of Tibet orbivirus, TIBOV, from Culicoides collected in Yunnan, China[J]. PLoS One, 2015, 10(8):e0136257.
[23] WANG J L, LI H C, HE Y W, et al. Isolation and genetic characterization of Mangshi Virus:a newly discovered seadornavirus of the reoviridae family found in Yunnan Province, China[J]. PLoS One, 2015, 10(12):e0143601.