H6N1亚型禽流感对哺乳动物的传播与致病机制研究
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摘要
流感病毒分为A、B、C三个属，唯有A型流感病毒属的病毒能引起流感大流行。低致病性H6亚型禽流感病毒在北美和亚洲鸟群中普遍存在，而且其感染宿主范围很广，能够直接感染鸡、小鼠和水貂等，甚至有感染人的报道，其流行和传播趋势呈上升趋势，对人类健康和公共卫生安全构成潜在威胁。
目前，国内外对人流感病毒、H5N1亚型高致病性禽流感病毒和H9N2亚型禽流感病毒研究较多，而对H6亚型低致病性禽流感病毒的研究相对较少，人们往往会因为其对禽的低致病性，而忽略其对人类健康的潜在危害性。H6亚型禽流感病毒A/teal/HongKong/W312/97(tl/HK/97)与1997年大流行的H5N1流感病毒有很高的同源性，这说明低致病性的H6亚型的流感病毒具有与其它亚型的流感病毒发生重配，且能突破物种的种间障碍在人群大流行的潜在能力。
本研究重点探讨了H6亚型低致病性禽流感病毒对哺乳动物的致病性、在哺乳动物间进行水平传播的能力以及致病性变异的分子机制。首先，我们对本实验室保存的一株野鸭源A/H6N1亚型禽流感病毒(A/Mallard/SanJiang/275/2007，简称A/H6N1)进行全基因组测序和遗传进化分析，结果发现，A/H6N1具有低致病性禽流感病毒所特有的分子特征。然后，我们分别以小鼠、豚鼠和犬作为模式动物，对A/H6N1的生物学特性进行进一步的分析研究，结果发现，该病毒能够感染小鼠、豚鼠和犬，但不会导致感染动物死亡；该病毒也不能在豚鼠间进行水平传播。同时，为了评估H6亚型禽流感病毒的潜在传播性，我们将A/H6N1与曾在2009年引起全球流感大流行的甲型H1N1流感病毒(简称A/H1N1)同时感染豚鼠，让两者在豚鼠体内进行自然重配，并采用细胞生物学和分子生物学的方法筛选H6亚型重配病毒，结果发现，A/H6N1和A/H1N1之间具有良好的遗传兼容性，两者同时感染豚鼠能够产生具有稳定遗传表型的子代H6亚型重配病毒，且子代H6亚型重配病不具备在豚鼠间进行水平传播的能力。最后，为研究H6亚型禽流感病毒致病性变异分子机制，我们将A/H6N1感染BALB/c小鼠，经肺脏连续传代获得3株致病性显著增强的鼠适应株；通过全基因组测序和序列比对，初步筛选出3个可能决定A/H6N1致病性的毒力决定子；应用反向遗传技术、点突变技术和动物实验，最终确定导致A/H6N1致病性增强的关键性氨基酸位点。本研究获得以下结果：
1.本研究完成对一株野鸭源A/H6N1亚型禽流感病毒(A/Mallard/SanJiang/275/2007，简称A/H6N1)的全基因组测序，并对该株病毒进行了遗传进化分析。结果发现，该株病毒遗传背景较复杂；
2.本试验对A/H6N1亚型禽流感病毒的基本生物学特性进行了研究，并成功构建了A/H6N1亚型禽流感病毒BALB/c小鼠感染模型和A/H6N1亚型禽流感病毒犬感染模型，初步发现A/H6N1亚型禽流感病毒能够有效感染多种哺乳动物，具有跨种传播的潜力；
3.本研究成功构建了A/H6N1亚型禽流感病毒反向遗传操作平台，为研究A/H6N1亚型禽流感病毒传播性和致病性分子机制夯实了基础；
4.本研究以豚鼠为哺乳动物模型，对A/H6N1亚型禽流感病毒的传播性和遗传兼容性进行了初步探索，结果发现：A/H6N1亚型禽流感病毒不能通过直接接触途径在豚鼠间进行有效传播；经三轮噬斑纯化获得的H6/H1重配病毒（血凝素为H6）不能通过直接接触途径在豚鼠间进行有效传播；A/H6N1亚型禽流感病毒与A/H1N1人流感病毒能共同感染哺乳动物并发生基因重配，说明这两种亚型的流感病毒在哺乳动物体内具有良好的遗传兼容性。以上结果说明，H6亚型禽流感病毒虽然具有在人间大流行的潜力，但是还需要不断的演化和适应。
5.本研究成功筛选出影响A/H6N1亚型禽流感病毒致病性的关键性氨基酸位点：小鼠是研究流感病毒致病性的理想动物模型，本研究将A/H6N1亚型禽流感病毒在BALB/c小鼠肺脏连续传代获得3株A/H6N1亚型禽流感病毒的变异株，并对3株变异株进行了全基因组测序和生物学特性分析初步筛选出可能导致变异株致病性增强的关键性氨基酸位点；然后通过反向遗传技术、点突变技术和小鼠感染模型确定PB2-E627K和PA-T97I是导致A/H6N1亚型禽流感病毒对小鼠致病性增强的关键性氨基酸位点。
There are three types of influenza viruses (A, B and C).The type A can cause influenza pandemic. Low pathogenic avian influenza virus H6subtype is one of the most commonly recognized subtypes in birds in North America and Europe. This subtype has a broad host range, the epidemic and spread show upward. It can infect chickens, mice and mink directly, even can infect people. Thus, H6subtype AIV could constitute a potential pandemic threat to public health.
