我国部分地区鸡源H9N2亚型AIV的系统进化和HA抗原分析以及AIV NA分型方法的研究
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摘要
禽流感(Avian Influenza, AI)是由禽流感病毒(Avian Influenza Virus, AIV)引起的多种动物易感的传染病,是危害世界养禽业的重要传染病之一。人类历史上多次发生流感的流行毒株均与禽源流感病毒有关系。二十世纪末相继发生了H5,H7和H9亚型的AIV由禽类直接传染给人类的事件。因此对AIV在不同种类的宿主中的流行情况进行持续的追踪和监测,具有重要的公共卫生学意义。
本文对我国部分养禽密集地区的H9N2亚型AIV流行株进行了分子流行病学监测和血凝素(Hemaglutinin, HA)抗原性分析,明确了这些毒株的进化特点。并针对目前因缺乏AIV的神经氨酸酶(Neuraminidase, NA)快速分型方法而导致的AIV监测中病毒NA分型困难的实际问题,建立了用于区分AIV 9个NA亚型的荧光定量RT-PCR方法。主要研究内容如下：
1.我国部分地区鸡源H9N2亚型禽流感病毒的系统进化和抗原性分析
H9N2是目前国内鸡群中流行最为广泛的禽流感病毒亚型,给我国的养鸡业造成了重要危害。相比于高致病性的H5N1亚型AIV, H9N2病毒因其致病力较低而易于在鸡群中持续感染和更广泛的传播,从而在流感病毒的基因重组中发挥着重要作用。1997年,香港禽流感感染人事件之后,我国加强了AIV的流行病学调查研究。但对近年来H9N2流感病毒在鸡群中的系统进化情况,尚缺乏系统和全面的研究资料。本研究中,我们对1998-2008年间分离自山东,河南,河北,天津和江苏北部的发病鸡群中的17株H9N2亚型禽流感病毒进行了全基因组的序列测定和血凝素抗原的分析,揭示了该地区鸡源H9N2病毒的系统进化特征。
分别研究了17株AIV H9N2分离株各基因片段的基因类型(gene type)以及病毒的基因型(genotype)。对17株H9N2病毒的8个基因片段(PB2, PB1, PA, HA, NP, NA,M和NS)分别进行系统进化分析表明,各分离毒株的HA基因片段均隶属于H9基因系统进化树的A/chicken/Beijing/1/94分支(BJ 94分支)；NS基因片段均隶属于NS基因系统进化树的A等位基因群的BJ 94分支；M基因隶属于该基因进化树的BJ 94分支或A/quail/HongKong/G 1/97分支(G1分支)；NA基因隶属于N2基因进化树的BJ 94分支或A/chicken/Hong Kong/G9/97分支(G9分支)；PB2,PB1和NP基因隶属于相应基因系统进化树的BJ 94分支或A/chicken/Shanghai/F/98分支(F98分支)；PA基因隶属于该基因进化树的BJ 94分支,或者F 98分支,或者A/duck/Hong Kong/Y439/97分支(Y439分支)。对AIV各分离毒株的基因型(Genotype)分析表明,这17株病毒共划分为7种不同的基因型：A, H, M, N,0, P和Q。其中M-Q共5个基因型是首次报道的H9N2流感病毒基因型。以基因型A和H的H9N2病毒为进化母本,这些新基因型的H9N2分离株经历了一重到四重的基因重组。通过鸡源H9N2毒株基因型的比较分析,我们发现本研究中新基因型的H9N2病毒分离株,并非来源于华南地区已报道的H9N2流行株向北方地区的传播,而很可能来自不同流感病毒毒株的基因重组。
对17个H9N2分离毒株的推导氨基酸序列的分子特征分析表明,有15个分离毒株的血凝素(HA)存在7个潜在的糖基化位点,而另外两个毒株(CK SD B1 98和CK BJ L1 05)潜在糖基化位点的数目为6个(298-300AA糖基化位点丧失)；17株H9N2分离毒株的NA基因,其中4个隶属于G9分支者(CK ZB B1 08,CK HN L1 08, CK HN L2 08和CK HN L3 08)编码469AA的NA蛋白。而其余13个隶属于BJ 94分支者,则编码466AA的NA蛋白(其茎部的62-64位氨基酸缺失)；有2株病毒(CKSD B4 07和CK ZB B1 08)的M2蛋白存在S31N的变异,而其余15株分离毒的M2蛋白不存在该位点的变异。这一变异是具有金刚烷胺抗药性的流感病毒的分子特征。
血凝素是禽流感病毒诱导动物机体产生中和抗体的主要蛋白抗原,也是病毒毒力和宿主特异性的主要决定因素。上述分子流行病学的研究表明,本文中的H9N2分离毒株,其HA基因同源性很高(90.2%-100%),在系统进化上均属于H9基因的BJ 94分支。选取分离自2002-2008年间的12个H9N2亚型病毒株(分别属于A, H, N,O和Q基因型)分别制备灭活疫苗,接种SPF鸡,于隔离饲养器中饲养,制备阳性血清。然后以这12株H9N2病毒作抗原,与各毒株的阳性血清进行交叉血凝抑制试验(hemagglutination-inhibition test, HI)。结果表明：各分离株的血清能够抑制所有12株AIV的血凝活性,虽然其抑制价存在差异。进一步的R值分析表明,交叉HI效价差异不显著,即：这12株H9N2分离株的HA的血凝活性没有显著差异。该结果表明这些H9N2分离毒株HA基因的点突变并未造成血凝素抗原的明显变异。
此外,本研究的17个分离毒株的PB1-F2基因编码6种不同长度的蛋白。该基因所编码蛋白长度的多态性促使我们进一步调查了国内不同亚型和宿主来源的A型流感病毒(Influenza A virus, IAV)中PB1-F2基因的流行情况。本研究从Genbank数据库下载了禽源、猪源和人源H5N1和H9N2亚型AIV、人源和猪源H1N1和H3N2流感病毒的PB1-F2基因共计337个,系统的对其进行了分子进化分析。结果表明流感病毒PB1-F2基因的进化与其HA基因的进化相关联：H1和H3亚型病毒的PB1-F2基因在系统进化上具有独立的分支,明显区别与H5和H9亚型的AIV的PB 1.F2基因的进化；IAV因宿主来源的不同,编码功能性的PB1-F2蛋白的比率也存在差异。对PB1-F2流行特征的分析有利于对该蛋白功能及作用机理的深入研究。
