一株牛流行热病毒的分离与鉴定
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摘要
牛流行热病毒(BFFV)是引发世界范围内牛流行热最主要病原之一,给奶牛业的发展造成严重危害。本研究通过RT-PCR对疑似牛流行热奶牛抗凝血样品进行检测,选取阳性样品颅内接种3日龄乳鼠,7日后乳鼠死亡,鼠脑组织通过PCR扩增出BFFV目的条带,乳鼠颅内重复接种3次,乳鼠发病死亡时间明显提前。选取阳性脑组织接种BHK-21细胞,盲传3代后出现明显细胞病变,特异性引物扩增获得目的条带,测序结果与牛流行热病毒序列一致,表明成功分离到一株牛流行热病毒。
Bovine ephemeral fever virus is one of the most pathogens leading to the bovine ephemeral fever worldwide, which has caused serious harm to the development of dairy industry. In this study, the anticoagulant sample of suspected bovine ephemeral fever from Henan were tested by RT-PCR, then positive samples was inoculated intracerebrally into sucking mice with 3 days of age. After 7 days, the suckling mice died. The target band of BFFV was identified by PCR from rat brain. After repeated intracranial inoculation for 3 times, the death time of sucking mice were significantly advanced. Followed by inoculation of positive samples into BHK-21 monolayer cells, the CPE was obvious after three blind inoculation. The target band were obtained with the specific primers, and the sequencing was consistent with the bovine ephemeral fever sequence. The results showed that a bovine ephemeral fever virus was successfully isolated.
Bovine ephemeral fever virus is one of the most pathogens leading to the bovine ephemeral fever worldwide, which has caused serious harm to the development of dairy industry. In this study, the anticoagulant sample of suspected bovine ephemeral fever from Henan were tested by RT-PCR, then positive samples was inoculated intracerebrally into sucking mice with 3 days of age. After 7 days, the suckling mice died. The target band of BFFV was identified by PCR from rat brain. After repeated intracranial inoculation for 3 times, the death time of sucking mice were significantly advanced. Followed by inoculation of positive samples into BHK-21 monolayer cells, the CPE was obvious after three blind inoculation. The target band were obtained with the specific primers, and the sequencing was consistent with the bovine ephemeral fever sequence. The results showed that a bovine ephemeral fever virus was successfully isolated.
引文
[1] Hayama Y, Moriguchi S, Yanase T, et al. Epidemiological analysis of bovine ephemeral fever in 2012—2013 in the subtropical islands of Japan[J]. BMC Veterinary Research, 2016, 12(1): 47.
[2] He C Q, Liu Y X, Wang H M, et al. New genetic mechanism, origin and population dynamic of bovine ephemeral fever virus[J]. Veterinary Microbiology, 2016, 182: 50-56.
[3] Liu D, Li K, Zhang L, et al. Seroprevalence investigation of bovine ephemeral fever in yaks in Tibetan Plateau of China from 2012 to 2015[J]. Tropical Animal Health & Production, 2016, 49(1): 1-4.
[4] Walker P J, Klement E. Epidemiology and control of bovine ephemeral fever[J]. Veterinary Research, 2015, 46: 124.
[5] 孟克那生. 牛流行热的发病特点及防治办法[J]. 中国动物保健, 2016, 18(3): 40-41.
[6] Cheng C Y, Shih W L, Huang W R, et al. Bovine ephemeral fever virus uses a clathrin-mediated and dynamin 2-dependent endocytosis pathway that requires Rab5 and Rab7 as well as microtubules[J]. Journal of Virology, 2012, 86(24): 13653-13661.
[7] 王洪梅, 侯佩莉, 杨宏军, 等. 牛流行热病毒山东地方株的分离与鉴定[J]. 山东农业科学, 2013, 45(2): 31-33, 37.
[8] Uren M F. Bovine ephemeral fever[J]. Australian Veterinary Journal, 2010, 66(8): 233-236.
[9] Tonbak S, Berber E, Yoruk M D, et al. A large-scale outbreak of bovine ephemeral fever in Turkey, 2012[J]. Journal of Veterinary Medical Science, 2013, 75(11): 1511-1514.
[10] 黄德萱. 牛流行热病毒抗原捕获ELISA诊断方法的建立与应用[D]. 北京:中国农业科学院, 2017.
[11] Li Z, Zheng F, Gao S, et al. Large-scale serological survey of bovine ephemeral fever in China[J]. Veterinary Microbiology, 2015, 176(1/2): 155-160.
[12] St George T D, Standfast H A, Dyce A L. The isolation of ephemeral fever from mosquitoes in Australia[J]. Australian Veterinary Journal, 1976, 52(5): 242.
[13] Young P L, Spradbrow P B. The role of neutrophils in bovine ephemeral fever virus infection of cattle[J]. Journal of Infectious Diseases, 1980, 142(1): 50-55.
[14] 江船. 牛流行热病毒感染宿主细胞差异表达miRNA的筛选及其鉴定[D]. 济南:山东大学, 2018.
[2] He C Q, Liu Y X, Wang H M, et al. New genetic mechanism, origin and population dynamic of bovine ephemeral fever virus[J]. Veterinary Microbiology, 2016, 182: 50-56.
[3] Liu D, Li K, Zhang L, et al. Seroprevalence investigation of bovine ephemeral fever in yaks in Tibetan Plateau of China from 2012 to 2015[J]. Tropical Animal Health & Production, 2016, 49(1): 1-4.
[4] Walker P J, Klement E. Epidemiology and control of bovine ephemeral fever[J]. Veterinary Research, 2015, 46: 124.
[5] 孟克那生. 牛流行热的发病特点及防治办法[J]. 中国动物保健, 2016, 18(3): 40-41.
[6] Cheng C Y, Shih W L, Huang W R, et al. Bovine ephemeral fever virus uses a clathrin-mediated and dynamin 2-dependent endocytosis pathway that requires Rab5 and Rab7 as well as microtubules[J]. Journal of Virology, 2012, 86(24): 13653-13661.
[7] 王洪梅, 侯佩莉, 杨宏军, 等. 牛流行热病毒山东地方株的分离与鉴定[J]. 山东农业科学, 2013, 45(2): 31-33, 37.
[8] Uren M F. Bovine ephemeral fever[J]. Australian Veterinary Journal, 2010, 66(8): 233-236.
[9] Tonbak S, Berber E, Yoruk M D, et al. A large-scale outbreak of bovine ephemeral fever in Turkey, 2012[J]. Journal of Veterinary Medical Science, 2013, 75(11): 1511-1514.
[10] 黄德萱. 牛流行热病毒抗原捕获ELISA诊断方法的建立与应用[D]. 北京:中国农业科学院, 2017.
[11] Li Z, Zheng F, Gao S, et al. Large-scale serological survey of bovine ephemeral fever in China[J]. Veterinary Microbiology, 2015, 176(1/2): 155-160.
[12] St George T D, Standfast H A, Dyce A L. The isolation of ephemeral fever from mosquitoes in Australia[J]. Australian Veterinary Journal, 1976, 52(5): 242.
[13] Young P L, Spradbrow P B. The role of neutrophils in bovine ephemeral fever virus infection of cattle[J]. Journal of Infectious Diseases, 1980, 142(1): 50-55.
[14] 江船. 牛流行热病毒感染宿主细胞差异表达miRNA的筛选及其鉴定[D]. 济南:山东大学, 2018.