Very little is known about the replicative capacity, immunogenicity, and correlates of protective immunity for H6AIV in mammals. People often because of its low pathogenicity ignore the potential harm to human health. The high level of homology of the internal protein genes of H6N1A/teal/Hong Kong/W312/97(teal/HK/97)-like viruses to those of the1997human H5N1viruses raises concerns about W312-like H6viruses. The continuing prevalence of H6viruses and frequently reasserts in avian populations highlight the potential for H6viruses and H6reassortants to cross the species barrier to infect humans and cause human-to-human transmission.
In this study, we focus on the molecular mechanism of transmission and pathogenicity of H6subtype AIV to mammals. First, we analy the whole genome sequencing and evolution of the A/H6N1subtype avian influenza virus (A/Mallard/SanJiang/275/2007) and the biological characteristics of H6subtype AIV using mice and dogs for mammalian model. Second, H6subtype AIV cannot spread effectively in mammals, in order to study the transmission about H6subtype AIV further and potential pandemic threat to public health, we observe the reassortment of H1N1AIV which pandemic in2009and H6subtype AIV in mammals. Third, in order to investigate the genetic basis of H6subtype AIV pathogenicity in mammals, we generated a mouse-adapted H6subtype AIV that possessed significantly higher virulence than wide-type virus. Increased virulence was detectable after8sequential lung passages in mice. The amino acid which is critical for the increased pathogenicity of H6subtype AIV is detected in vivo and in vitro. This research obtains the following results:
1. This study successfully sequence the completed genomes of A/H6N1subtype avian influenza virus (A/Mallard/SanJiang/275/2007) from duck and analy the origin and evolution of the strain, the source of this strain is complicated and easy to reassortant with other subtype influenza virus. Also, we found the mammalian model of A/H6N1subtype AIV by mice and dogs, analy the biological characteristics of A/H6N1subtype AIV. The A/H6N1subtype AIV can infect mammals and has the potential to cross the species barrier.
2. We successfully establish the A/H6N1subtype AIV reverse genetics system, which settled the foundation for further research on pathogenicity and transmission mechanism.
3. The transmission of A/H6N1subtype avian influenza virus is studied:The A/H6N1subtype AIV cannot spread effectively in mammals by the guinea pig model. A/H6N1AIV and A/H1N1have good genetic compatibility in mammals, can reassortant in mammals. The H6subtype reassortant virus also cannot effectively contact transmission in mammals. This suggest the A/H6N1subtype AIV has the potential to spread, but needs the continue evolution and adaptation.
4. The pathogenicity of A/H6N1subtype avian influenza virus is studied:Mice are ideal animal models for investigating pathogenic mechanisms of influenza viruses. We obtain3strains mouse-adapted H6subtype AIV after8lung passages in mice and analyze the whole genome sequence and biological characteristics. Two amino acid substitutions can enhance pathogenicity in the genome of H6subtype AIV. Assessments of replication in mice showed that PB2-E627K and PA-T97I increased virus replication, The PB2-E627K and PA-T97I amino acid substitution enhanced viral polymerase activity and replication. Thus, our results show that the combination of PB2-E627K and PA-T97I are critical for the pathogenicity of H6subtype AIV in mammalian host.
