上海地区流感流行与甲型流感病毒变异情况的研究
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摘要
流行性感冒(简称流感)是由流感病毒引起的具有高度传染性的急性呼吸道传染病。甲型流感病毒感染宿主范围广、病毒很容易发生变异,因此,甲型流感病毒每年都会在全球引起不同规模的季节性流行,以及约10-15年发生一次大流行。每年,全球发生的季节性流感导致25～50万人死亡,给公共卫生带来很大的挑战。对易感人群接种相应的流感疫苗是目前预防流感的重要措施,但是,WHO选取研制的流感疫苗株会因为流感病毒的变异而失去对人群的保护。因此,本研究采用现场流行病学、实验病毒学和分子生物学方法对流感病毒变异、人群对不同甲亚型病毒的免疫保护状况,流感季节性流行、亚型分布和以时间、地区序列分析流感病毒的基因进化特征进行研究,主要包括以下4个方面内容：
1.上海地区流感季节性流行与亚型分布,对2009～2010年上海地区流感病原学监测。2009年冬春季(1-25周)季节性H1N1和A／H3N2共同流行,季节性H1N1占优势,流行高峰期为1-2月份之间,在流行期末期有B型流感病毒分离。2009年夏季8月始分离到甲型H1N1流感病毒,并与A／H3N2在人群中同时流行,10月份(40周)以后的分离株主要是甲型H1N1,偶尔分离到季节性H1N1和B型流感病毒,夏秋季(26～52周)流感高峰期为8-9月份,且流行强度高于冬春季,流行持续时间长,在年末(52周)还有阳性分离株,分离株基本是甲型H1N1。这是因为甲型H1N1流感病毒在基因和抗原性上与季节性H1N1不相关联,人群的既往免疫不能有效保护。2010年1-10周,B型流感病毒与甲型H1N1病毒在人群中同时流行,期间甲型H1N1病毒活动性相对减弱,而B型流感病毒数量逐步上升,直到2010年第5周采集的标本分离到的主要是B型流感病毒。这与甲型H1N1流感病毒在人群中流行和甲型H1N1病毒疫苗接种有关。
2.检测2008～2009年上海地区人群甲型流感病毒HAI抗体水平,了解人群既往感染的免疫保护状况并探讨其流行趋势。结果显示,2008年上海地区一般人群对A／广东罗湖／219／2006(H1N1)抗原的HAI抗体阳性率为38.8%(134／345),对A／江西东湖／312／2006(H3N2)HAI抗体阳性率为42.3%(115／272)；2009年上海地区一般人群对疫苗株A/Brisbane/59/2007 (H1N1)、A/Brisbane/10/2007(H3N2)有一定的免疫屏障,HAI抗体阳性率分别为70.8%(638／901)及43.5%(392／901)。2008年,上海地区一般人群和职业人群中H5亚型HAl抗体阳性率分别为0.3%(1／345)和4%(8／198),H9亚型HAI抗体阳性率分别为2.6%(9／345)和17.7%(35／198)；2009年,两组人群的H5亚型HAI抗体阳性率分别为0.3%(1／332)和4.2%(15／356),H9亚型HAI抗体阳性率分别为2.4%(8／332)和34.6%(123／356),可见人群中特别是禽畜密切接触的职业人群中存有禽流感H5、H9抗体,职业人群H5、H9抗体阳性率增高,仍为由禽至人的传播,存在人能感染H5、H9亚型禽流感病毒,但没有人-人间传播的迹象。2009年甲型H1N1流感暴发并在全世界流行,我们检测了采自2009年1月上海地区一般人群血清甲型H1N1抗体水平,显示对甲型H1N1的疫苗株抗原HAI抗体阳性率为9.2%(37／404),表明上海地区人群对于甲型H1N1流感病毒普遍缺乏免疫保护,人群对甲型H1N1流感病毒普遍易感。但不同年龄组人群对甲型H1N1流感病毒HAI抗体阳性率有差别,以老年组(≥60岁)血清抗体阳性率最高,为25.0%(21／84),其次是成年组(25岁～),血清抗体阳性率为10.0%(8／80),5-25岁年龄组血清抗体阳性率较低为2.5%(2／80)。随着甲型H1N1在上海地区流行,人群甲型H1N1抗体水平增强,2009年10月中旬,5-15岁年龄组血清抗体阳性率为26.5%(53／200)。表明在疫苗接种前,人群已经开始对甲型H1N1流感病毒产生免疫保护。
3.选择一定条件甲型流感病毒进行HA、NA、PB2、M等全基因测序,并与WHO疫苗推荐株序列比较,分析甲型流感病毒基因进化。2005～2009年,季节性H1N1亚型HA基因进化树呈同一主干发展,相同年份序列相近,成簇分布；不同年份之间侧枝较长,表明序列变异较大。与A/NewCaledonia/20/1999 (H1N1)(2000～2007年的疫苗推荐株)、A/SolomonIslands/3/2006(H1N1) (2007～2008年的疫苗推荐株)相比,A/Brisbane/59/2007(H1N1) (2008～2010年的疫苗推荐株)的侧枝长度最短,表明A/Brisbane/59/2007(H1N1)对同期上海地区人群流感流行的保护性较好。但2008年夏秋季分离株及部分2009年冬春季分离株单独形成一个分枝,与疫苗推荐株A/Brisbane/59/2007 (H1N1)距离相对较远,HA基因序列变异较大。基因推断的氨基酸序列比较显示,变异位点涉及3个抗原决定区域,特有的变异位点多达十余个。
2004～2009年,A／H3N2的HA基因进化树呈一个主干向上发展,侧枝很短的特征。进化树的主干很长,基本按照毒株出现的时间先后发展；但侧枝较短。相同年份毒株序列相近聚集成簇,不同年份的新旧毒株序列之间相互穿插,比较季节性H1N1流感病毒遗传继承关系更为明显。A/Brisbane/10/2007(H3N2)作为2008～2010的疫苗推荐株,距离2008～2009流感流行株主干侧枝长度较短,表明其与大部分同期流行株序列相近。基因推导氨基酸序列比较显示,变异位点位于三至四个抗原决定区域。