2.禽流感病毒NA亚型分型的荧光定量RT-PCR方法研究
禽流感病毒的分子生物学检测方法为其流行病学监测提供了敏感而快速的技术手段。目前已发展了多种针对AIV的型特异性抗原(M或NP)基因以及常见的HA和NA亚型鉴定的分子检测方法,但是对全部9种NA亚型进行分型鉴定的分子生物学方法则鲜见报道。本研究应用Primer Hunter软件分别设计了针对9种NA亚型的AIV引物,使用SYBR Green I为检测染料,以体外转录的N1-N9亚型的RNA标准品为检测模板,建立了用于NA分型的荧光定量RT-PCR(real time RT-PCR, RRT-PCR)方法。在上述基础上,设计了引物池(primer pool)实验,进行4个反应即可鉴别AIV的9种不同的NA亚型。所建立的RRT-PCR方法敏感性高,每个反应可以检测到351-8000拷贝,或者4-62fg的不同NA亚型的AIV RNA。RRT-PCR方法的特异性强,各NA亚型的引物仅与相同亚型的AIV鸡胚尿囊液样品的RNA发生反应,而与其它NA亚型或者几种重要禽病病毒的RNA没有交叉反应。本研究为AIV尿囊液样品的NA亚型鉴定提供了一种敏感而特异的检测方法。
Avian Influenza (AI) is a disease caused by Avian Influenza Virus (AIV). Multiple species of animals are susceptible to AIV infection. AI is also a big problem for the global poultry industry. Furthermore, the end of last century saw the infection of AIVs of several subtypes (H5, H7 and H9) in humans, which demonstrate the capability of AIV to transmit directly from poultry to human being and arise a great public health concern. Therefore, it's crucial to take long-time surveillance of AIV to trace its evolution and prevalence in diiferent hosts. In this study, H9N2 subtype AIVs were collected from several intensive farming districts in China. Molecular epidemiology and HA antigenicity analysis were conducted to illuminate the genesis and evolution of these isolates. To meet the practical need for NA subtyping in AIV surveillance, one step real-time RT-PCR were developed for NA differentiation. The major work is as following:
1. Phylogenetic and antigenic analysis of chicken H9N2 AIVs in China
H9N2 subtype is the most prevalent Avian Influenza virus in poultry in mainland China. Infection of chickens with H9N2 virus may cause respiratory syndrome, reduced egg production and variable rate of morbidity and mortality. The epidemiologic study of AIV in multiple poultry species was reinforced after human infection cases of H5N1 in Hongkong in 1997. However, systematical surveillance information for the evolution of H9N2 viruses in chicken flocks in northern and eastern China is limited, especially in the last few years, when the intensive farming accounts for more than 50 percent of the country's total chicken farming. In this study, seventeen H9N2 viruses isolated from northern and eastern China were analyzed phylogenetically and antigenically to reveal the genesis and evolution properties of H9N2 viruses in this region.