目前，国内外对人流感病毒、H5N1亚型高致病性禽流感病毒和H9N2亚型禽流感病毒研究较多，而对H6亚型低致病性禽流感病毒的研究相对较少，人们往往会因为其对禽的低致病性，而忽略其对人类健康的潜在危害性。H6亚型禽流感病毒A/teal/HongKong/W312/97(tl/HK/97)与1997年大流行的H5N1流感病毒有很高的同源性，这说明低致病性的H6亚型的流感病毒具有与其它亚型的流感病毒发生重配，且能突破物种的种间障碍在人群大流行的潜在能力。
本研究重点探讨了H6亚型低致病性禽流感病毒对哺乳动物的致病性、在哺乳动物间进行水平传播的能力以及致病性变异的分子机制。首先，我们对本实验室保存的一株野鸭源A/H6N1亚型禽流感病毒(A/Mallard/SanJiang/275/2007，简称A/H6N1)进行全基因组测序和遗传进化分析，结果发现，A/H6N1具有低致病性禽流感病毒所特有的分子特征。然后，我们分别以小鼠、豚鼠和犬作为模式动物，对A/H6N1的生物学特性进行进一步的分析研究，结果发现，该病毒能够感染小鼠、豚鼠和犬，但不会导致感染动物死亡；该病毒也不能在豚鼠间进行水平传播。同时，为了评估H6亚型禽流感病毒的潜在传播性，我们将A/H6N1与曾在2009年引起全球流感大流行的甲型H1N1流感病毒(简称A/H1N1)同时感染豚鼠，让两者在豚鼠体内进行自然重配，并采用细胞生物学和分子生物学的方法筛选H6亚型重配病毒，结果发现，A/H6N1和A/H1N1之间具有良好的遗传兼容性，两者同时感染豚鼠能够产生具有稳定遗传表型的子代H6亚型重配病毒，且子代H6亚型重配病不具备在豚鼠间进行水平传播的能力。最后，为研究H6亚型禽流感病毒致病性变异分子机制，我们将A/H6N1感染BALB/c小鼠，经肺脏连续传代获得3株致病性显著增强的鼠适应株；通过全基因组测序和序列比对，初步筛选出3个可能决定A/H6N1致病性的毒力决定子；应用反向遗传技术、点突变技术和动物实验，最终确定导致A/H6N1致病性增强的关键性氨基酸位点。本研究获得以下结果：
1.本研究完成对一株野鸭源A/H6N1亚型禽流感病毒(A/Mallard/SanJiang/275/2007，简称A/H6N1)的全基因组测序，并对该株病毒进行了遗传进化分析。结果发现，该株病毒遗传背景较复杂；
2.本试验对A/H6N1亚型禽流感病毒的基本生物学特性进行了研究，并成功构建了A/H6N1亚型禽流感病毒BALB/c小鼠感染模型和A/H6N1亚型禽流感病毒犬感染模型，初步发现A/H6N1亚型禽流感病毒能够有效感染多种哺乳动物，具有跨种传播的潜力；
3.本研究成功构建了A/H6N1亚型禽流感病毒反向遗传操作平台，为研究A/H6N1亚型禽流感病毒传播性和致病性分子机制夯实了基础；
4.本研究以豚鼠为哺乳动物模型，对A/H6N1亚型禽流感病毒的传播性和遗传兼容性进行了初步探索，结果发现：A/H6N1亚型禽流感病毒不能通过直接接触途径在豚鼠间进行有效传播；经三轮噬斑纯化获得的H6/H1重配病毒（血凝素为H6）不能通过直接接触途径在豚鼠间进行有效传播；A/H6N1亚型禽流感病毒与A/H1N1人流感病毒能共同感染哺乳动物并发生基因重配，说明这两种亚型的流感病毒在哺乳动物体内具有良好的遗传兼容性。以上结果说明，H6亚型禽流感病毒虽然具有在人间大流行的潜力，但是还需要不断的演化和适应。
5.本研究成功筛选出影响A/H6N1亚型禽流感病毒致病性的关键性氨基酸位点：小鼠是研究流感病毒致病性的理想动物模型，本研究将A/H6N1亚型禽流感病毒在BALB/c小鼠肺脏连续传代获得3株A/H6N1亚型禽流感病毒的变异株，并对3株变异株进行了全基因组测序和生物学特性分析初步筛选出可能导致变异株致病性增强的关键性氨基酸位点；然后通过反向遗传技术、点突变技术和小鼠感染模型确定PB2-E627K和PA-T97I是导致A/H6N1亚型禽流感病毒对小鼠致病性增强的关键性氨基酸位点。
There are three types of influenza viruses (A, B and C).The type A can cause influenza pandemic. Low pathogenic avian influenza virus H6subtype is one of the most commonly recognized subtypes in birds in North America and Europe. This subtype has a broad host range, the epidemic and spread show upward. It can infect chickens, mice and mink directly, even can infect people. Thus, H6subtype AIV could constitute a potential pandemic threat to public health.