2009年甲型H1N1的HA、NA、PB2基因进化树都呈现与季节性H1N1、A／H3N2 HA进化树相似的模式,不同区域、不同月份分离株互有穿插,呈现一定的变异继承关系。12月份以来的一些分离株侧枝较长,与主枝干距离较远,有一定的变异。HA基因推断的氨基酸序列比较显示,一些氨基酸位点已发生变异,大部分位于HA1片段上,但都不位于抗原决定区域,也没有增减潜在的糖基化位点。分离株NA基因推断的氨基酸序列中,第274位氨基酸均为H,未检测到耐奥司他韦的变异(Y),对病毒聚合酶PB2序列分析,PB2位点第627位和701位氨基酸分别是E和D,是禽源流感病毒PB2蛋白的特征。
4.时间序列分析流感病毒抗原变异状况,采用金黄地鼠作为动物模型,选取2005～2009年上海地区散发和聚集性事件中的流行株,制备免疫血清,并进行交叉HAI实验,研究甲型流感病毒HA抗原的变异情况。结果显示,上海地区H3甲型流感病毒在05年以后到现在流行株抗原进化相对比较缓慢,而甲型流感H1亚型08年下半年流行株较之以前的疫苗株和流行株均发生了抗原变异。甲型H1N1流感病毒HA抗原与人季节性H1N1流感病毒抗原有很大的差异。
Influenza is a highly contagious acute respiratory disease caused by influenza virus. As the wide host range and high genetic variation, influenza A virus causes global seasonal epidemics with different scales every year and pandemic per 10 to 15 years.250000 to 500000 people were killed worldwide each year, which brought great challenges to the public health. Vaccination on susceptible people is an important measure to prevent influenza, but flu vaccine will soon lose the protection for people because of frequency virus mutation. Therefore, we study the flu virus variation, immune status against influenza virus in the population, seasonal epidemics and subtype distribution of influenza and time and area series analysis on evolutionary characteristics of influenza virus genes via field epidemiology, laboratory virology and molecular biology, as the following four aspects:
1. Influenza activity was surveilled during 2009 to 2010 in Shanghai to study seasonal epidemics and subtype distributions. Seasonal H1N1 and A/H3N2 co-circulated in winter and spring of 2009 (from 1st to 25th week) with seasonal H1N1 being dominated, and the peak occurred from January to February, while influenza B viruses were isolated in the end. Swine origin influenza viruses (A-H1N1) began to co-circulated with seasonal A/H3N2 in August, and then became dominating since October (the 40th week) while seasonal influenza B virus and H1N1 were isolated occasionally. the peak of summer and autumn of 2009 (from 26th to 52nd week) was August to September, and the influenza activity was stronger and lasted longer with positive isolates, mostly A-H1N1 isolates, in the 52nd week, This is because influenza virus A-H1N1 was not associated with seasonal H1N1 in gene and antigen and immunity of population early could not effectively protect from it. From the 1st to 10th week in 2010, though A-H1N1 still co-circulated with influenza B virus, A-H1N1 activity was weaker relatively, and the number of influenza B virus gradually increased until the 5th week of 2010 and finally became dominating. It might relate to the epidemic and vaccination of A-H1N1 in population.
