某马场H3N8亚型马流感病毒的分离鉴定及对犬易感性的实验
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摘要
2007年12月,北京某马场共有23匹马,一周内有21匹相继发病,根据流行病学调查和临床症状初步疑似马流感。采集病马的鼻汁,经处理后接种SPF鸡胚,分离到一株马流感病毒,经经HI和基因序列分析试验鉴定为H3N8亚型马流感病毒,毒株命名为A/Equine/Huabei/2/2007(H3N8),简称EHB07,鸡胚半数感染量为106.5EID50/0.1ml。确诊本次疫情由H3N8亚型马流感病毒感染引起。EHB07的HA基因与鸭源毒株A/Duck/Ukraine/1963(H3N8)、A/Nanchang/1681/1992(H3N8)和中国吉林分离株A/Equine/Jilin/1/1989(H3N8)的同源性较低(本毒株为迄今为止唯一的一株从马体分离到的禽源流感毒株)(Guo Y,1992),分别为76.85,77.1%和77.8%,与其它马源毒株的同源性较高,为90.4%-99.1%,其中与2005年意大利巴里分离毒株A/Equine/Bari/2005(H3N8)的同源性最高,达99.1%。与日本茨城分离毒株已发表的部分序列比较发现,二者的同源性为97.63%。EHB07的NA基因和2007年日本分离株A/Equine/Ibaraki/1/07(H3N8)处于同一分支内,亲缘关系最近,同源率97.3%；与鸭源毒株和中国吉林分离的马源毒株差异较大,同源性差,同源率为74.8%-77.2%,其中与吉林株的核苷酸同源率为74.9%。除此以外,本实验毒株与世界上分离的其它马源毒株的同源率为90.0%-98.7%,与2002年美国肯塔基州的分离毒株同源性最高,达98.7%。因此,也说明本实验分析的毒株起源于马,而非禽类。与1992年本地发生的马流感疫情相比,本次疫情具有流行范围小、传播速度慢、病情缓和、容易治疗、恢复迅速的特点。
2004年-2006年美国连续发生犬流感疫情,而且初步研究确定犬流感是由H3N8亚型马流感病毒感染引起,受到全世界的关注。为了确定A/Equine/Huabei/2/2007(H3N8, F4)毒株对犬的易感性,我们选择14只宠物犬进行了攻毒试验。以105.5EID50/只(条)的剂量经滴鼻点眼和静脉注射的途径进行攻毒,攻毒后定期检查犬的临床症状表现、体温、血常规、血清生化并采集鼻咽拭子,并按照试验设计于攻毒后6天、14天分别剖杀试验犬,采集脏器病料进行病毒分离和病理组织学观察。结果所有攻毒犬无任何临床感染的症状,体温、血细胞数量、生化指标均无任何异常,病例组织学观察未见明显病变,病毒分离除了有一条金色寻回猎犬的鼻咽拭子分离到病毒外其余均为阴性,抗体检测仅有一条金色寻回猎犬犬在14天的血清中检测到很低的抗体(1：20)。初步试验结果表明本次试验用犬对F4代A/Equine/Huabei/2/2007(H3N8)不易感。
Due to the invasion of foreign epidemic, suspected equine influenza outbreak occurred in a horse farm of Beijing in December,2007. Among the total 23 horses of the farm,21 were attacked by disease constantly within a week which was identified as equine influenza preliminarily according to epidemiological investigation and clinical symptoms. Inoculated the collected horse swab after being treated with SPF chicken embryos, an equine influenza virus strain was isolated and identified as Subtype H3N8 equine influenza virus with half the infection amount of chicken embryos of 106.5EID50/0.1ml. The strain was named A/Equine/Huabei/2/2007 (H3N8) and referred to as EHB07. Initial diagnosis showed that this epidemic was caused by the infection of Subtype H3N8 equine influenza virus. Compared with the horse flu outbreak occurred locally in 1992, this epidemic was characterized by small-area and slow spread, eased symptoms, easy treatment, and quick recovery.
