H5N1亚型禽流感病毒对家鸭致病力分子机制的研究
摘要
水禽包括家鸭是H5N1亚型流感病毒的储存宿主,早期病毒不致死水禽,感染病毒的水禽自身也不表现出明显的临床症状,但可以长时间的带毒并向外界排毒,具有潜在感染其他宿主的危险。先前H5N1亚型禽流感病毒和水禽一直保持着和谐关系,但是随着病毒的不断进化,对水禽高致病力的毒株也随之出现。如2002年首次报道在香港出现致死水禽的H5N1亚型毒株,2005年青海湖大规模爆发H5N1亚型禽流感致死候鸟事件等。本实验选取13株2001-2005年部分H5N1分离株,以家鸭为水禽的模型,分析这些毒株对家鸭的致病力。结果发现这13株病毒对家鸭表现出不同的致病性,致死率从0%-100%不等,表明在这期间我国H5N1毒株对家鸭的致病性表现出多样性的特点。由于低致病性和中等致病性毒株的存在,因此在我国家鸭仍然是H5N1重要的储存宿主之一,在流行病学具有重要意义。
为了解H5N1亚型禽流感病毒对水禽致病力改变的内在分子基础,我们选取了两株模式病毒。A/Goose/HB65对家鸭表现为典型的弱毒株,106EID50剂量感染家鸭后,家鸭并不表现出可见的临床症状,其DLD50>108.5 EID50。相反A/Duck/HB49对家鸭表现出明显的高致病性,106EID50剂量感染家鸭后,全部感染鸭在感染后3-5天内全部死亡,而且表现出明显的神经症状,其DLD50,102.5 EID50。进一步比较两株病毒在家鸭体内复制情况,发现两株病毒在肺脏、肾脏及胰腺都可以很好的复制,可以达到很高病毒滴度;但是病毒在脑内滴度差异显著, A/Duck/HB49可以达到107.6EID50/ml而A/Goose/HB65滴度仅为102.5EID50/ml。因此推测H5N1亚型禽流感病毒对家鸭产生高致病性原因是病毒侵害中枢神经系统。比较A/Goose/HB65和A/Duck/HB49基因组的相似性,发现它们各基因片段相似性均达到99%以上。因此A/Goose/HB65和A/Duck/HB49是研究H5N1亚型禽流感病毒对家鸭致病力分子基础的理想模式病毒。
选择了A/Goose/HB65和A/Duck/HB49作为本研究的模式病毒,我们分别建立了这两株病毒的反向遗传操作系统。为了弄清决定这两株病毒的致病力的主要基因,我们拯救出一系列单基因替换病毒,并将这些病毒做家鸭感染实验。结果发现PA基因决定了A/Goose/HB65和A/Duck/HB49对家鸭的致病性。在A/Goose/HB65背景下,将65PA替换为49PA,拯救出病毒GS/HB65-49PA,其致病力显著增强。其DLD50,103.5 EID50,相对于亲本病毒A/Goose/HB65(DLD50>108.5 EID50)其致病力增强了10万倍以上,病毒在脑内滴度也显著提高,分别为GS/HB65,102.5EID50/ml而GS/HB65-49PA,105.2EID50/ml;相反当在A/Duck/HB49背景下,将49PA替换为65PA时,拯救出病毒DK/HB49-65PA,其致病力下降了100倍(DK/HB49,102.5 EID50 VS DK/HB49-56PA,104.5 EID50)。相对于亲本病毒,感染DK/HB49-65PA病毒家鸭脑组织滴度也有所下降。为进一步在PA上寻找其影响致病力的关键位点,我们拯救出一系列PA突变的病毒,并且做了感染家鸭实验。结果发现PA224和383位氨基酸共同决定了这两株病毒对家鸭的致病力。PA蛋白S224P和N383D的突变可以显著增强GS/HB65对家的致病力(增强10万倍以上);P224S和D383N的突变可以降低DK/HB49对家鸭的致病力(降低100倍)。
PA224和383位氨基的改变是怎样影响病毒对家鸭的致病力?我们对其可能的机制进行深入探讨。分别在聚合酶活性、PA蛋白在细胞核内聚集、PA蛋白自身稳定性及PA和DK-CLE蛋白的相互作用方面进行研究。结果发现PA蛋白383位点的改变影响H5N1亚型流感病毒聚合酶活性,同时也影响了PA和PB1蛋白在细胞核内的聚集。而224位点则不影响这两个方面。S224P改变导致PA蛋白丢失潜在磷酸化位点,但具体对PA功能的影响,仍有待进一步的研究。此外PA蛋白224和383位改变并不影响自身的稳定性,也不影响和DK-CLE蛋白的相互作用。
总之,本实验以2001-2005年间13株H5N1亚型禽流感病毒为研究对象,从中筛选出2株研究病毒对家鸭致病力分子机制的模式病毒。利用反向遗传操作技术,首次证明PA是决定H5N1亚型禽流感病毒对家鸭致病力的主要基因。通过定点突变,首次发现PA蛋白224和383位氨基酸是影响病毒对家鸭致病力的关键位点,并对这两个位点的改变是如何影响致病力的可能机理做了深入的探讨。
Wild waterfowl, including ducks are natural hosts of H5N1 influenza virus. Early strains rarely caused disease in natural hosts. The infected hosts showed no sign of disease, but could shed virus to enviroment which other annimals can be easily infected. However with the evolution of H5N1 virus, reemerging H5N1 influenza virus in HK in 2002 are highly pathogenic to wild migratory birds and resident waterfowl. Late in 2005 an outbreak of H5N1 virus infection occurred among migratory birds at Qinhai Lake in china. However the molecular basis for H5N1 virus transforming from low pathogenicty to higy pathogenicity for waterflow is still unkwon.
