禽流感病毒H9N2亚型ZZ1株的分离鉴定及其免疫原性初步研究
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摘要
禽流感(Avian Influenza,AI)是由正粘病毒科、流感病毒属A型流感病毒(Avian Influenza Viruses,AIV)引起的一种急性、高度接触性传染病。
目前在我国,多种亚型的禽流感病毒已在我国诸多地区被分离到,H9N2亚型低致病性禽流感更是时有发生,给我国养禽业带来了严重的经济损失。近年来,河南及周边地区多家养鸡场出现疑似禽流感H9亚型病例,临床症状主要表现为鸡只发生不同程度的呼吸道症状,死亡率10%以下,产蛋鸡产蛋率下降,给养鸡业造成了严重的经济损失。此文的目的在于查明病因,找出致病病原,并进行免疫原性的初步研究,为利用禽流感病毒H9N2亚型地方分离进行H9亚型禽流感的防治提供一定的理论参考依据。
我们采用常规方法和分子生物学方法对河南及周边地区部分发病鸡场疑似H9亚型低致病性禽流感的病例进行了病原分离及部分病原生物学特性的研究。
无菌采集发病鸡的气管、肺脏、肾脏等组织,经过研磨和无菌过滤等处理后接种9日龄鸡胚,采用鸡胚盲传分离病毒,测定血凝效价,利用血清中和和终点稀释方法进行纯化。
理化特性试验表明,尿囊液对去污剂等脂溶剂的灭活比较敏感,福尔马林、乙醚可破坏其感染性,病毒不耐热,不耐强酸强碱。
尿囊液送哈尔滨兽医研究所进行禽流感亚型鉴定,鉴定结果表明分离毒株为H9亚型禽流感病毒。将分离毒株命名为A/Chicken/henan/ZZ1/06,简称ZZ1株。参考已发表的基因序列,合成禽流感病毒NA基因引物,并扩增目的片段。对分离毒株的NA基因进行了克隆和测序,并进行了遗传进化分析,序列分析结果表明,禽流感病毒H9亚型ZZ1株属于N2亚型。
将禽流感病毒H9N2亚型ZZ1株病毒灭活后加弗氏佐剂作为抗原接种SPF鸡,试验鸡不仅产生了较高的抗体水平,而且能够抵抗禽流感病毒H9N2亚型ZZ1病毒株的攻击,表明禽流感病毒H9N2亚型ZZ1株具有较好的免疫原性。
将灭活后加有弗氏佐剂的禽流感病毒H9N2亚型ZZ1株接种SPF鸡,每周测定相应的H9亚型禽流感抗体水平。在接种灭活后加有弗氏佐剂的禽流感H9N2亚型ZZ1株后,H9亚型禽流感抗体上升很快,在接种后第14天HI效价即可达到6.0(Log2)以上,在接种后第21天HI效价即可达到8.0(Log2)以上,HI抗体效价基本呈线性增长;在接种后第35天HI效价即可接近峰值9.0(Log2);接种后第28天～49天HI效价处于8.0(Log2)～9.0(Log2)水平,直至第98天仍可以维持在6.0(Log2)以上。
用灭活后加有弗氏佐剂的禽流感病毒H9N2亚型ZZ1毒株免疫罗曼鸡,在接种灭活后加有弗氏佐剂的禽流感H9N2亚型ZZ1株后,H9亚型禽流感抗体上升很快,在接种后第14天HI效价即可达到6.0(Log2)以上,在接种后第21天内以上,H9亚型禽流感抗体HI抗体效价基本呈线性增长;在接种后第21天HI效价即可接近峰值9.0(Log2);接种后第28天～49天HI效价处于9.0(Log2)左右水平,直至第84天仍可以维持在6.0(Log2)以上。从整体来看,灭活后加有弗氏佐剂的禽流感H9N2亚型ZZ1株接种罗曼鸡后可以产生明显的H9亚型禽流感抗体,且抗体峰值水平较接种SPF鸡略高,持续时间与接种SPF鸡相当。
本论文试验为利用禽流感H9N2亚型地方分离株进行H9亚型禽流感的防治提供了一定的理论参考依据。
Avian Influenza virus by the Division are sticky, influenza virus type A influenza virus (Avian Influenza Viruses, AIV) caused an acute, highly contagious diseases.Hemagglutinin(HA) and Neuraminidase(NA) of influenza viruses are easy to have antigenic drift and antigenic shift,and new pathogenic subtype which is able to infect humans can be formed by such viral mutations. Genetic reassembly which always happens in H5N1 subtype accelerates the threat of avian influenza A viruses to human being.