During 2004 and 2006, canine influenza occurred consecutively in the United States; preliminary study confirmed that it was caused by the infection of Subtype H3N8 equine influenza virus, which caused worldwide attention. To determine the susceptibility of A/Equine/Huabei/2/2007 (H3N8) strain on dogs,14 pet dogs were selected and tapped Challenge Test. The challenge routes were intranasal-and-eyed infection and intravenous injection with the amount of attack agents 105.5EID50. After the challenge, checked regularly the dogs'clinical symptoms, temperature, blood, serum biochemical and collected nasopharyngeal swabs; 6 to 14days later, killed and dissected all the dogs, collected viscera materials for virus isolation and histological observation. The result was that all dogs inoculated showed no clinical symptom of infection, no abnormal situation in body temperature, blood cells and biochemical parameters, and no obvious lesions through histological observation. Except that virus was isolated from the nasopharyngeal swabs of a Golden Retriever among the dogs, all viruses isolated were negative. In antibody test, only a Golden Retriever was found of low antibodies in the 14-day serum (1:20). Based on the results above, it could be affirmed preliminarily that this experimental strain had no obvious infectious on dogs.
2004年-2006年美国连续发生犬流感疫情,而且初步研究确定犬流感是由H3N8亚型马流感病毒感染引起,受到全世界的关注。为了确定A/Equine/Huabei/2/2007(H3N8, F4)毒株对犬的易感性,我们选择14只宠物犬进行了攻毒试验。以105.5EID50/只(条)的剂量经滴鼻点眼和静脉注射的途径进行攻毒,攻毒后定期检查犬的临床症状表现、体温、血常规、血清生化并采集鼻咽拭子,并按照试验设计于攻毒后6天、14天分别剖杀试验犬,采集脏器病料进行病毒分离和病理组织学观察。结果所有攻毒犬无任何临床感染的症状,体温、血细胞数量、生化指标均无任何异常,病例组织学观察未见明显病变,病毒分离除了有一条金色寻回猎犬的鼻咽拭子分离到病毒外其余均为阴性,抗体检测仅有一条金色寻回猎犬犬在14天的血清中检测到很低的抗体(1：20)。初步试验结果表明本次试验用犬对F4代A/Equine/Huabei/2/2007(H3N8)不易感。
Due to the invasion of foreign epidemic, suspected equine influenza outbreak occurred in a horse farm of Beijing in December,2007. Among the total 23 horses of the farm,21 were attacked by disease constantly within a week which was identified as equine influenza preliminarily according to epidemiological investigation and clinical symptoms. Inoculated the collected horse swab after being treated with SPF chicken embryos, an equine influenza virus strain was isolated and identified as Subtype H3N8 equine influenza virus with half the infection amount of chicken embryos of 106.5EID50/0.1ml. The strain was named A/Equine/Huabei/2/2007 (H3N8) and referred to as EHB07. Initial diagnosis showed that this epidemic was caused by the infection of Subtype H3N8 equine influenza virus. Compared with the horse flu outbreak occurred locally in 1992, this epidemic was characterized by small-area and slow spread, eased symptoms, easy treatment, and quick recovery.
During 2004 and 2006, canine influenza occurred consecutively in the United States; preliminary study confirmed that it was caused by the infection of Subtype H3N8 equine influenza virus, which caused worldwide attention. To determine the susceptibility of A/Equine/Huabei/2/2007 (H3N8) strain on dogs,14 pet dogs were selected and tapped Challenge Test. The challenge routes were intranasal-and-eyed infection and intravenous injection with the amount of attack agents 105.5EID50. After the challenge, checked regularly the dogs'clinical symptoms, temperature, blood, serum biochemical and collected nasopharyngeal swabs; 6 to 14days later, killed and dissected all the dogs, collected viscera materials for virus isolation and histological observation. The result was that all dogs inoculated showed no clinical symptom of infection, no abnormal situation in body temperature, blood cells and biochemical parameters, and no obvious lesions through histological observation. Except that virus was isolated from the nasopharyngeal swabs of a Golden Retriever among the dogs, all viruses isolated were negative. In antibody test, only a Golden Retriever was found of low antibodies in the 14-day serum (1:20). Based on the results above, it could be affirmed preliminarily that this experimental strain had no obvious infectious on dogs.
