CN111057682A - 一株禽源h9n2亚型禽流感毒株分离鉴定及应用 - Google Patents
一株禽源h9n2亚型禽流感毒株分离鉴定及应用 Download PDFInfo
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Abstract
本发明涉及动物病毒学领域,本发明提供一株禽源H9N2亚型禽流感毒株分离鉴定及应用。该H9N2亚型禽流感毒株已于2019年07月02日保藏至中国普通微生物菌种保藏管理中心,保藏号为CGMCC No.18172。本发明从分子水平阐述分离毒株的抗原变异情况;甲醛灭活后,与605佐剂制备灭活疫苗,免疫21日龄SPF鸡,通过血清学方法和免疫攻毒方法对免疫效果进行评价,证实该毒株保护效果较好,可用作禽流感H9N2亚型疫苗候选毒株。本发明为禽流感防控提供思路,为禽流感候选毒株筛选提供技术教导,具有重要的公共卫生意义。
Description
技术领域
本发明涉及动物病毒学领域,本发明提供一株禽源H9N2亚型禽流感病毒毒株的生物特性研究及在疫苗开发上的应用。
技术背景
禽流感病毒(Avian influenza virus,AIV)隶属于正粘病毒科A型流感病毒属,核酸为单股负链RNA,总长度约为13.6kb,共含有8个节段,可编码11个蛋白。由于单股负链RNA中HA节段和NA节段最易发生变异这一特性,不同病毒侵入同一宿主时,极易发生不同病毒之间的基因重排,增加病毒变异几率,甚至可能产生新型的流感病毒,改变病毒的宿主侵袭范围及病毒的毒力。按照AIV对家禽致病程度,可分为高致病性(High Pathogenic AvianInfluenza virus,HPAIV)和低致病性(Low Pathogenic Avian Influenza virus,LPAIV),感染家禽的HPAIV 和LPAIV分别以H5、H7和H9亚型为主。
目前全世界范围的禽群中广泛存在AIV,H9N2亚型发病率占禽流感总发病率90%以上,虽然仅有不到30%的致死率,却可导致呼吸道症状、蛋鸡的产蛋下降,并使鸡群易继发严重的呼吸道疾病,影响家禽生产性能。该病毒自1966年在北美的火鸡体内首次分离,1992 年传入我国。该病毒的流行不仅对各国养殖业造成严重损失,还对人类健康构成巨大威胁,积极开展禽流感流行病学调查,对H9N2的分子进化进行长期、持续性的跟踪与监控,并选用保护效果较好的疫苗候选株制备免疫保护效果较好的疫苗,已成为防治H9N2爆发和流行的关键,具有重要的公共卫生学意义。
发明内容
本发明提供一株H9N2亚型禽流感病毒疫苗候选毒株,阐明HA与NA两个基因片段序列及其与国内经典的疫苗病毒株的系统进化差异;提供该毒株的分离、鉴定和纯化方法;提供利用该毒株制备预防禽流感H9N2灭活苗方法及疫苗判定指标;明确该毒株作为疫苗候选毒株的可行性。
发明人使用分离、鉴定并纯化好的H9N2亚型禽流感病毒,对以下生物学特性进行确定:
1.遗传进化分析:参考Genbank公布H9N2亚型序列,设计HA、NA片段全长引物,并对流感毒株HA、NA片段进行扩增、测序并构建进化图谱。结果表明,HA、NA基因属于 H9,4.2(Y280/G9-Like)谱系。
2.生长特性:病毒在SPF鸡胚中可稳定高滴度生长,血凝价稳定在10Log2左右。
3.HA抗原相关性分析:使用605佐剂,分别制备8株AIV分离毒株灭活疫苗,免疫 21日龄SPF鸡用于制备单因子血清,检测HA抗原相关性均大于0.81,可作为H9N2亚型禽流感流感疫苗候选毒株。
4.疫苗免疫后保护效果评估:本发明使用AIV-BZ株制备灭活苗免疫21日龄SPF鸡用血清学方法与免疫攻毒方法评估保护效果,结果表明,本发明使用毒株制备灭活油苗具有很好保护效果。
5.本发明对禽流感毒株A/chicken/Shandong/BZ/2018(H9N2)(AIV-BZ株)进行生物保藏,保藏信息如下:
保藏时间:2019年07月02日
保藏单位名称:中国普通微生物菌种保藏管理中心(CGMCC)
保藏单位地址:北京市朝阳区北辰西路1号院3号
保藏编号:CGMCC No.18172
分类株命名:禽流感A型流感病毒H9N2亚型
本发明提供一种H9N2亚型禽流感病毒AIV-BZ株,保藏编号为CGMCC No.18172。
在一个方面中,其在鸡胚中可高滴度增殖,血凝价稳定在210。
本发明提供一种禽流感灭活疫苗,其包含灭活后的所述H9N2亚型禽流感病毒AIV-BZ 株,灭活后病毒液血凝价HA不低于9Log2。