Very little is known about the replicative capacity, immunogenicity, and correlates of protective immunity for H6AIV in mammals. People often because of its low pathogenicity ignore the potential harm to human health. The high level of homology of the internal protein genes of H6N1A/teal/Hong Kong/W312/97(teal/HK/97)-like viruses to those of the1997human H5N1viruses raises concerns about W312-like H6viruses. The continuing prevalence of H6viruses and frequently reasserts in avian populations highlight the potential for H6viruses and H6reassortants to cross the species barrier to infect humans and cause human-to-human transmission.
In this study, we focus on the molecular mechanism of transmission and pathogenicity of H6subtype AIV to mammals. First, we analy the whole genome sequencing and evolution of the A/H6N1subtype avian influenza virus (A/Mallard/SanJiang/275/2007) and the biological characteristics of H6subtype AIV using mice and dogs for mammalian model. Second, H6subtype AIV cannot spread effectively in mammals, in order to study the transmission about H6subtype AIV further and potential pandemic threat to public health, we observe the reassortment of H1N1AIV which pandemic in2009and H6subtype AIV in mammals. Third, in order to investigate the genetic basis of H6subtype AIV pathogenicity in mammals, we generated a mouse-adapted H6subtype AIV that possessed significantly higher virulence than wide-type virus. Increased virulence was detectable after8sequential lung passages in mice. The amino acid which is critical for the increased pathogenicity of H6subtype AIV is detected in vivo and in vitro. This research obtains the following results:
1. This study successfully sequence the completed genomes of A/H6N1subtype avian influenza virus (A/Mallard/SanJiang/275/2007) from duck and analy the origin and evolution of the strain, the source of this strain is complicated and easy to reassortant with other subtype influenza virus. Also, we found the mammalian model of A/H6N1subtype AIV by mice and dogs, analy the biological characteristics of A/H6N1subtype AIV. The A/H6N1subtype AIV can infect mammals and has the potential to cross the species barrier.
2. We successfully establish the A/H6N1subtype AIV reverse genetics system, which settled the foundation for further research on pathogenicity and transmission mechanism.
3. The transmission of A/H6N1subtype avian influenza virus is studied:The A/H6N1subtype AIV cannot spread effectively in mammals by the guinea pig model. A/H6N1AIV and A/H1N1have good genetic compatibility in mammals, can reassortant in mammals. The H6subtype reassortant virus also cannot effectively contact transmission in mammals. This suggest the A/H6N1subtype AIV has the potential to spread, but needs the continue evolution and adaptation.
4. The pathogenicity of A/H6N1subtype avian influenza virus is studied:Mice are ideal animal models for investigating pathogenic mechanisms of influenza viruses. We obtain3strains mouse-adapted H6subtype AIV after8lung passages in mice and analyze the whole genome sequence and biological characteristics. Two amino acid substitutions can enhance pathogenicity in the genome of H6subtype AIV. Assessments of replication in mice showed that PB2-E627K and PA-T97I increased virus replication, The PB2-E627K and PA-T97I amino acid substitution enhanced viral polymerase activity and replication. Thus, our results show that the combination of PB2-E627K and PA-T97I are critical for the pathogenicity of H6subtype AIV in mammalian host.
引文
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