H5N1禽流感和鹅Ⅰ型副粘病毒分离株的生物学特性及感染性的研究
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摘要
对2006年从中国某地区分离到的一株H5N1亚型的禽流感病毒株(命名为A//LF3/2006,实验室编号AI0624)进行了生物学特性及其感染性的研究。经过血凝和血凝抑制试验初步鉴定、病毒分离、半数感染量(EID50)、鸡胚平均致死时间(MDT)、病毒静脉内接种指数(IVPI)测定,确定了该毒株的毒力。进一步对A/CHICKEN/LF3/06毒株进行了HA、NA和NS三段基因序列的测定,绘制该病毒的遗传进化树,比较其与国内外流行相关毒株的遗传演化关系,尤其是与近几年香港、东南亚爆发禽流感致人死亡事件的代表毒株的亲缘关系,分析该毒株的遗传变异情况,对于弄清我国流行的高致病性禽流感毒株的遗传进化研究有重要意义。
该毒株EID50为10-7.625/0.1ml,IVPI为3；MDT为50.4h,且死亡率为100%,均证明该毒株是强毒。根据Genbank中已发表的禽流感HA、NA和NS三个基因片段起始密码子前和终止密码子后的非编码区和上下游区段的序列设计了两对引物,对该毒株的HA、NA和NS三个基因片段进行RT-PCR扩增、重组、测序,结果表明：该毒株HA基因裂解位点存在有6个碱性氨基酸残基：PLRERRRK-R*G,具有高致病力禽流感毒株的HA裂解位点的特征,与IVPI测定结果相吻合。试验毒株A/CHICKEN/LF3/06属于欧亚群系,其HA、NA和NS三个基因核苷酸的同源性及分子进化关系与中国A/duck/Fujian/1734/05、A/human/Zhejiang/16/06和马来西亚A/chicken/Malaysia/5223/07三株H5N1亚型禽流感毒株亲缘关系相对更近,并且不同于96广东禽流感、97香港禽流感、中东、非洲禽流感的独立进化分支,由此推测此分离株可能与A/duck/Fujian/1734/05和A/human/Zhejiang-/16/06两株病毒有着共同的祖先。
通过A/Chicken/LF3/2006毒株对鸡、小鼠和兔子的感染性试验,研究鸡源的此毒株是否能够感染哺乳动物,探讨病毒的跨种间动物传播机制。实验表明该鸡源毒株对于鸡是高致病性,也可以感染老鼠和兔子：该毒株经呼吸道感染后,对肺组织、脑组织有一定的嗜性；在大剂量攻毒时,对小鼠和兔子的体重影响都较大,中小等剂量攻毒时,对体重的影响较小；对兔子体温有影响；实验过程中小鼠和兔子均有死亡；小鼠和兔子在较高浓度感染时体内均可产生抗体,在中低浓度就几乎没有抗体产生；攻毒试验证明该病毒有明显的剂量效应。说明该毒株有可能直接感染哺乳动物发病,故推断该毒株经哺乳动物体内复制增殖后,对哺乳动物存在一定致病性。因此,可推测此株病毒还是有潜在的感染人的可能性。
通过对此株H5N1亚型高致病力禽流感病毒的生物学特性和感染性的系统研究,为今后进一步研究我国H5N1亚型禽流感病毒的致病机制、病毒的结构与功能奠定实验和理论基础,同时也为我国H5N1亚型高致病力禽流感的遗传与演化研究提供了实验数据。
同时,通过RT-PCR技术对一株鹅Ⅰ型副粘病毒(G)编码的6种主要结构蛋白,即核蛋白(NP)、磷蛋白(P)、基质蛋白(M)、血凝素-神经氨酸酶蛋白(HN)、融合蛋白(F)和巨蛋白(L)分别进行扩增,然后将其克隆到PMD18-T载体后进行序列测定和拼接；并将克隆到的6个基因片段与国内外已报道的Ⅰ型副粘病毒基因序列进行比较分析。结果表明：我们克隆到的G株鹅副粘病毒F基因与La Sota株同源性最高,同源率为99.1%,与其它各毒株各基因同源性均较低。
采用HA/HI试验、病毒分离和RT-PCR方法对该毒株分离地区的野生老鼠进行禽流感流行病学调查,可为我国的养禽业提供数据,也解决存在的当地野生老鼠是否感染该病毒的疑问。
An H5N1 avian influenza virus was isolated from China in a certain area at 2006 (named as A/CHICKEN/LF3/06, LAB No. AI0624) was studied the biological characteristics and the infection in this paper. After preliminary identified by HA and HI,virus isolation,50% egg infections dose (EID50), mean death test (MDT) in eggs and Intravenous patho-genicity index (IVPI) determine, its virulence was understanded. In order to demostrate the phylogenetic relationships between the A/CHICKEN/LF3/06 strain and the other isolates reported in the world, especially strains isolated from chickens and human in HongKong and in Southeast-Asian countries outbreak of AIV, Then the HA,NA and NS genes of the A/CHICKEN/LF3/06 strain was sequenced, molecular evolutionarily analyzed and drawed Phylogenetic tree, finally we determine its pathogenicity to mammiferous.