引文
1.陈晶宇,刘锦旭,崔卫东等。马流行性感冒病毒的分离和鉴定[J]黑龙江畜牧兽医,1990,(1)：
2.丁壮,周昌芳,李建华。马病防治手册[M]。北京：金盾出版社,2006：60-63。
3.郭巍,戴伶俐,李雪峰等。马流感病毒多重RT-PCR检测方法的建立,动物医学进展,2008,29(11)：28-31。
4.郭巍,闫岩,王英原等。新疆地区-株马流感病毒的分离鉴定[J],中国预防兽医学报,2008,30(8)：584-587
5.郭元吉,程小雯著。流行性感冒及其实验技术[M]。北京：中国三峡出版社,1997
6.贾晓庆,唐泰山,黄金华。马流感病毒RT-PCR检测方法的建立[J]。畜牧与兽医,2009,41(2)：63-66
7.贾斌,王晓钧,相文华等。我国马流感病毒不同分离株实验感染SPF鸡的研究[J],中国预防兽医学报,1999,21(1)：11-13
8.侯云德。分子病毒学[M]。北京：学院出版社,1990,313-326
9.李德庆,时智元,蔡相明。三阳清解汤治疗马流感68例[J]。中国兽医科技,2002,32(8)：42-43。
10.李雪峰,戴伶俐,郭巍等。H3N8亚型马流感病毒内蒙古株NS基因测定与序列分折[J],黑龙江畜牧兽医,2009,6：87-89
11.刘清海,孙洪升,车秀华。马流感的研究现状[J]。畜牧兽医科技信息,2005,6：19-21.
12.苏德昌,董泽生。应用中药荆防败毒散加减治疗马流感的体会[J]。青海畜牧业,2004,2：47
13.孙高超,毕可东,张娜。马流感简介[J]。动物医学进展,2008,29(8)：115-116。
14.田建国,叶丛华,张文东等。云南地区马流感病毒检测及其HA基因序列分析[J]。畜牧与兽医,2009,41(6)：22-26
15.王秀荣,杨建德,许景君等。马流感A/马/京防/74-1(H7N7)毒株HA基因的序列分析[J]。中国预防兽医学报,2004,26(5)：332-335
16.杨建德,相文华,薛飞等。我国H3N8马流感病毒血凝素基因分子进化树的分析[J]中国生物工程杂志,2003,(06).
17.杨建德,薛飞,王晓钧等。马流感病毒(A/equine/Qinghai/1/94)核蛋白基因的序列测定及同源性分析[J]中国预防兽医学报,2003,(02)
18.杨建德,王文军,薛飞等。马流感病毒(A/Equine/Qinghai/1/94)神经氨酸酶基因的克隆与同源性分析[J]中国预防兽医学报,2003,(05)
19.杨建德,相文华。我国马流感的研究现状[J]黑龙江畜牧兽医,2002,(03).
20.殷震,刘景华等。动物病毒学[M],第二版,北京：科学出版社。1997,704-735
21.张利峰,张鹤,晓谷强等。马流感血凝及血凝抑制试验的研究与应用,[J],检验检疫科学,2003(13)1：30-32。
22.赵文成,许娇娜。我国马流感的研究现状[J]。畜牧兽医科技信息,2008,6：5-7
23.左铃兰,江国林。大理州马流行性感冒的防治[J]。云南畜牧兽医,2009,3：28-29.
24. Chamber T M。用DFA试验快速诊断马流感[J]。国外兽医学,畜禽传染病,1995,15(2)：40-42。
25. Damiani A M, Scicluna M T, Ciabatti I, et al. Genetic characterization of equine influenza viruses isolated in Italy between 1999 and 2005[J].Virus Res,2008,131,131 (1):100-105.
26. Guo Y, Wang M, Kawaka Y, et al. Characterization of a new avian-like influenza A virus from horses in China.Virology,1992,1881,188(1):245-255.
27. Global surveillance network for equine influenza.http://www.equiflunet.org.uk.
28. Martella V, Elia G, Decaro N, et al. An outbreak of equine influenza virus in vaccinated horses in Italy is due to an H3N8 stain closely related to recent North American representatives of the Florida sub-lineage[J].Vet Microbial,2007,121,121:56-63
29. Newton J R, Daly J M, Spencer L et. al. Description of the outbreak of equine influenza(H3N8)in the United Kingdom in 2003, during which recently vaccinated horses in New market dev-eloped respiratory disease [J].Vet Dec,2007,158,158 (6):185-192.
30. OIE. Equine influenza.Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, part3. http://www.oie.org:2004,
31. Reeve-Johnson L. Equine influenza in Australia [J].Vet Rec,2007,161,161 (18):635.
32. Chris Olsen. Canine Influenza. North Carolina University College of Veterinary Medicine. http://www.ncvdl.com/.2006.
33. Control of Canine Influenza in Dogs-Questions, Answers, and Interim Guidelines October 17,2005. www.vetmed.ufl.edu/pr.