According to the pathogenicity,Pathogenic avian influenza be divided into highly pathogenic avian influenza(HPAI) and low pathogenic avian influenza(LPAI),generally.Highly pathogenic avian influenza is a devastating blow on the poultry industry, and low pathogenic avian influenza is often complicated by secondary infection or other bacterial or viral diseases, which may caused the death of a large number of chickens. at the same time ,AI constitutes a serious threat to the public health of human beings.
Vaccine means the most important measure to combat Avian influenza outbreak for a long time,But, because of high frequency variation of avian influenza virus antigen, which resulted in the effective prevention and control difficult.Because of broad area, large quantity of birds, frequent circulation of poultry product, it is difficult to remove completely from the external environment after once the bird flu happened, The antigenic drift and antigenic shift of AIV makes it be possible that low virulence influenza shift to HPAI, and which makes potential menace to the avian industry in our country.
At present in our country, multi-subtype avian influenza virus (AIV) has been isolated from many areas,and low-pathogenic avian influenza has occurred from time to time, which to result in China's poultry industry suffered heavy financial losses. In recent years, avian inflenza still occur from time to time after routine immunization program in some farms of Henan Province, which result in serious economic losses, and caused great public health risk . Objective This study was conducted to identify the exact cause of the disease, and to provide scientific data for the control of avian influenza. In this study, isolation of pathogen and study on some biological characteristics was performed in several farms by routine methods and molecular biological methods.
Trachea, kidney, and lung samples collected from suspect AI outbreaks were made to suspensions which were inoculated into the allantoic of SPF embryo. The third passage virus produced gross lesions to chicken embryos,and allantoic fluid can agglutinate a 1% suspension of fowl erythrocytes. The allantoic fluid neutralize by ND positive serum and inoculated 9-day-old chick embryo, the collection allantoic fluid can still agglutinate 1% suspension of fowl erythrocytes.
Physical and chemical characteristics of the pilot showed that the allantoic fluid of fat, such as solvent detergent inactivated more sensitive, formalin, ether could undermine its infectious, and it is intolerance to heat, strong acid strong base.
Strain isolated was Named A/Chicken/henan/ZZ1/06, referred to as strain ZZ1. Allantoic fluid sent to the Harbin Veterinary Research Institute to identify avian influenza subtypes.The result showed that which belonged to H9 subtype strain. NA gene primers were synthesized and the objective fragment was amplified with by RT-PCR.The NA gene order was sequenced and analysis of genetic evolution.
Antigen? were prepared comprise Inactivation ZZ1 strain. were inoculated into SPF chickens and Roman chinckens.Antibody levels of H9 subtype AI were measured weekly.The results showed that vaccination with Inactivation ZZ1 strain inactivated Antigen, not only had higher antibody levels ,but also resistance to the challenges with ZZ1 strain,which showed that the Inactivation ZZ1 strain have good immunogenicity.