EID50, IVPI, MDT were10-7.625/0.lml,3and 50.4h respectively, and the mortality is 100%. Accordingly, this virus is a H5N1 High Pathogenic Avian Influenza Virus.4 primers was designed based on genes of the HA,NA and NS genes of AIV reported at Genbank, then the genes of this virus was RT-PCR, cloned, sequenced and molecular evolutionarily analyzed, the result showed:the cleavage site of HA gene is PLRERRRK-R*G that is the sign of High Pathogenic Avian Influenza Virus and its same with the result of IVPI.Virus in this study was closely related with A/duck/Fujian/1734/05,A/human/Zhejiang/16/06 and A/chicken/Malaysia/5223/07 which isolated from duck in Fujian, China in 2005,from human in Zhejiang,China in 2006 and from chicken in Malaysia in 2007.But, it was comparatively far related with A/chicken/Hong Kong/220/97 and A/goose/Guangdong/1/96, which isolated from chickens in HongKong during the 1997 outbreak of AIV and from gooses in Guangdong, China in 1996.Therefore, I suppose that A/CHICKEN/LF3/06 in this study possibly has same ancestor with A/duck/Fujian/1734/05 and A/human/Zhejiang/16/06.
The virus intranasal inoculated BALB/c mice experiment showed:in the high-dose infection, the mice had larger affection on weight loss, in the medium-dose to low-dose infection, the mice had less affection on weight loss.But during the experiment, there were mice dead, So this virus is a H5N1 higher pathogenic to mice. Through respiratory tract infections, the virus showed certain tropism on the lung tissue, the brain tissue. In addition, Mice infected intranasally with high-dose have produced antibody, with medium-dose to low-dose have no produced antibody. The experiment has proved the virus has significant dose effect.Concluded that after the strain reproduce in the mammalian, there is a certain pathogenicity on mammals, the strain might be directly infected mammalian. Therefore, It can be speculated this strain has potential infection possibility to people.
At the same time,Reverse Transcription-Polymerase Chain Reaction (RT-PCR) was used to amplify the six major encoding structure protein of an A goose paramyxovirus typeⅠ(G).The cDNAs were then cloned to PMD18-T vector and sequenced. These sequences were compared with paramyxovirus typeⅠhome and abroad reported. The results show that the F gene was comparatively far related with La Sota, Rate of homologous was 99.1%,and other genetic homology of the strains were low.
In conclusion, the research is good for the further study of the molecular evolution, structure, and function and pathogenicity mechanism of H5N1 AIV.
该毒株EID50为10-7.625/0.1ml,IVPI为3；MDT为50.4h,且死亡率为100%,均证明该毒株是强毒。根据Genbank中已发表的禽流感HA、NA和NS三个基因片段起始密码子前和终止密码子后的非编码区和上下游区段的序列设计了两对引物,对该毒株的HA、NA和NS三个基因片段进行RT-PCR扩增、重组、测序,结果表明：该毒株HA基因裂解位点存在有6个碱性氨基酸残基：PLRERRRK-R*G,具有高致病力禽流感毒株的HA裂解位点的特征,与IVPI测定结果相吻合。试验毒株A/CHICKEN/LF3/06属于欧亚群系,其HA、NA和NS三个基因核苷酸的同源性及分子进化关系与中国A/duck/Fujian/1734/05、A/human/Zhejiang/16/06和马来西亚A/chicken/Malaysia/5223/07三株H5N1亚型禽流感毒株亲缘关系相对更近,并且不同于96广东禽流感、97香港禽流感、中东、非洲禽流感的独立进化分支,由此推测此分离株可能与A/duck/Fujian/1734/05和A/human/Zhejiang-/16/06两株病毒有着共同的祖先。
通过A/Chicken/LF3/2006毒株对鸡、小鼠和兔子的感染性试验,研究鸡源的此毒株是否能够感染哺乳动物,探讨病毒的跨种间动物传播机制。实验表明该鸡源毒株对于鸡是高致病性,也可以感染老鼠和兔子：该毒株经呼吸道感染后,对肺组织、脑组织有一定的嗜性；在大剂量攻毒时,对小鼠和兔子的体重影响都较大,中小等剂量攻毒时,对体重的影响较小；对兔子体温有影响；实验过程中小鼠和兔子均有死亡；小鼠和兔子在较高浓度感染时体内均可产生抗体,在中低浓度就几乎没有抗体产生；攻毒试验证明该病毒有明显的剂量效应。说明该毒株有可能直接感染哺乳动物发病,故推断该毒株经哺乳动物体内复制增殖后,对哺乳动物存在一定致病性。因此,可推测此株病毒还是有潜在的感染人的可能性。
通过对此株H5N1亚型高致病力禽流感病毒的生物学特性和感染性的系统研究,为今后进一步研究我国H5N1亚型禽流感病毒的致病机制、病毒的结构与功能奠定实验和理论基础,同时也为我国H5N1亚型高致病力禽流感的遗传与演化研究提供了实验数据。
同时,通过RT-PCR技术对一株鹅Ⅰ型副粘病毒(G)编码的6种主要结构蛋白,即核蛋白(NP)、磷蛋白(P)、基质蛋白(M)、血凝素-神经氨酸酶蛋白(HN)、融合蛋白(F)和巨蛋白(L)分别进行扩增,然后将其克隆到PMD18-T载体后进行序列测定和拼接；并将克隆到的6个基因片段与国内外已报道的Ⅰ型副粘病毒基因序列进行比较分析。结果表明：我们克隆到的G株鹅副粘病毒F基因与La Sota株同源性最高,同源率为99.1%,与其它各毒株各基因同源性均较低。