The seventeen H9N2 viruses were collected in 1998-2008 during avian influenza (AI) outbreaks in Beijing, Tianjin, Hebei, Henan, Shandong, and Jiangsu provinces of northern and eastern China. To systematically track the genesis and evolution of H9N2 viruses in this region, whole genome sequences of these isolates were obtained and their phylogenetic properties were determined. Phylogenetic analysis revealed several newly emerged lineages of gene segments in addition to the A/chicken/Beijing/1/94 (BJ 94)-like and A/chicken/Shanghai/F/98(F 98)-like lineages, which are prevailing in northern and eastern China according to the previous reports. Reassortments among these gene segments generated five novel genotypes of H9N2 viruses (M, N,O, P and Q) that have not been reported before in China. The emerging genotypes of H9N2 viruses in this region indicate that H9N2 virus genes undergo active evolution, particularly their internal genes, which raises concern for their likely contribution to gene reassortment and production of AIVs with new properties. Our study provides valuable insight into the prevalence of H9N2 viruses in northern and eastern China and demonstrates the need of long-term monitoring of the evolution of H9N2 AIV.
Hemagglutinin (HA) is the main antigen of AIV to induce neutralizing antibody in infected animals. HA is also the main determinants of virulence and host-specificity of AIV. In this section, Hyperimmune antisera against each of 12 H9N2 viruses were prepared. Antigenic analysis between these 12 H9N2 isolates was performed by cross HI test with the above panel of monofactorial sera. The test was repeated three times and the mean values were used for antigenic analysis. HA antigenic correlation between two certain viruses were calculated according to the formula R=√r1×r2. The cross HI test results of 12 selected viruses demonstrated that the rooster antisera panel successfully inhibitded each of the 12 isolates in HI test, although the titration varied when a certain serum was reacted with different isolates. Statistical analysis about the correlation of two certain HA antigens were performed and the results indicated that there is no significant antigenic difference between HA of the 12 viruses.The result also demonstrated that the point mutation of these HA gene havn't significantly influenced the antigenicity of HA proteins.