34. Ed Dubovi Sept. Ruben Donis,26 issue of Science Express, the online version of Science magazine
35. Tara Anderson 2008, ScienceDaily (Mar.20,2008)
36. P.C.Crawford, Edward J. Dubovi, William L. Castleman, Iain Stephenson, Transmission of Equine Influenza Virus to Dogs Science 21 October 2005:Vol.310. no.5747:482-485
37. Kyoung-Jin Yoon, Vickie L. Cooper, Kent J. Schwartz, Influenza Virus Infection in Racing Greyhounds
38. R.Newton, A. Cooke, D.Elton, N.Bryant, A. Rash, S. et al. Canine influenza virus: cross-species transmission from horses, Vet Rec.2007,161:142-143
39. Daly, Janet M.; Blunden, Anthony S.; MacRae, Shona; Transmission of equine influenza virus to English foxhounds Emerg Infect Dis.2008,14(3):461-464.
40. Takashi Yamanaka, Manabu Nemoto, Koji Tsujimura, Takashi Kondo Interspecies transm ission of equine influenza virus (H3N8) to dogs by close contact with experimentally infected horses. Available online 21 June 2009.http://www.sciencedirect.com
41. Sigfrido Burgos and Sergio A. Burgos.. Reports of Avian Influenza H5N1 in Cats and dogs. International Journal of Poultry Science 2007,6(12):1003-1005.
42. Matthias Giese, Timm C. Harder, Jens P. Teifke, et al. Experimental infection and natural
contact exposure of dogs with avian influenza virus (H5N1). Emerging infectious disease, 2008.14(2):308-310.
43. Riks Mass, Mirriam Tacken, Lisette Ruuls, et al. Avian influenza (H5N1) susceptibility and receptors in dogs. Emerging infectious disease,2007.13(8):1219-1221.
44. Daesub Song, Chulseung Lee, Bokyu Kang. Emerging infectious disease, www.cdc.gov/eid 2009.15(1):56-58.
45. Song D, Kang B, Lee C, Jung K, Ha G, Kang D, et al. Transmission of avian infl uenza virus (H3N2) to dogs. Emerg Infect Dis.2008,14(7)41-46
2.丁壮,周昌芳,李建华。马病防治手册[M]。北京：金盾出版社,2006：60-63。
3.郭巍,戴伶俐,李雪峰等。马流感病毒多重RT-PCR检测方法的建立,动物医学进展,2008,29(11)：28-31。
4.郭巍,闫岩,王英原等。新疆地区-株马流感病毒的分离鉴定[J],中国预防兽医学报,2008,30(8)：584-587
5.郭元吉,程小雯著。流行性感冒及其实验技术[M]。北京：中国三峡出版社,1997
6.贾晓庆,唐泰山,黄金华。马流感病毒RT-PCR检测方法的建立[J]。畜牧与兽医,2009,41(2)：63-66
7.贾斌,王晓钧,相文华等。我国马流感病毒不同分离株实验感染SPF鸡的研究[J],中国预防兽医学报,1999,21(1)：11-13
8.侯云德。分子病毒学[M]。北京：学院出版社,1990,313-326
9.李德庆,时智元,蔡相明。三阳清解汤治疗马流感68例[J]。中国兽医科技,2002,32(8)：42-43。
10.李雪峰,戴伶俐,郭巍等。H3N8亚型马流感病毒内蒙古株NS基因测定与序列分折[J],黑龙江畜牧兽医,2009,6：87-89
11.刘清海,孙洪升,车秀华。马流感的研究现状[J]。畜牧兽医科技信息,2005,6：19-21.
12.苏德昌,董泽生。应用中药荆防败毒散加减治疗马流感的体会[J]。青海畜牧业,2004,2：47
13.孙高超,毕可东,张娜。马流感简介[J]。动物医学进展,2008,29(8)：115-116。
14.田建国,叶丛华,张文东等。云南地区马流感病毒检测及其HA基因序列分析[J]。畜牧与兽医,2009,41(6)：22-26
15.王秀荣,杨建德,许景君等。马流感A/马/京防/74-1(H7N7)毒株HA基因的序列分析[J]。中国预防兽医学报,2004,26(5)：332-335
16.杨建德,相文华,薛飞等。我国H3N8马流感病毒血凝素基因分子进化树的分析[J]中国生物工程杂志,2003,(06).
17.杨建德,薛飞,王晓钧等。马流感病毒(A/equine/Qinghai/1/94)核蛋白基因的序列测定及同源性分析[J]中国预防兽医学报,2003,(02)
18.杨建德,王文军,薛飞等。马流感病毒(A/Equine/Qinghai/1/94)神经氨酸酶基因的克隆与同源性分析[J]中国预防兽医学报,2003,(05)
19.杨建德,相文华。我国马流感的研究现状[J]黑龙江畜牧兽医,2002,(03).