The present studies characterised pathogenicity of 13 strains of H5N1 influenza viruses for ducks which islolated from china during 2001 to 2005. The results showed there are highly, moderately or low pathogenic for ducks. This indicated diverse pathogenicity among H5N1 viruses for ducks in china. So domestic ducks will still play an important role in epidemiology of H5N1 avian influenza virus in china. To understand the genetic determinant of virulence for duks, two viruses whose virulence differ a lot, but with higly similarity of their genome were selected as model viruses. DK/HB49 was highly pathogenic for ducks with its DLD50, 2.5lgEID50. All the infected duks were killed within 3-5 days p.i. with dosage of 106 EID50; On the contraty, GS/HB65 was low pathogenic for ducks with its DLD50, >8.5 lgEID50. All the infected ducks did not show any clinic signs. Comparing the virus titre level in different organs, it was found a distict titre differnce in brain of the ducks between incoluated with DK/HB49 and GS/HB65(107.6EID50/ml VS 102.5EID50/ml), when the ducks inoculated with dosage of 106 EID50. But no statistical titre difference present in lung, pancreas, and kidney. So we hypothesized both two type viruses (highly pathogenic and low pathogenic) could enter duck brain, whlie virulent virus could replicated efficiently in duck brain cells, but low pathogenic vrius cannot. That was main reason for causing infected ducks to death.
Then we established reverse genetic system for DK/HB49 and GS/HB65, and rescued a series of reassortant and mutant viruses. Further these viruses were tested their virulence for ducks. At last we found PA gene contribute the virulence of these two viruses. The virulence of the reassortant bearing the PA gene from DK/HB49 in the GS/HB65 background was increased 105-fold relative to that of the GS/HB65 virus. Whlie at background of DK/HB49 only 65PA was substituted for 49PA, the pathogenicity of the reassortant virus was discreased by 102-fold. The virus titer in the brian of ducks infected with DK/HB49 was enhanced greatly compared with those ducks infected with GS/HB65. In order to pinpoint the key amino acids that affected virulence, we generated many PA mutant viruses and tested them on ducks. These results indicated the combination of the two amino acid mutations at positions 224 and 383 of PA protein determined the pathogenicity of GS/HB65 and DK/HB49.
We have found the two amino acid mutations at positions 224 and 383 of PA involved in pathogenicity of these two viruses. What is the exact mechnism? With this question we performed mini-genome assay to detect the change of polymerase activity when mutant PA at positions 224 or /and 383 were introduced. It showed substitution of amino acid at position 383 changed the vrial polymerase dramatically both in 293T and in DF1 cells . The amino acid at position 383 in the PA protein also affects the transport of PA and PB1 into the nucleus of DF1 cells. Substitution of amino acid at position S224P of PA protein result in loss of an potential phsophorylation site . However, the mechanism underlying how the S224P mutation in the PA protein affects the virulence of the H5N1 virus in ducks remains to be determined. By the way substitution of amino acid at position 224 or/and 383 did not affect PA itself stablity and interation with DK-CLE.