Seventeen H9N2 isolates encode PB1-F2 protein of 5 different lengths. Molecular characterization of PB1-F2 gene of chicken H9N2 subtype avian influenza viruses (AIVs) in China was analyzed and the prevalence of the PB1-F2 genes of influenza A viruses (IAVs) prevailed in China was further investigated.337 PB1 genes of IAVs derived from avian (H5N1 and H9N2 subtypes), pig (H5N1, H9N2, H1N1 and H3N2 subtypes) and human beings (H5N1,H9N2, H1N1 and H3N2 subtypes) in China were downloaded from Genbank database and analyzed. The deduced sequences of PB1-F2 proteins have multifold lengths and the ratio of PB1-F2 genes encoding full-length PB1-F2 protein differed in the HA subtype and host origin of IAV isolates. Through this study, the molecular epidemiologic characterization of PB1-F2 gene of IAVs in China was illuminated, which can also works as the foundation of PB1-F2 gene function research in the infection process of IAVs.
The HA proteins of fifteen H9N2 isolates contain 7 potential glycosylation sites, while the HA of CK SD B198 and CK BJ L105 have I at position 300 (C to T mutation at position 899 of their HA genes) and result in the loss of one potential glycosylation site. The four H9N2 isolates that belong to the G9-like lineage (CK ZB B1 08, CK HN L1 08, CK HN L208 and CK HN L308) possess full-length NA protein (469AA), while the NA of the Y280-like lineage contain deletion of three amino acids (62-64 AA) at the stalk region. An analysis of the viral amino acid sequence of M2 protein has revealed substitution of S3 IN in two isolates, which is the molecular characterization of amantadine resistance in AIVs.
2. Development of Real Time RT-PCR Assays for Neuraminidase Subtyping of Avian Influenza Virus
In this study, nine pairs of neuramidinase (NA) subtype-specific primers were designed with Primer Hunter software and successfully used in real time RT-PCR with four primer-pool reactions to differentiate nine NA subtypes of AIV. The RRT-PCR assays are sensitive and can detect in vitro transcribed RNA of different NA subtypes ranging from 351 to 8000 copies per reaction, or 4-62fg of total RNA of AIV per reaction. The assays possess good specificity. There is no cross reaction between RNA of different NA subtypes in RRT-PCR with each subtype-specific primers. No ampliation were displayed when detect RNA of IBV, IBDV, NDV with the primer pools, either. This study validated the powerful function of Primer Hunter for the design of subtyping primers and also introduced a sensitive and specific method for NA subtyping in AIV epidemiological surveillance.
本文对我国部分养禽密集地区的H9N2亚型AIV流行株进行了分子流行病学监测和血凝素(Hemaglutinin, HA)抗原性分析,明确了这些毒株的进化特点。并针对目前因缺乏AIV的神经氨酸酶(Neuraminidase, NA)快速分型方法而导致的AIV监测中病毒NA分型困难的实际问题,建立了用于区分AIV 9个NA亚型的荧光定量RT-PCR方法。主要研究内容如下：
1.我国部分地区鸡源H9N2亚型禽流感病毒的系统进化和抗原性分析
