鸽副黏病毒灭活疫苗候选株aYQ的生物学特性研究
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摘要
评价鸽副黏病毒(PPMV)致弱毒株aYQ的生物学特性,为研制鸽副黏病毒灭活疫苗奠定基础。将PPMV aYQ株经SPF鸡胚连续传代,建立种子批。测定各代次毒种的血凝效价(HA)和鸡胚半数感染量(EID50),并进行纯净性检验;测定毒种鸡胚最小致死量的平均死亡时间(MDT)、1日龄鸡脑内接种致病指数(ICPI)和6周龄鸡静脉接种致病指数(IVPI);测定毒种对鸡胚和鸽的毒力、遗传稳定性和抗原相关性。PPMV aYQ株经SPF鸡胚传代至F10,HA效价为1∶256～1∶512,病毒含量为10-8.5 EID50/0.1mL～10-8.9 EID50/0.1mL,毒种各代次均无细菌、霉菌、支原体以及外源病毒污染;PPMV aYQ株的ICPI和IVPI结果均为0,MDT测定结果大于120h;以103倍稀释的PPMV aYQ株病毒液接种10日龄SPF鸡胚96h无死亡,无眼观病变。以106EID50/羽的剂量人工感染鸽,未见异常症状;PPMV aYQ株各代次F基因均未发生基因突变,F蛋白裂解位点氨基酸序列均为-112GRQGRL 117-;交叉血凝抑制试验和交叉中和试验结果表明,PPMV aYQ株与NDV La Sota株间存在明显的抗原性差异。鸽副黏病毒弱毒株aYQ能够在SPF鸡胚上稳定传代,且生物学特性稳定,免疫原性良好,可以作为鸽源副黏病毒灭活疫苗的毒种。
The objective of this study was to evaluate the biological characteristics of the aYQ strain of pigeon paramyxovirus and to lay the foundation for the development of the pigeon-paramyxovirus inactivated vaccine.PPMV aYQ strain were passaged continuously in SPF chicken embryos to establish a seed batch.HA and EID50 were determined and the purity of each strain was tested;The virulence of the aYQ strain was evaluated by MDT,ICPI and IVPI.Pathogenicity of aYQ strain to chicken embryos and pigeons,genetic stability and antigen relevance were determined.PPMV aYQ strain was passaged to 10 generations in SPF chicken embryos in the absence of bacteria,mold,mycoplasma and exogenous viruses.The HA titers of various generation viruses were 1∶256-1∶512and the virus contents were 10-8.5-10-8.9 EID50/0.1mL.Both ICPI and IVPI of PPMV aYQ strain were 0,and the MDT was more than 120h;The 10-day-old SPF chicken embryos inoculated with 103-fold diluted PPMV aYQ strain survived at 96 hpost inoculation.Pigeons which were infected artificially at a dose of 106EID50 with PPMV aYQ strain showed no apparent clinical symptoms;The results of sequence analysis showed that there was no gene mutation in F gene of PPMV aYQ strain,and the deduced amino acid sequences were-112 GRQGRL 117-.The results of cross-hemagglutination inhibition test and cross-neutralization test showed that there was obvious antigenic difference between PPMV aYQ strain and NDV La Sota strain.The aYQ strain can be passaged in SPF chicken embryos with stable genetic and biological characteristics.It indicated that the PPMV aYQ strain possess satisfied immunogenicity,and can be used as the preferred pigeon paramyxovirus inactivated vaccine candidate strain.
The objective of this study was to evaluate the biological characteristics of the aYQ strain of pigeon paramyxovirus and to lay the foundation for the development of the pigeon-paramyxovirus inactivated vaccine.PPMV aYQ strain were passaged continuously in SPF chicken embryos to establish a seed batch.HA and EID50 were determined and the purity of each strain was tested;The virulence of the aYQ strain was evaluated by MDT,ICPI and IVPI.Pathogenicity of aYQ strain to chicken embryos and pigeons,genetic stability and antigen relevance were determined.PPMV aYQ strain was passaged to 10 generations in SPF chicken embryos in the absence of bacteria,mold,mycoplasma and exogenous viruses.The HA titers of various generation viruses were 1∶256-1∶512and the virus contents were 10-8.5-10-8.9 EID50/0.1mL.Both ICPI and IVPI of PPMV aYQ strain were 0,and the MDT was more than 120h;The 10-day-old SPF chicken embryos inoculated with 103-fold diluted PPMV aYQ strain survived at 96 hpost inoculation.Pigeons which were infected artificially at a dose of 106EID50 with PPMV aYQ strain showed no apparent clinical symptoms;The results of sequence analysis showed that there was no gene mutation in F gene of PPMV aYQ strain,and the deduced amino acid sequences were-112 GRQGRL 117-.The results of cross-hemagglutination inhibition test and cross-neutralization test showed that there was obvious antigenic difference between PPMV aYQ strain and NDV La Sota strain.The aYQ strain can be passaged in SPF chicken embryos with stable genetic and biological characteristics.It indicated that the PPMV aYQ strain possess satisfied immunogenicity,and can be used as the preferred pigeon paramyxovirus inactivated vaccine candidate strain.