本发明提供一种所述禽流感灭活疫苗的制备方法,采用所述H9N2亚型禽流感病毒AIV-BZ株接种SPF鸡胚,培养96-120小时,经0.1-0.3%甲醛灭活后与605佐剂按照4:6比例混合制备而成。
本发明提供一种禽流感病毒HA蛋白,其氨基酸序列如SEQ ID NO:1所示。
本发明提供一种禽流感病毒HA基因,其编码所述禽流感病毒HA蛋白。
本发明提供一种扩增禽流感病毒HA基因的引物对,上游引物为AGCAAAAGCAGGAGTAAAAAT,下游引物为AGTAGAAACAAGGAGTTTTTTC。
本发明提供一种禽流感病毒NA蛋白,其氨基酸序列如SEQ ID NO:2所示。
本发明提供一种禽流感病毒NA蛋白,其编码所述禽流感病毒NA蛋白。
本发明提供一种扩增禽流感病毒HA基因的引物对,上游引物为AGCAAAAGCAGGGGAATT,下游引物为AGTAGAAAACAAGGGTGTTTTT。
本申请的有益效果:本发明分离得到一株H9N2亚型禽流感病毒株 A/chicken/Shandong/BZ/2018(H9N2)(ShanDong/BZ)。该病毒AIV-BZ株与其余7株H9N2分离株HA抗原相关性均大于0.81,表明该病毒株与其余流行毒株HA抗原相关性较好,可作为 H9N2亚型禽流感疫苗的候选毒株。使用该病毒AIV-BZ株对免疫后的实验组、商品苗与空白对照组进行攻毒实验,发现AIV-BZ株制备灭活苗免疫后14天即可产生免疫保护性抗体,有效保护临床毒株攻击,且保护效果优于当前商品苗(SS株)。由此可见,本发明为禽流感防控提供保护效果较好的疫苗候选株,具有重要的公共卫生意义。
附图说明
图1:H9N2 HA基因氨基酸进化树,分离株A/chicken/Shandong/BZ/2018属于 H9.4.2(Y280/G9-Like)谱系
图2:H9N2 NA基因氨基酸进化树,分离株A/chicken/Shandong/BZ/2018属于 H9.4.2(Y280/G9-Like)谱系
具体实施方式
实施例1病毒分离鉴定
取发病鸡咽喉腔及泄殖腔拭子于含有青、链霉素双抗(600IU/ml)的PBS缓冲液中处理2小时,充分捻转、挤压,保留液体部分8000rpm离心10min,取上清液0.22um过滤处理,接种9-11日龄SPF鸡胚0.2ml/枚,收集24h后死胚尿囊液,用RT-PCR及血凝试验鉴定其纯净性。对混合感染样品尿囊液1:1加入特异性阳性血清中和,结合鸡胚终点稀释法继续传代。直至尿囊液连续两代检测H9N2血凝价不低于5Log2,AIV其它亚型及NDV血凝价不高于3Log2;同时RT-PCR检测H9为阳性,其余检测均为阴性,鉴定引物如表1。
表1鉴别及测序引物
结果显示:连续传代3-5代后,RT-PCR检测,仅H9亚型为阳性,H5、H7、NDV及IBV 均为阴性;H9血凝价均不低于5Log2,AIV其它亚型及NDV血凝价不高于3Log2。纯化好H9 尿囊液连续传代10-12代,HA血凝价稳定维持在10Log2左右,-80℃冻存备用。
实施例2病毒HA、NA基因扩增及序列测定
1.引物合成
根据Genbank公布的序列,用Primer5软件设计HA、NA特异性全长引物,如表2。
表2鉴别及测序引物
2.目的基因扩增及序列测定
本发明使用纯化后可在SPF鸡胚稳定增殖的尿囊液毒作为核酸提取样品,按照RNApure超纯总RNA快速提取试剂盒使用说明操作。所提取RNA按照EasyScript One-StepEnzyme Mix推荐方法进行RT-PCR扩增。1%琼脂糖凝胶电泳证明扩增出目的条带后,将RT-PCR扩增产物送北京生工生物公司进行测序。得到HA基因氨基酸序列如Seq ID No.1所示;得到NA 基因氨基酸序列如Seq ID No.2所示。测序结果,与Genbank公布的H9N2相关序列进行比对,利用Mega 6分生软件构建进化树,该分离株HA、NA基因氨基酸进化树均属于 H9,4.2(Y280/G9-Like)谱系,如图1、图2所示。
实施例3病毒HA抗原性分析
1.单因子血清制备
将2018年分离的包括本发明中涉及的AIV-BZ株在内共计8株H9N2流行毒株,按照灭活毒液:605佐剂4:6比例混合制备灭活疫苗,免疫21日龄SPF鸡,隔离器中饲养14天采血分离血清,制备单因子血清。
2.交叉HI试验
分别制备上述8株病毒的4单位病毒液,与上述8种单因子血清进行交叉HI试验,检测HA抗原交叉反应性。重复三次取均值用于确定上述H9N2病毒HA抗原之间的相关性R。