H9N2是目前国内鸡群中流行最为广泛的禽流感病毒亚型,给我国的养鸡业造成了重要危害。相比于高致病性的H5N1亚型AIV, H9N2病毒因其致病力较低而易于在鸡群中持续感染和更广泛的传播,从而在流感病毒的基因重组中发挥着重要作用。1997年,香港禽流感感染人事件之后,我国加强了AIV的流行病学调查研究。但对近年来H9N2流感病毒在鸡群中的系统进化情况,尚缺乏系统和全面的研究资料。本研究中,我们对1998-2008年间分离自山东,河南,河北,天津和江苏北部的发病鸡群中的17株H9N2亚型禽流感病毒进行了全基因组的序列测定和血凝素抗原的分析,揭示了该地区鸡源H9N2病毒的系统进化特征。
分别研究了17株AIV H9N2分离株各基因片段的基因类型(gene type)以及病毒的基因型(genotype)。对17株H9N2病毒的8个基因片段(PB2, PB1, PA, HA, NP, NA,M和NS)分别进行系统进化分析表明,各分离毒株的HA基因片段均隶属于H9基因系统进化树的A/chicken/Beijing/1/94分支(BJ 94分支)；NS基因片段均隶属于NS基因系统进化树的A等位基因群的BJ 94分支；M基因隶属于该基因进化树的BJ 94分支或A/quail/HongKong/G 1/97分支(G1分支)；NA基因隶属于N2基因进化树的BJ 94分支或A/chicken/Hong Kong/G9/97分支(G9分支)；PB2,PB1和NP基因隶属于相应基因系统进化树的BJ 94分支或A/chicken/Shanghai/F/98分支(F98分支)；PA基因隶属于该基因进化树的BJ 94分支,或者F 98分支,或者A/duck/Hong Kong/Y439/97分支(Y439分支)。对AIV各分离毒株的基因型(Genotype)分析表明,这17株病毒共划分为7种不同的基因型：A, H, M, N,0, P和Q。其中M-Q共5个基因型是首次报道的H9N2流感病毒基因型。以基因型A和H的H9N2病毒为进化母本,这些新基因型的H9N2分离株经历了一重到四重的基因重组。通过鸡源H9N2毒株基因型的比较分析,我们发现本研究中新基因型的H9N2病毒分离株,并非来源于华南地区已报道的H9N2流行株向北方地区的传播,而很可能来自不同流感病毒毒株的基因重组。
对17个H9N2分离毒株的推导氨基酸序列的分子特征分析表明,有15个分离毒株的血凝素(HA)存在7个潜在的糖基化位点,而另外两个毒株(CK SD B1 98和CK BJ L1 05)潜在糖基化位点的数目为6个(298-300AA糖基化位点丧失)；17株H9N2分离毒株的NA基因,其中4个隶属于G9分支者(CK ZB B1 08,CK HN L1 08, CK HN L2 08和CK HN L3 08)编码469AA的NA蛋白。而其余13个隶属于BJ 94分支者,则编码466AA的NA蛋白(其茎部的62-64位氨基酸缺失)；有2株病毒(CKSD B4 07和CK ZB B1 08)的M2蛋白存在S31N的变异,而其余15株分离毒的M2蛋白不存在该位点的变异。这一变异是具有金刚烷胺抗药性的流感病毒的分子特征。
血凝素是禽流感病毒诱导动物机体产生中和抗体的主要蛋白抗原,也是病毒毒力和宿主特异性的主要决定因素。上述分子流行病学的研究表明,本文中的H9N2分离毒株,其HA基因同源性很高(90.2%-100%),在系统进化上均属于H9基因的BJ 94分支。选取分离自2002-2008年间的12个H9N2亚型病毒株(分别属于A, H, N,O和Q基因型)分别制备灭活疫苗,接种SPF鸡,于隔离饲养器中饲养,制备阳性血清。然后以这12株H9N2病毒作抗原,与各毒株的阳性血清进行交叉血凝抑制试验(hemagglutination-inhibition test, HI)。结果表明：各分离株的血清能够抑制所有12株AIV的血凝活性,虽然其抑制价存在差异。进一步的R值分析表明,交叉HI效价差异不显著,即：这12株H9N2分离株的HA的血凝活性没有显著差异。该结果表明这些H9N2分离毒株HA基因的点突变并未造成血凝素抗原的明显变异。
此外,本研究的17个分离毒株的PB1-F2基因编码6种不同长度的蛋白。该基因所编码蛋白长度的多态性促使我们进一步调查了国内不同亚型和宿主来源的A型流感病毒(Influenza A virus, IAV)中PB1-F2基因的流行情况。本研究从Genbank数据库下载了禽源、猪源和人源H5N1和H9N2亚型AIV、人源和猪源H1N1和H3N2流感病毒的PB1-F2基因共计337个,系统的对其进行了分子进化分析。结果表明流感病毒PB1-F2基因的进化与其HA基因的进化相关联：H1和H3亚型病毒的PB1-F2基因在系统进化上具有独立的分支,明显区别与H5和H9亚型的AIV的PB 1.F2基因的进化；IAV因宿主来源的不同,编码功能性的PB1-F2蛋白的比率也存在差异。对PB1-F2流行特征的分析有利于对该蛋白功能及作用机理的深入研究。
2.禽流感病毒NA亚型分型的荧光定量RT-PCR方法研究
禽流感病毒的分子生物学检测方法为其流行病学监测提供了敏感而快速的技术手段。目前已发展了多种针对AIV的型特异性抗原(M或NP)基因以及常见的HA和NA亚型鉴定的分子检测方法,但是对全部9种NA亚型进行分型鉴定的分子生物学方法则鲜见报道。本研究应用Primer Hunter软件分别设计了针对9种NA亚型的AIV引物,使用SYBR Green I为检测染料,以体外转录的N1-N9亚型的RNA标准品为检测模板,建立了用于NA分型的荧光定量RT-PCR(real time RT-PCR, RRT-PCR)方法。在上述基础上,设计了引物池(primer pool)实验,进行4个反应即可鉴别AIV的9种不同的NA亚型。所建立的RRT-PCR方法敏感性高,每个反应可以检测到351-8000拷贝,或者4-62fg的不同NA亚型的AIV RNA。RRT-PCR方法的特异性强,各NA亚型的引物仅与相同亚型的AIV鸡胚尿囊液样品的RNA发生反应,而与其它NA亚型或者几种重要禽病病毒的RNA没有交叉反应。本研究为AIV尿囊液样品的NA亚型鉴定提供了一种敏感而特异的检测方法。