In summary, we compared two naturally isolated H5N1 avian influenza viruses that differ in their lethality in ducks, and demonstrated that the polymerase PA plays an important role in the increased virulence of the H5N1 DK/49 virus in ducks. We report for the first time that the two amino acids 224P and 383D in PA are critical and have cumulative effects on the highly lethal phenotype of the virus to ducks. The amino acid at position 338 in PA plays an important role in the activity of the polymerase and in the accumulation of the polymerase PA and PB1 subunits in the nucleus of virus-infected cells. Our results demonstrate that PA is a virulence factor of H5N1 avian influenza viruses.
为了解H5N1亚型禽流感病毒对水禽致病力改变的内在分子基础,我们选取了两株模式病毒。A/Goose/HB65对家鸭表现为典型的弱毒株,106EID50剂量感染家鸭后,家鸭并不表现出可见的临床症状,其DLD50>108.5 EID50。相反A/Duck/HB49对家鸭表现出明显的高致病性,106EID50剂量感染家鸭后,全部感染鸭在感染后3-5天内全部死亡,而且表现出明显的神经症状,其DLD50,102.5 EID50。进一步比较两株病毒在家鸭体内复制情况,发现两株病毒在肺脏、肾脏及胰腺都可以很好的复制,可以达到很高病毒滴度;但是病毒在脑内滴度差异显著, A/Duck/HB49可以达到107.6EID50/ml而A/Goose/HB65滴度仅为102.5EID50/ml。因此推测H5N1亚型禽流感病毒对家鸭产生高致病性原因是病毒侵害中枢神经系统。比较A/Goose/HB65和A/Duck/HB49基因组的相似性,发现它们各基因片段相似性均达到99%以上。因此A/Goose/HB65和A/Duck/HB49是研究H5N1亚型禽流感病毒对家鸭致病力分子基础的理想模式病毒。
选择了A/Goose/HB65和A/Duck/HB49作为本研究的模式病毒,我们分别建立了这两株病毒的反向遗传操作系统。为了弄清决定这两株病毒的致病力的主要基因,我们拯救出一系列单基因替换病毒,并将这些病毒做家鸭感染实验。结果发现PA基因决定了A/Goose/HB65和A/Duck/HB49对家鸭的致病性。在A/Goose/HB65背景下,将65PA替换为49PA,拯救出病毒GS/HB65-49PA,其致病力显著增强。其DLD50,103.5 EID50,相对于亲本病毒A/Goose/HB65(DLD50>108.5 EID50)其致病力增强了10万倍以上,病毒在脑内滴度也显著提高,分别为GS/HB65,102.5EID50/ml而GS/HB65-49PA,105.2EID50/ml;相反当在A/Duck/HB49背景下,将49PA替换为65PA时,拯救出病毒DK/HB49-65PA,其致病力下降了100倍(DK/HB49,102.5 EID50 VS DK/HB49-56PA,104.5 EID50)。相对于亲本病毒,感染DK/HB49-65PA病毒家鸭脑组织滴度也有所下降。为进一步在PA上寻找其影响致病力的关键位点,我们拯救出一系列PA突变的病毒,并且做了感染家鸭实验。结果发现PA224和383位氨基酸共同决定了这两株病毒对家鸭的致病力。PA蛋白S224P和N383D的突变可以显著增强GS/HB65对家的致病力(增强10万倍以上);P224S和D383N的突变可以降低DK/HB49对家鸭的致病力(降低100倍)。
PA224和383位氨基的改变是怎样影响病毒对家鸭的致病力?我们对其可能的机制进行深入探讨。分别在聚合酶活性、PA蛋白在细胞核内聚集、PA蛋白自身稳定性及PA和DK-CLE蛋白的相互作用方面进行研究。结果发现PA蛋白383位点的改变影响H5N1亚型流感病毒聚合酶活性,同时也影响了PA和PB1蛋白在细胞核内的聚集。而224位点则不影响这两个方面。S224P改变导致PA蛋白丢失潜在磷酸化位点,但具体对PA功能的影响,仍有待进一步的研究。此外PA蛋白224和383位改变并不影响自身的稳定性,也不影响和DK-CLE蛋白的相互作用。
总之,本实验以2001-2005年间13株H5N1亚型禽流感病毒为研究对象,从中筛选出2株研究病毒对家鸭致病力分子机制的模式病毒。利用反向遗传操作技术,首次证明PA是决定H5N1亚型禽流感病毒对家鸭致病力的主要基因。通过定点突变,首次发现PA蛋白224和383位氨基酸是影响病毒对家鸭致病力的关键位点,并对这两个位点的改变是如何影响致病力的可能机理做了深入的探讨。
Wild waterfowl, including ducks are natural hosts of H5N1 influenza virus. Early strains rarely caused disease in natural hosts. The infected hosts showed no sign of disease, but could shed virus to enviroment which other annimals can be easily infected. However with the evolution of H5N1 virus, reemerging H5N1 influenza virus in HK in 2002 are highly pathogenic to wild migratory birds and resident waterfowl. Late in 2005 an outbreak of H5N1 virus infection occurred among migratory birds at Qinhai Lake in china. However the molecular basis for H5N1 virus transforming from low pathogenicty to higy pathogenicity for waterflow is still unkwon.