抗原相关性的判定标准如下:
抗原间的相关性R=√(R1xR2),
R:2株病毒间的抗原性差异
R1:甲病毒对乙血清的HI滴度/甲病毒对甲血清的HI滴度
R2:乙病毒对甲血清的HI滴度/乙病毒对乙血清的HI滴度
如果R=1,表明两个毒株间抗原性相同;如果0.67≤R≤1.5,表明两个毒株间抗原性无明显差异;如果0.5≤R≤0.67,表明两个毒株间抗原性有小的差异;如果R<0.5,表明两个毒株间抗原性的明显差异;R值越小,抗原性差异越大。结果如下表3,表4:
表3不同H9N2毒株交叉凝集试验结果
表4不同H9N2毒株HA抗原相关性结果
8株H9N2流行毒株HI交叉试验结果以及HA抗原相关性分析结果见表3、表4。由结果可知,分离株A/chicken/Shandong/BZ/2018(H9N2)(ShanDong/BZ)与其余7株H9N2分离株HA抗原相关性均大于0.81,表明该分离株与其余流行毒株HA抗原相关性较好,可作为H9N2亚型禽流感疫苗的候选毒株。
实施例4免疫保护效果评估
1.AIV-BZ株灭活疫苗制备与检验
本发明使用AIV-BZ株稳定传代SPF鸡胚毒,加入终浓度0.2%甲醛,37℃灭活24h,期间震动4-6次。灭活后病毒液且血凝价HA不低于9Log2;按现行《中国兽药典》检验,均无菌生长;取灭活鸡胚液连续盲传SPF鸡胚两代,检测其HA均为阴性,方可用于制备疫苗。
灭活毒液与605佐剂按照4:6比例均匀混合制备灭活疫苗,搅拌速度为800rpm,加入终浓度为0.005%硫柳汞钠作为防腐剂。制备好疫苗按照按现行《中国兽药典》进行性状、无菌检验,均合格。
2.血清学实验
取21日龄SPF鸡,选取10只作为实验组,颈部皮下注射AIV-BZ株制备疫苗,0.3ml/只;10只免疫商品疫苗(AIV-SS株)作为对照组,颈部皮下免疫0.3ml/只;另取5只作空白对照。于接种后14天采血并分离血清,检测抗体。免疫组14天H9抗体值均在8Log2以上,高于商品苗组(SS株)免疫结果。见表5。
表5疫苗免疫SPF鸡14天后抗体结果
3.攻毒保护试验
如上述血清学实验,免疫后21天,分别用AIV-BZ株对实验组、商品苗与空白对照组进行翅静脉攻毒,攻毒剂量为0.2ml(106EID50)。攻毒后5天分别采集喉头和泄殖腔棉拭子,接种10日龄SPF鸡胚,每只接5枚;培养观察96小时后,收集尿囊液,检测阳性率;如为阴性,收集尿囊液再次接种SPF鸡胚,HA大于4Log2判为阳性。实验组10只全部为阴性,商品苗组8只为阴性,对照组5只全部为阳性,见表6。说明AIV-BZ株制备灭活苗免疫后14 天可产生免疫保护性抗体,有效保护临床毒株攻击,且保护效果优于当前商品苗(SS株)。
表6疫苗免疫SPF鸡21天后攻毒保护结果
序列表
<110> 北京华夏兴洋生物科技有限公司
北京生泰尔科技股份有限公司
<120> 一株禽源H9N2亚型禽流感毒株分离鉴定及应用
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Claims (10)
1.一种H9N2亚型禽流感病毒AIV-BZ株,保藏编号为CGMCC No.18172。
2.如权利要求1所述的H9N2亚型禽流感病毒AIV-BZ株,其在鸡胚中可高滴度增殖,血凝价稳定在210。
3.一种禽流感灭活疫苗,其包含灭活后的如权利要求1所述的H9N2亚型禽流感病毒AIV-BZ株,灭活后病毒液血凝价HA不低于9Log2。
4.一种如权利要求3所述的禽流感灭活疫苗的制备方法,采用如权利要求1或2所述的H9N2亚型禽流感病毒AIV-BZ株接种SPF鸡胚,培养96-120小时,经0.1%-0.3%甲醛灭活后与605佐剂按照4:6比例混合制备而成。
5.一种禽流感病毒HA蛋白,其氨基酸序列如SEQ ID NO:1所示。
6.一种禽流感病毒HA基因,其编码如权利要求5所述的禽流感病毒HA蛋白。
7.一种扩增禽流感病毒HA基因的引物对,上游引物为AGCAAAAGCAGGGGAATT,下游引物为AGTAGAAAACAAGGGTGTTTTT。
8.一种禽流感病毒NA蛋白,其氨基酸序列如SEQ ID NO:2所示。
9.一种禽流感病毒NA蛋白,其编码如权利要求8所述的禽流感病毒NA蛋白。
10.一种扩增禽流感病毒NA基因的引物对,上游引物为AGCAAAAGCAGGAGTAAAAAT,下游引物为AGTAGAAACAAGGAGTTTTTTC。
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