Avian Influenza (AI) is a disease caused by Avian Influenza Virus (AIV). Multiple species of animals are susceptible to AIV infection. AI is also a big problem for the global poultry industry. Furthermore, the end of last century saw the infection of AIVs of several subtypes (H5, H7 and H9) in humans, which demonstrate the capability of AIV to transmit directly from poultry to human being and arise a great public health concern. Therefore, it's crucial to take long-time surveillance of AIV to trace its evolution and prevalence in diiferent hosts. In this study, H9N2 subtype AIVs were collected from several intensive farming districts in China. Molecular epidemiology and HA antigenicity analysis were conducted to illuminate the genesis and evolution of these isolates. To meet the practical need for NA subtyping in AIV surveillance, one step real-time RT-PCR were developed for NA differentiation. The major work is as following:
1. Phylogenetic and antigenic analysis of chicken H9N2 AIVs in China
H9N2 subtype is the most prevalent Avian Influenza virus in poultry in mainland China. Infection of chickens with H9N2 virus may cause respiratory syndrome, reduced egg production and variable rate of morbidity and mortality. The epidemiologic study of AIV in multiple poultry species was reinforced after human infection cases of H5N1 in Hongkong in 1997. However, systematical surveillance information for the evolution of H9N2 viruses in chicken flocks in northern and eastern China is limited, especially in the last few years, when the intensive farming accounts for more than 50 percent of the country's total chicken farming. In this study, seventeen H9N2 viruses isolated from northern and eastern China were analyzed phylogenetically and antigenically to reveal the genesis and evolution properties of H9N2 viruses in this region.
The seventeen H9N2 viruses were collected in 1998-2008 during avian influenza (AI) outbreaks in Beijing, Tianjin, Hebei, Henan, Shandong, and Jiangsu provinces of northern and eastern China. To systematically track the genesis and evolution of H9N2 viruses in this region, whole genome sequences of these isolates were obtained and their phylogenetic properties were determined. Phylogenetic analysis revealed several newly emerged lineages of gene segments in addition to the A/chicken/Beijing/1/94 (BJ 94)-like and A/chicken/Shanghai/F/98(F 98)-like lineages, which are prevailing in northern and eastern China according to the previous reports. Reassortments among these gene segments generated five novel genotypes of H9N2 viruses (M, N,O, P and Q) that have not been reported before in China. The emerging genotypes of H9N2 viruses in this region indicate that H9N2 virus genes undergo active evolution, particularly their internal genes, which raises concern for their likely contribution to gene reassortment and production of AIVs with new properties. Our study provides valuable insight into the prevalence of H9N2 viruses in northern and eastern China and demonstrates the need of long-term monitoring of the evolution of H9N2 AIV.