2. HAI antibody levels against influenza A virus were detected from 2008 to 2009 in Shanghai population to understand the immune level due to past infection and to forecast the flu epidemic trends. The result showed the positive rate of antibodies to influenza virus A/Guangdong Luohu/219/06(H1N1) is 38.8%(134/345),to A/ Jiangxi Donghu/312/06(H3N2) is 42.3%(115/272)in the general population in 2008; the Shanghai general population have a certain immune barrier to A/Brisbane/59/2007 (H1N1) and A/Brisbane/10/2007 (H3N2) with 70.8%(638/901) and 43.5%(392/901) positive rate of HAI antibody respectively during 2009. In 2008, the positive rate of antibodies to influenza A virus subtype H5 is 0.3%(1/345) and 4%(8/198) in the general population and the contacted population respectively, and the positive rate of antibodies to influenza A virus subtype H9 is 2.6%(9/345) and 17.7%(35/198) respectively; during 2009 the positive rate of antibodies to H5 is 0.3%(1/332) and 4.2 %(15/356), to H9 is 2.4%(8/332) and 34.6%(123/356) in these two groups respectively. There is antibody to avian flu H5 and H9 in people, especially in the contacted population, and the increase of H5 and H9 positive rate indicated possible infected from avian, but no sign of human-human transmission. A-H1N1 influenza viruses outbroke and got pandemic in the world, we tested the antibody levels to A-H1N1 flu in serum collected from the Shanghai general population in January 2009. The positive rate of antibody of A-H1N1 among the whole population in Shanghai was 9.2%(37/404), revealed the whole population in Shanghai generally lacked the immunity against A-H1N1 and were highly sensitive to A-H1N1. Among various age groups the positive rate of antibody of the≥60 age group was the highest with 25%(21/84), then the 25-year group with 10%(8/80), the 5 to 25-year group was lower with 2.5%(2/80).With the epidemic of A-H1N1 in Shanghai, the antibody levels increased in population, until mid-October 2009, the positive rate of antibody of 5 to 15-year group was 26.5%(53/200), showed that people have begun to obtained the immunity against A-H1N1 before vaccination was applied widely.
3. We sequenced HA, NA, PB2 and M genes of some influenza A isolates and compared with the WHO reference vaccine strains subsequently, to analyze the genetic evolution of influenza A. During 2005 to 2009, HA genes of seasonal H1N1 were rooted in one trunk, and the sequences of the same year were clustered as similar to each other, but the branches between years were longer revealing notable variation. Compared to A/New Caledonia/20/1999 (H1N1) (recommend as vaccine from 2000 to 2007), A/Solomon slands/3/2006(H1N1) (recommend as vaccine from 2007 to 2008) and A/Brisbane/59/2007(H1N1) (recommend as vaccine from 2008 to 2010) had shorter branch, which indicated the two latter would have better protection to the population of Shanghai. However, all strains isolated in summer and autumn of 2008 and some in winter and spring of 2009 formed a separate branch, and were relatively far away from A/Brisbane/59/2007 (H1N1), which revealed that some large variations on HA gene. Amino acid sequence comparison showed that the variable sites involved in three antigenic regions, and the unique variable sites were up to a dozen.
The phylogenetic tree of A/H3N2 in 2004～2009 was characterized as one long trunk with very short brunches. The interlaced influenza viruses isolated from different years made the more remarkable successive connection compared to the seasonal H1N1. A/Brisbane/10/2007(H3N2) recommended as vaccine of 2008 to 2010 had shorter branch, which showed similar to most influenza viruses and better protection to the population of Shanghai. But mutations were found in some HA amino acid sites, and mostly involved in three or four antigenic regions.