The present studies characterised pathogenicity of 13 strains of H5N1 influenza viruses for ducks which islolated from china during 2001 to 2005. The results showed there are highly, moderately or low pathogenic for ducks. This indicated diverse pathogenicity among H5N1 viruses for ducks in china. So domestic ducks will still play an important role in epidemiology of H5N1 avian influenza virus in china. To understand the genetic determinant of virulence for duks, two viruses whose virulence differ a lot, but with higly similarity of their genome were selected as model viruses. DK/HB49 was highly pathogenic for ducks with its DLD50, 2.5lgEID50. All the infected duks were killed within 3-5 days p.i. with dosage of 106 EID50; On the contraty, GS/HB65 was low pathogenic for ducks with its DLD50, >8.5 lgEID50. All the infected ducks did not show any clinic signs. Comparing the virus titre level in different organs, it was found a distict titre differnce in brain of the ducks between incoluated with DK/HB49 and GS/HB65(107.6EID50/ml VS 102.5EID50/ml), when the ducks inoculated with dosage of 106 EID50. But no statistical titre difference present in lung, pancreas, and kidney. So we hypothesized both two type viruses (highly pathogenic and low pathogenic) could enter duck brain, whlie virulent virus could replicated efficiently in duck brain cells, but low pathogenic vrius cannot. That was main reason for causing infected ducks to death.
Then we established reverse genetic system for DK/HB49 and GS/HB65, and rescued a series of reassortant and mutant viruses. Further these viruses were tested their virulence for ducks. At last we found PA gene contribute the virulence of these two viruses. The virulence of the reassortant bearing the PA gene from DK/HB49 in the GS/HB65 background was increased 105-fold relative to that of the GS/HB65 virus. Whlie at background of DK/HB49 only 65PA was substituted for 49PA, the pathogenicity of the reassortant virus was discreased by 102-fold. The virus titer in the brian of ducks infected with DK/HB49 was enhanced greatly compared with those ducks infected with GS/HB65. In order to pinpoint the key amino acids that affected virulence, we generated many PA mutant viruses and tested them on ducks. These results indicated the combination of the two amino acid mutations at positions 224 and 383 of PA protein determined the pathogenicity of GS/HB65 and DK/HB49.
We have found the two amino acid mutations at positions 224 and 383 of PA involved in pathogenicity of these two viruses. What is the exact mechnism? With this question we performed mini-genome assay to detect the change of polymerase activity when mutant PA at positions 224 or /and 383 were introduced. It showed substitution of amino acid at position 383 changed the vrial polymerase dramatically both in 293T and in DF1 cells . The amino acid at position 383 in the PA protein also affects the transport of PA and PB1 into the nucleus of DF1 cells. Substitution of amino acid at position S224P of PA protein result in loss of an potential phsophorylation site . However, the mechanism underlying how the S224P mutation in the PA protein affects the virulence of the H5N1 virus in ducks remains to be determined. By the way substitution of amino acid at position 224 or/and 383 did not affect PA itself stablity and interation with DK-CLE.
In summary, we compared two naturally isolated H5N1 avian influenza viruses that differ in their lethality in ducks, and demonstrated that the polymerase PA plays an important role in the increased virulence of the H5N1 DK/49 virus in ducks. We report for the first time that the two amino acids 224P and 383D in PA are critical and have cumulative effects on the highly lethal phenotype of the virus to ducks. The amino acid at position 338 in PA plays an important role in the activity of the polymerase and in the accumulation of the polymerase PA and PB1 subunits in the nucleus of virus-infected cells. Our results demonstrate that PA is a virulence factor of H5N1 avian influenza viruses.
引文
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