引文
[1]陆承平.兽医微生物学[M].5版.北京:中国农业出版社,2013.
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[4]符子华,龚国华,易忠,等.三株鸽新城疫病毒新疆分离株F基因序列分析及遗传起源探讨[J].中国预防兽医学报,2007(10):777-782.
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[7]杨冰,张波,符子华,等.鸽新城疫油佐剂灭活苗的研制与应用[J].新疆农业科学,2005,42(5):359-362.
[8]叶贺佳,仇微红,李敏,等.鸽新城疫灭活疫苗的研制及免疫效果研究[J].中国家禽,2012,34(16):20-24.
[9]赵宝华,傅元华,范建华,等.鸽新城疫油乳剂灭活疫苗的研制[J].江苏农业学报,2010,26(6):1293-1297.
[10] Aldous E W,Fuller C M,Mynn J K,et al.A molecular epidemiological investigation of isolates of the variant avian paramyxovirus type 1virus(PPMV-1)responsible for the 1978to present panzootic in pigeons[J].Avian Pathol,2004,33(2):258-269.
[11] Wakamatsu N,King D J,Kapczynski D R,et al.Experimental pathogenesis for chickens,turkeys,and pigeons of exotic Newcastle disease virus from an outbreak in California during2002-2003[J].Vet Pathol,2006,43(6):925-933.
[12] OIE.Manual of Diagnostic Tests and Vaccines for Terrestrisal Animal(Mammals,Birds and Bees)[M].//5th ed.Paris,France:World Organisation for Animal Health(OIE),2004.
[13]李仕超,仇旭升,刘开春,等.六株鸽源新城疫病毒的分离、鉴定及生物信息学分析[J].中国动物传染病学报,2014(5):1-9.
[14] Pedersen J C,Senne D A,Woolcock P R,et al.Phylogenetic relationships among virulent Newcastle disease virus isolates from the 2002-2003outbreak in California and other recent outbreaks in North America[J].J Clin Microbiol,2004,42(5):2329-2334.
[15]张俊平,杨惠萍,蒋凤英,等.鸽副黏病毒Ⅰ型(川沙株)的分离与鉴定[J].中国兽药杂志,2013(11):13-17.
[16] Kim L M,King D J,Guzman H,et al.Biological and phylogenetic characterization of pigeon paramyxovirus serotype 1circulating in wild North American pigeons and doves[J].J Clin Microbiol,2008,46(10):3303-3310.
[17]许信刚,李勇,张耀相,等.鸽新城疫病毒分离株的部分生物学特性鉴定及疫苗免疫试验[J].中国兽医科技,2001(3):22-23.
[18]黎俊君,何琴,焦培荣,等.四株广东鸽源新城疫病毒的生物学特性[J].中国兽医科学,2011(7):677-683.
[19]胡北侠,杨少华,张会娟,等.鸽源新城疫病毒全基因组序列分析及致病性研究[J].畜牧兽医学报,2014,45(2):255-261.
[20]刘华雷,王永坤,严维巍,等.禽副粘病毒Ⅰ型(APMV-1)分子生物学研究进展[J].动物医学进展,2001,22(1):16-21.
[21]韦平,韦天超,杨宗维,等.禽Ⅰ型副粘病毒各种禽源分离株毒力及其相关基因的研究[J].病毒学报,2005,21(1):35-42.
[22]单松华,邵朝纲,邹键,等.鸽基因Ⅶ型新城疫病毒的分离鉴定[J].病毒学报,2003,19(4):360-364.
[23] Dortmans J C,Koch G,Rottier P J,et al.Virulence of Newcastle disease virus:what is known so far?[J].Vet Res,2011,42:122.
[24]Ujvári D,Wehmann E,Herczeg J,et al.Identification and subgrouping of pigeon type Newcastle disease virus strains by restriction enzyme cleavage site analysis[J].J Virol Meth,2006,131(2):115-121.
[25] Guo H,Liu X,Han Z,et al.Phylogenetic analysis and comparison of eight strains of pigeon paramyxovirus type 1(PPMV-1)isolated in China between 2010and 2012.[J].Arch Virol,2013,158(6):1121-1131.
[26] Wang J,Liu H,Liu W,et al.Genomic characterizations of six pigeon paramyxovirus type 1viruses isolated from live bird markets in China during 2011to 2013[J].PLoS One,2015,10(4):e124261.
[27] Alexander D J.Newcastle disease and other avian paramyxoviruses[J].Rev Sci Et Tech,2000,19(2):443.
[28] Scott P C,Wilson T B,Walker C.Serological and growth rate responses to the use of chicken Newcastle disease vaccines in pigeons[J].Australian Vet J,2013,91(12):525-530.
[2]贺奋义.鸽新城疫疫苗的研制及其F基因的克隆表达和生物活性研究[D].甘肃兰州:甘肃农业大学,2012.