Hemagglutinin (HA) is the main antigen of AIV to induce neutralizing antibody in infected animals. HA is also the main determinants of virulence and host-specificity of AIV. In this section, Hyperimmune antisera against each of 12 H9N2 viruses were prepared. Antigenic analysis between these 12 H9N2 isolates was performed by cross HI test with the above panel of monofactorial sera. The test was repeated three times and the mean values were used for antigenic analysis. HA antigenic correlation between two certain viruses were calculated according to the formula R=√r1×r2. The cross HI test results of 12 selected viruses demonstrated that the rooster antisera panel successfully inhibitded each of the 12 isolates in HI test, although the titration varied when a certain serum was reacted with different isolates. Statistical analysis about the correlation of two certain HA antigens were performed and the results indicated that there is no significant antigenic difference between HA of the 12 viruses.The result also demonstrated that the point mutation of these HA gene havn't significantly influenced the antigenicity of HA proteins.
Seventeen H9N2 isolates encode PB1-F2 protein of 5 different lengths. Molecular characterization of PB1-F2 gene of chicken H9N2 subtype avian influenza viruses (AIVs) in China was analyzed and the prevalence of the PB1-F2 genes of influenza A viruses (IAVs) prevailed in China was further investigated.337 PB1 genes of IAVs derived from avian (H5N1 and H9N2 subtypes), pig (H5N1, H9N2, H1N1 and H3N2 subtypes) and human beings (H5N1,H9N2, H1N1 and H3N2 subtypes) in China were downloaded from Genbank database and analyzed. The deduced sequences of PB1-F2 proteins have multifold lengths and the ratio of PB1-F2 genes encoding full-length PB1-F2 protein differed in the HA subtype and host origin of IAV isolates. Through this study, the molecular epidemiologic characterization of PB1-F2 gene of IAVs in China was illuminated, which can also works as the foundation of PB1-F2 gene function research in the infection process of IAVs.
The HA proteins of fifteen H9N2 isolates contain 7 potential glycosylation sites, while the HA of CK SD B198 and CK BJ L105 have I at position 300 (C to T mutation at position 899 of their HA genes) and result in the loss of one potential glycosylation site. The four H9N2 isolates that belong to the G9-like lineage (CK ZB B1 08, CK HN L1 08, CK HN L208 and CK HN L308) possess full-length NA protein (469AA), while the NA of the Y280-like lineage contain deletion of three amino acids (62-64 AA) at the stalk region. An analysis of the viral amino acid sequence of M2 protein has revealed substitution of S3 IN in two isolates, which is the molecular characterization of amantadine resistance in AIVs.
2. Development of Real Time RT-PCR Assays for Neuraminidase Subtyping of Avian Influenza Virus
In this study, nine pairs of neuramidinase (NA) subtype-specific primers were designed with Primer Hunter software and successfully used in real time RT-PCR with four primer-pool reactions to differentiate nine NA subtypes of AIV. The RRT-PCR assays are sensitive and can detect in vitro transcribed RNA of different NA subtypes ranging from 351 to 8000 copies per reaction, or 4-62fg of total RNA of AIV per reaction. The assays possess good specificity. There is no cross reaction between RNA of different NA subtypes in RRT-PCR with each subtype-specific primers. No ampliation were displayed when detect RNA of IBV, IBDV, NDV with the primer pools, either. This study validated the powerful function of Primer Hunter for the design of subtyping primers and also introduced a sensitive and specific method for NA subtyping in AIV epidemiological surveillance.
引文
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