采用HA/HI试验、病毒分离和RT-PCR方法对该毒株分离地区的野生老鼠进行禽流感流行病学调查,可为我国的养禽业提供数据,也解决存在的当地野生老鼠是否感染该病毒的疑问。
An H5N1 avian influenza virus was isolated from China in a certain area at 2006 (named as A/CHICKEN/LF3/06, LAB No. AI0624) was studied the biological characteristics and the infection in this paper. After preliminary identified by HA and HI,virus isolation,50% egg infections dose (EID50), mean death test (MDT) in eggs and Intravenous patho-genicity index (IVPI) determine, its virulence was understanded. In order to demostrate the phylogenetic relationships between the A/CHICKEN/LF3/06 strain and the other isolates reported in the world, especially strains isolated from chickens and human in HongKong and in Southeast-Asian countries outbreak of AIV, Then the HA,NA and NS genes of the A/CHICKEN/LF3/06 strain was sequenced, molecular evolutionarily analyzed and drawed Phylogenetic tree, finally we determine its pathogenicity to mammiferous.
EID50, IVPI, MDT were10-7.625/0.lml,3and 50.4h respectively, and the mortality is 100%. Accordingly, this virus is a H5N1 High Pathogenic Avian Influenza Virus.4 primers was designed based on genes of the HA,NA and NS genes of AIV reported at Genbank, then the genes of this virus was RT-PCR, cloned, sequenced and molecular evolutionarily analyzed, the result showed:the cleavage site of HA gene is PLRERRRK-R*G that is the sign of High Pathogenic Avian Influenza Virus and its same with the result of IVPI.Virus in this study was closely related with A/duck/Fujian/1734/05,A/human/Zhejiang/16/06 and A/chicken/Malaysia/5223/07 which isolated from duck in Fujian, China in 2005,from human in Zhejiang,China in 2006 and from chicken in Malaysia in 2007.But, it was comparatively far related with A/chicken/Hong Kong/220/97 and A/goose/Guangdong/1/96, which isolated from chickens in HongKong during the 1997 outbreak of AIV and from gooses in Guangdong, China in 1996.Therefore, I suppose that A/CHICKEN/LF3/06 in this study possibly has same ancestor with A/duck/Fujian/1734/05 and A/human/Zhejiang/16/06.
The virus intranasal inoculated BALB/c mice experiment showed:in the high-dose infection, the mice had larger affection on weight loss, in the medium-dose to low-dose infection, the mice had less affection on weight loss.But during the experiment, there were mice dead, So this virus is a H5N1 higher pathogenic to mice. Through respiratory tract infections, the virus showed certain tropism on the lung tissue, the brain tissue. In addition, Mice infected intranasally with high-dose have produced antibody, with medium-dose to low-dose have no produced antibody. The experiment has proved the virus has significant dose effect.Concluded that after the strain reproduce in the mammalian, there is a certain pathogenicity on mammals, the strain might be directly infected mammalian. Therefore, It can be speculated this strain has potential infection possibility to people.
At the same time,Reverse Transcription-Polymerase Chain Reaction (RT-PCR) was used to amplify the six major encoding structure protein of an A goose paramyxovirus typeⅠ(G).The cDNAs were then cloned to PMD18-T vector and sequenced. These sequences were compared with paramyxovirus typeⅠhome and abroad reported. The results show that the F gene was comparatively far related with La Sota, Rate of homologous was 99.1%,and other genetic homology of the strains were low.
In conclusion, the research is good for the further study of the molecular evolution, structure, and function and pathogenicity mechanism of H5N1 AIV.
引文
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