[3]王伟伟.江苏省新城疫病毒分子流行病学研究及鸽源分离株基因组序列的分析[D].江苏扬州:扬州大学,2009.
[4]符子华,龚国华,易忠,等.三株鸽新城疫病毒新疆分离株F基因序列分析及遗传起源探讨[J].中国预防兽医学报,2007(10):777-782.
[5]邹永新,李少方,廖新权,等.广东部分地区鸽禽Ⅰ型副黏病毒分子流行病学研究[J].中国预防兽医学报,2009(10):756-760.
[6]刘业兵,田春利,胡艳丽,等.鸽Ⅰ型副黏病毒的分离鉴定及生物学特性研究[J].中国家禽,2006,28(24):63-65.
[7]杨冰,张波,符子华,等.鸽新城疫油佐剂灭活苗的研制与应用[J].新疆农业科学,2005,42(5):359-362.
[8]叶贺佳,仇微红,李敏,等.鸽新城疫灭活疫苗的研制及免疫效果研究[J].中国家禽,2012,34(16):20-24.
[9]赵宝华,傅元华,范建华,等.鸽新城疫油乳剂灭活疫苗的研制[J].江苏农业学报,2010,26(6):1293-1297.
[10] Aldous E W,Fuller C M,Mynn J K,et al.A molecular epidemiological investigation of isolates of the variant avian paramyxovirus type 1virus(PPMV-1)responsible for the 1978to present panzootic in pigeons[J].Avian Pathol,2004,33(2):258-269.
[11] Wakamatsu N,King D J,Kapczynski D R,et al.Experimental pathogenesis for chickens,turkeys,and pigeons of exotic Newcastle disease virus from an outbreak in California during2002-2003[J].Vet Pathol,2006,43(6):925-933.
[12] OIE.Manual of Diagnostic Tests and Vaccines for Terrestrisal Animal(Mammals,Birds and Bees)[M].//5th ed.Paris,France:World Organisation for Animal Health(OIE),2004.
[13]李仕超,仇旭升,刘开春,等.六株鸽源新城疫病毒的分离、鉴定及生物信息学分析[J].中国动物传染病学报,2014(5):1-9.
[14] Pedersen J C,Senne D A,Woolcock P R,et al.Phylogenetic relationships among virulent Newcastle disease virus isolates from the 2002-2003outbreak in California and other recent outbreaks in North America[J].J Clin Microbiol,2004,42(5):2329-2334.
[15]张俊平,杨惠萍,蒋凤英,等.鸽副黏病毒Ⅰ型(川沙株)的分离与鉴定[J].中国兽药杂志,2013(11):13-17.
[16] Kim L M,King D J,Guzman H,et al.Biological and phylogenetic characterization of pigeon paramyxovirus serotype 1circulating in wild North American pigeons and doves[J].J Clin Microbiol,2008,46(10):3303-3310.
[17]许信刚,李勇,张耀相,等.鸽新城疫病毒分离株的部分生物学特性鉴定及疫苗免疫试验[J].中国兽医科技,2001(3):22-23.
[18]黎俊君,何琴,焦培荣,等.四株广东鸽源新城疫病毒的生物学特性[J].中国兽医科学,2011(7):677-683.
[19]胡北侠,杨少华,张会娟,等.鸽源新城疫病毒全基因组序列分析及致病性研究[J].畜牧兽医学报,2014,45(2):255-261.
[20]刘华雷,王永坤,严维巍,等.禽副粘病毒Ⅰ型(APMV-1)分子生物学研究进展[J].动物医学进展,2001,22(1):16-21.
[21]韦平,韦天超,杨宗维,等.禽Ⅰ型副粘病毒各种禽源分离株毒力及其相关基因的研究[J].病毒学报,2005,21(1):35-42.
[22]单松华,邵朝纲,邹键,等.鸽基因Ⅶ型新城疫病毒的分离鉴定[J].病毒学报,2003,19(4):360-364.
[23] Dortmans J C,Koch G,Rottier P J,et al.Virulence of Newcastle disease virus:what is known so far?[J].Vet Res,2011,42:122.
[24]Ujvári D,Wehmann E,Herczeg J,et al.Identification and subgrouping of pigeon type Newcastle disease virus strains by restriction enzyme cleavage site analysis[J].J Virol Meth,2006,131(2):115-121.
[25] Guo H,Liu X,Han Z,et al.Phylogenetic analysis and comparison of eight strains of pigeon paramyxovirus type 1(PPMV-1)isolated in China between 2010and 2012.[J].Arch Virol,2013,158(6):1121-1131.
[26] Wang J,Liu H,Liu W,et al.Genomic characterizations of six pigeon paramyxovirus type 1viruses isolated from live bird markets in China during 2011to 2013[J].PLoS One,2015,10(4):e124261.
[27] Alexander D J.Newcastle disease and other avian paramyxoviruses[J].Rev Sci Et Tech,2000,19(2):443.
[28] Scott P C,Wilson T B,Walker C.Serological and growth rate responses to the use of chicken Newcastle disease vaccines in pigeons[J].Australian Vet J,2013,91(12):525-530.