20.殷震,刘景华等。动物病毒学[M],第二版,北京：科学出版社。1997,704-735
21.张利峰,张鹤,晓谷强等。马流感血凝及血凝抑制试验的研究与应用,[J],检验检疫科学,2003(13)1：30-32。
22.赵文成,许娇娜。我国马流感的研究现状[J]。畜牧兽医科技信息,2008,6：5-7
23.左铃兰,江国林。大理州马流行性感冒的防治[J]。云南畜牧兽医,2009,3：28-29.
24. Chamber T M。用DFA试验快速诊断马流感[J]。国外兽医学,畜禽传染病,1995,15(2)：40-42。
25. Damiani A M, Scicluna M T, Ciabatti I, et al. Genetic characterization of equine influenza viruses isolated in Italy between 1999 and 2005[J].Virus Res,2008,131,131 (1):100-105.
26. Guo Y, Wang M, Kawaka Y, et al. Characterization of a new avian-like influenza A virus from horses in China.Virology,1992,1881,188(1):245-255.
27. Global surveillance network for equine influenza.http://www.equiflunet.org.uk.
28. Martella V, Elia G, Decaro N, et al. An outbreak of equine influenza virus in vaccinated horses in Italy is due to an H3N8 stain closely related to recent North American representatives of the Florida sub-lineage[J].Vet Microbial,2007,121,121:56-63
29. Newton J R, Daly J M, Spencer L et. al. Description of the outbreak of equine influenza(H3N8)in the United Kingdom in 2003, during which recently vaccinated horses in New market dev-eloped respiratory disease [J].Vet Dec,2007,158,158 (6):185-192.
30. OIE. Equine influenza.Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, part3. http://www.oie.org:2004,
31. Reeve-Johnson L. Equine influenza in Australia [J].Vet Rec,2007,161,161 (18):635.
32. Chris Olsen. Canine Influenza. North Carolina University College of Veterinary Medicine. http://www.ncvdl.com/.2006.
33. Control of Canine Influenza in Dogs-Questions, Answers, and Interim Guidelines October 17,2005. www.vetmed.ufl.edu/pr.
34. Ed Dubovi Sept. Ruben Donis,26 issue of Science Express, the online version of Science magazine
35. Tara Anderson 2008, ScienceDaily (Mar.20,2008)
36. P.C.Crawford, Edward J. Dubovi, William L. Castleman, Iain Stephenson, Transmission of Equine Influenza Virus to Dogs Science 21 October 2005:Vol.310. no.5747:482-485
37. Kyoung-Jin Yoon, Vickie L. Cooper, Kent J. Schwartz, Influenza Virus Infection in Racing Greyhounds
38. R.Newton, A. Cooke, D.Elton, N.Bryant, A. Rash, S. et al. Canine influenza virus: cross-species transmission from horses, Vet Rec.2007,161:142-143
39. Daly, Janet M.; Blunden, Anthony S.; MacRae, Shona; Transmission of equine influenza virus to English foxhounds Emerg Infect Dis.2008,14(3):461-464.
40. Takashi Yamanaka, Manabu Nemoto, Koji Tsujimura, Takashi Kondo Interspecies transm ission of equine influenza virus (H3N8) to dogs by close contact with experimentally infected horses. Available online 21 June 2009.http://www.sciencedirect.com
41. Sigfrido Burgos and Sergio A. Burgos.. Reports of Avian Influenza H5N1 in Cats and dogs. International Journal of Poultry Science 2007,6(12):1003-1005.
42. Matthias Giese, Timm C. Harder, Jens P. Teifke, et al. Experimental infection and natural
contact exposure of dogs with avian influenza virus (H5N1). Emerging infectious disease, 2008.14(2):308-310.
43. Riks Mass, Mirriam Tacken, Lisette Ruuls, et al. Avian influenza (H5N1) susceptibility and receptors in dogs. Emerging infectious disease,2007.13(8):1219-1221.
44. Daesub Song, Chulseung Lee, Bokyu Kang. Emerging infectious disease, www.cdc.gov/eid 2009.15(1):56-58.
45. Song D, Kang B, Lee C, Jung K, Ha G, Kang D, et al. Transmission of avian infl uenza virus (H3N2) to dogs. Emerg Infect Dis.2008,14(7)41-46