目前在我国,多种亚型的禽流感病毒已在我国诸多地区被分离到,H9N2亚型低致病性禽流感更是时有发生,给我国养禽业带来了严重的经济损失。近年来,河南及周边地区多家养鸡场出现疑似禽流感H9亚型病例,临床症状主要表现为鸡只发生不同程度的呼吸道症状,死亡率10%以下,产蛋鸡产蛋率下降,给养鸡业造成了严重的经济损失。此文的目的在于查明病因,找出致病病原,并进行免疫原性的初步研究,为利用禽流感病毒H9N2亚型地方分离进行H9亚型禽流感的防治提供一定的理论参考依据。
我们采用常规方法和分子生物学方法对河南及周边地区部分发病鸡场疑似H9亚型低致病性禽流感的病例进行了病原分离及部分病原生物学特性的研究。
无菌采集发病鸡的气管、肺脏、肾脏等组织,经过研磨和无菌过滤等处理后接种9日龄鸡胚,采用鸡胚盲传分离病毒,测定血凝效价,利用血清中和和终点稀释方法进行纯化。
理化特性试验表明,尿囊液对去污剂等脂溶剂的灭活比较敏感,福尔马林、乙醚可破坏其感染性,病毒不耐热,不耐强酸强碱。
尿囊液送哈尔滨兽医研究所进行禽流感亚型鉴定,鉴定结果表明分离毒株为H9亚型禽流感病毒。将分离毒株命名为A/Chicken/henan/ZZ1/06,简称ZZ1株。参考已发表的基因序列,合成禽流感病毒NA基因引物,并扩增目的片段。对分离毒株的NA基因进行了克隆和测序,并进行了遗传进化分析,序列分析结果表明,禽流感病毒H9亚型ZZ1株属于N2亚型。
将禽流感病毒H9N2亚型ZZ1株病毒灭活后加弗氏佐剂作为抗原接种SPF鸡,试验鸡不仅产生了较高的抗体水平,而且能够抵抗禽流感病毒H9N2亚型ZZ1病毒株的攻击,表明禽流感病毒H9N2亚型ZZ1株具有较好的免疫原性。
将灭活后加有弗氏佐剂的禽流感病毒H9N2亚型ZZ1株接种SPF鸡,每周测定相应的H9亚型禽流感抗体水平。在接种灭活后加有弗氏佐剂的禽流感H9N2亚型ZZ1株后,H9亚型禽流感抗体上升很快,在接种后第14天HI效价即可达到6.0(Log2)以上,在接种后第21天HI效价即可达到8.0(Log2)以上,HI抗体效价基本呈线性增长;在接种后第35天HI效价即可接近峰值9.0(Log2);接种后第28天～49天HI效价处于8.0(Log2)～9.0(Log2)水平,直至第98天仍可以维持在6.0(Log2)以上。
用灭活后加有弗氏佐剂的禽流感病毒H9N2亚型ZZ1毒株免疫罗曼鸡,在接种灭活后加有弗氏佐剂的禽流感H9N2亚型ZZ1株后,H9亚型禽流感抗体上升很快,在接种后第14天HI效价即可达到6.0(Log2)以上,在接种后第21天内以上,H9亚型禽流感抗体HI抗体效价基本呈线性增长;在接种后第21天HI效价即可接近峰值9.0(Log2);接种后第28天～49天HI效价处于9.0(Log2)左右水平,直至第84天仍可以维持在6.0(Log2)以上。从整体来看,灭活后加有弗氏佐剂的禽流感H9N2亚型ZZ1株接种罗曼鸡后可以产生明显的H9亚型禽流感抗体,且抗体峰值水平较接种SPF鸡略高,持续时间与接种SPF鸡相当。
本论文试验为利用禽流感H9N2亚型地方分离株进行H9亚型禽流感的防治提供了一定的理论参考依据。
Avian Influenza virus by the Division are sticky, influenza virus type A influenza virus (Avian Influenza Viruses, AIV) caused an acute, highly contagious diseases.Hemagglutinin(HA) and Neuraminidase(NA) of influenza viruses are easy to have antigenic drift and antigenic shift,and new pathogenic subtype which is able to infect humans can be formed by such viral mutations. Genetic reassembly which always happens in H5N1 subtype accelerates the threat of avian influenza A viruses to human being.
According to the pathogenicity,Pathogenic avian influenza be divided into highly pathogenic avian influenza(HPAI) and low pathogenic avian influenza(LPAI),generally.Highly pathogenic avian influenza is a devastating blow on the poultry industry, and low pathogenic avian influenza is often complicated by secondary infection or other bacterial or viral diseases, which may caused the death of a large number of chickens. at the same time ,AI constitutes a serious threat to the public health of human beings.
Vaccine means the most important measure to combat Avian influenza outbreak for a long time,But, because of high frequency variation of avian influenza virus antigen, which resulted in the effective prevention and control difficult.Because of broad area, large quantity of birds, frequent circulation of poultry product, it is difficult to remove completely from the external environment after once the bird flu happened, The antigenic drift and antigenic shift of AIV makes it be possible that low virulence influenza shift to HPAI, and which makes potential menace to the avian industry in our country.
At present in our country, multi-subtype avian influenza virus (AIV) has been isolated from many areas,and low-pathogenic avian influenza has occurred from time to time, which to result in China's poultry industry suffered heavy financial losses. In recent years, avian inflenza still occur from time to time after routine immunization program in some farms of Henan Province, which result in serious economic losses, and caused great public health risk . Objective This study was conducted to identify the exact cause of the disease, and to provide scientific data for the control of avian influenza. In this study, isolation of pathogen and study on some biological characteristics was performed in several farms by routine methods and molecular biological methods.
Trachea, kidney, and lung samples collected from suspect AI outbreaks were made to suspensions which were inoculated into the allantoic of SPF embryo. The third passage virus produced gross lesions to chicken embryos,and allantoic fluid can agglutinate a 1% suspension of fowl erythrocytes. The allantoic fluid neutralize by ND positive serum and inoculated 9-day-old chick embryo, the collection allantoic fluid can still agglutinate 1% suspension of fowl erythrocytes.
Physical and chemical characteristics of the pilot showed that the allantoic fluid of fat, such as solvent detergent inactivated more sensitive, formalin, ether could undermine its infectious, and it is intolerance to heat, strong acid strong base.
Strain isolated was Named A/Chicken/henan/ZZ1/06, referred to as strain ZZ1. Allantoic fluid sent to the Harbin Veterinary Research Institute to identify avian influenza subtypes.The result showed that which belonged to H9 subtype strain. NA gene primers were synthesized and the objective fragment was amplified with by RT-PCR.The NA gene order was sequenced and analysis of genetic evolution.
Antigen? were prepared comprise Inactivation ZZ1 strain. were inoculated into SPF chickens and Roman chinckens.Antibody levels of H9 subtype AI were measured weekly.The results showed that vaccination with Inactivation ZZ1 strain inactivated Antigen, not only had higher antibody levels ,but also resistance to the challenges with ZZ1 strain,which showed that the Inactivation ZZ1 strain have good immunogenicity.
引文
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51. Avian influenza[J]. Saudi Med J, 2006. 27(6): 923‐928.
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59. Zhou, J.Y., et al., Characterization of a highly pathogenic H5N1 influenza virus derived from bar‐headed geese in China[J]. J Gen Virol, 2006. 87(Pt 7): 1823‐1833.
60. Zhou, K., et al., RNA interference of avian influenza virus H5N1 by inhibiting viral mRNA with siRNA expression plasmids[J]. J Biotechnol, 2008. 135(2): 140‐144.
61. Philpott, M., B.C. Easterday, and V.S. Hinshaw, Neutralizing epitopes of the H5 hemagglutinin from a virulent avian influenza virus and their relationship to pathogenicity[J]. J Virol, 1989. 63(8): 3453‐3458.
62. Salomon, R.,R.G. Webster, The influenza virus enigma[J]. Cell, 2009. 136(3): 402‐410.
63.黄庚明,辛潮安.禽流感病毒血凝素(HA)基因的研究进展.[J].广东畜牧兽医科技,, 2001,26(2):3‐5.
64. Khan, O.A., et al., Isolation and identification of highly pathogenic avian influenza H5N1 virus from Houbara bustards (Chlamydotis undulata macqueenii) and contact falcons[J]. Avian Pathol, 2009. 38(1): 35‐39.
65. Walker J.A., K.Y., Importance of conserved amino acids at the cleavage site of the hemagglutinin of a virulent avian influenza A virus[J]. J Gen Virol., 1993,74(2):311‐4.
66. Vey M., O.M., Adler S.,et al., Hemagglutinin activation of pathogenic avian influenza viruses of serotypes H7 requires the protease recognition motif R‐X‐K/R‐R.[J]. Virology., 1992,188:408‐413.
67. Kawaoka Y., W.R.G., Interoplay between carbohydrate in the stalk and the length of theconnection peptid determines the cleavability of influenza virus hemagglutinin[J]. J Virol 1989. 63:3296‐3000.
68. Kawaoka Y., N.A., Alexander D.J.,et al, Molecular analyses of the hemagglutinin genes of H5 influenza viruses:Origin of a virulent turkey strain.[J]. Virology., 1987,158:218‐227.
69. Bosch, F.X., M.Orlich,H.D.Klenk and R.Rott., The structure of the hemagglutinin,a determinant for the pathogenicity of influenza viruses[J]. Virology,, 1979,95:197‐207.
70. Hay, A.J., The virus genome and its replication.[J]. in“Textbook of influenza”(Nicholson,K.G.,Wbster,R.G.,and Hay,A.J.,Eds.)Blackwell Science Ltd.,London., 1998:pp43‐53.
71. Colman, P.M., W.G.Laver,J.N.Varghese,A.T.Baker,P.A.Tulloch,G.M.Air and R.G.Webster., The three‐dimensional structure of complex of influenza virus neuraminidase and an antibody[J]. Nature, 1987,326:358‐363.
72. Crawford J., W.B., Vosnesenskcy A., et al, Baculovirus derived hemagglutinin vaccines protect against lethal influenza infections by avain H5 and H7 subtypes[J]. Vaccine, 1999,17:2265~2274.
73. Treanor JJ., T.E., Zebedee SL.,et al, Passively transferred monoclonal antibody to the M2 protein inhibits influenza A virus replication mice[J]. J Viro, 1999,64(3):1357~1375.
74.陈全姣,金梅林,陈焕春.禽流感疫苗研究进展[J].中国生物工程杂志, 2004,24(4),34‐37.
75.柴丽娜,杨大光,郝春丽,等.禽流感疫苗研究进展[J].上海畜牧兽医通讯, 2007(01). 12‐13.
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77. ParkinN, C.P., CoelinghK, Genetically engineered live attenuated influenza A virus vaccine candidates[J]. J Virol,1997,71:2772~2778.
78. Ulmer JB., D.J., Parker SE.,et al, Heterologous p rotection against influenza by injection of DNA encoding a viralp rotein[J]. Science, 1993,259:1745~1749.
79. Justew ID., W.R., Long2 term maintenance of B cell immunity of influenza virus hemagglutinin in mice following DNA2 based immunization[J]. Virology, 1996,224:10~17.
80. Pertmer TM, O.A., Moser JM,et al, DNA vaccines for influenza virus:differential effects of maternal antibody on immune responses to hemagglutinin and nucleoprotein[J]. J Virol, 2000,74:7787~7793.
81.海洋,赵玉军,张立鹏.禽流感疫苗的研究进展[J].上海畜牧兽医通讯, 2007(06). 39‐40.
82. Thomson SA., S.M., Medveczky J.,et al, Delivery of multiple CD8 cytotoxic T cell epitopes by DNA vaccination[J]. J Immuno, 1998,160:1717~1723.
83. SchlesingerS, D., Alphavirusvectorsforgene expression and vaccines[J]. Curr Opin Biotechno, 1999,10:434~439.
84. Dubenskey T W, L.M.H., Ulmer J B, Delivery systems for gene‐based vaccines[J]. Molecular Medicine, 2000,6:723~732.
85.信爱国,宋学林,高华峰.禽流感病毒免疫研究进展[J].上海畜牧兽医通讯, 2006(01). 16‐17.
86.毕英佐.H9N2亚型禽流感的流行现状和防治措施[J].家禽科学, 2007(11),3‐5.
87. JIN‐HUA LIU, K.O., AARON MWEENE, et al, Genetic Conservation of Hemagglutinin Gene of H9 Influenza Virus in Chicken Population in Mainland China[J]. Virus Genes, 2004.29:3 ,329‐334.
88. JIN‐HUA LIU, K.O., WEI‐MIN SHI, et al, Phylogenetic Analysis of Neuraminidase Gene of H9N2 Influenza Virus Prevalent in chickens in China during 1995‐2002[J]. Virus Genes 2003. 27(2): 197‐202.
89. Kobasa D., et al., Neuraminidase hemadsorption activity,conserved in avian influenza A viruses ,does no influence viral replicaion in ducks.[J]. J Virol, 1997. 71: 6706‐6713.
90.温志芬,陈瑞爱,唐满华,等.禽流感H9亚型灭活油乳剂疫苗不同剂量的免疫效果比较[J].中国兽医科技, 2003. 33(12): 65‐67.
91.吴庭才,张春杰,郭向鹏.禽流感母源抗体消长规律和免疫保护抗体临界值的测定[J].洛阳农业高等专科学校学报,2002. 22(3): 243‐244.