The phylogenetic trees of HA, NA and PB2 gene of the A-H1N1 strains isolated in 2009 had the similar model with the HA phylogenetic trees of seasonal H1N1 and A/H3N2, the interlaced influenza viruses isolated from different regions and months had some successive connection. Several isolates had some variations with the longer lateral branches far from the trunk since December. Mutations were found in some HA amino acid site, mostly in the HA1 segment, but none of them was in the antigenic determinant region and there was no change in potential glycosylation sites. None changes were observed in the 274 NA amino acid residue which was related to the drug resistance to oseltamivir. PB2 protein analysis showed that the 627 and 701 amino acid residues were Glutamic acid (Glu, E) and Aspartic acid (Asp, D) respectively, which were same encoded amino acid with avian flu PB2 protein.
4. We also did time series analysis of the antigenic variation of influenza virus. We chose golden hamster as the animal model and selected influenza virus isolated from sentry hospitals and influenza outbreaks of Shanghai during 2005 to 2009 to get strain specific antiserum, and carried out cross HAI test to study the HA antigenic variation. The results showed that the antigenic evolution of A/H3N2 strains slowed down relatively since 2005, and seasonal H1N1 isolated in first half of 2008 had some antigenic variations than the previous isolated strains and the vaccine strain, while the HA antigen of A-H1N1 strains were very different from human seasonal H1N1.
1.上海地区流感季节性流行与亚型分布,对2009～2010年上海地区流感病原学监测。2009年冬春季(1-25周)季节性H1N1和A／H3N2共同流行,季节性H1N1占优势,流行高峰期为1-2月份之间,在流行期末期有B型流感病毒分离。2009年夏季8月始分离到甲型H1N1流感病毒,并与A／H3N2在人群中同时流行,10月份(40周)以后的分离株主要是甲型H1N1,偶尔分离到季节性H1N1和B型流感病毒,夏秋季(26～52周)流感高峰期为8-9月份,且流行强度高于冬春季,流行持续时间长,在年末(52周)还有阳性分离株,分离株基本是甲型H1N1。这是因为甲型H1N1流感病毒在基因和抗原性上与季节性H1N1不相关联,人群的既往免疫不能有效保护。2010年1-10周,B型流感病毒与甲型H1N1病毒在人群中同时流行,期间甲型H1N1病毒活动性相对减弱,而B型流感病毒数量逐步上升,直到2010年第5周采集的标本分离到的主要是B型流感病毒。这与甲型H1N1流感病毒在人群中流行和甲型H1N1病毒疫苗接种有关。
2.检测2008～2009年上海地区人群甲型流感病毒HAI抗体水平,了解人群既往感染的免疫保护状况并探讨其流行趋势。结果显示,2008年上海地区一般人群对A／广东罗湖／219／2006(H1N1)抗原的HAI抗体阳性率为38.8%(134／345),对A／江西东湖／312／2006(H3N2)HAI抗体阳性率为42.3%(115／272)；2009年上海地区一般人群对疫苗株A/Brisbane/59/2007 (H1N1)、A/Brisbane/10/2007(H3N2)有一定的免疫屏障,HAI抗体阳性率分别为70.8%(638／901)及43.5%(392／901)。2008年,上海地区一般人群和职业人群中H5亚型HAl抗体阳性率分别为0.3%(1／345)和4%(8／198),H9亚型HAI抗体阳性率分别为2.6%(9／345)和17.7%(35／198)；2009年,两组人群的H5亚型HAI抗体阳性率分别为0.3%(1／332)和4.2%(15／356),H9亚型HAI抗体阳性率分别为2.4%(8／332)和34.6%(123／356),可见人群中特别是禽畜密切接触的职业人群中存有禽流感H5、H9抗体,职业人群H5、H9抗体阳性率增高,仍为由禽至人的传播,存在人能感染H5、H9亚型禽流感病毒,但没有人-人间传播的迹象。2009年甲型H1N1流感暴发并在全世界流行,我们检测了采自2009年1月上海地区一般人群血清甲型H1N1抗体水平,显示对甲型H1N1的疫苗株抗原HAI抗体阳性率为9.2%(37／404),表明上海地区人群对于甲型H1N1流感病毒普遍缺乏免疫保护,人群对甲型H1N1流感病毒普遍易感。但不同年龄组人群对甲型H1N1流感病毒HAI抗体阳性率有差别,以老年组(≥60岁)血清抗体阳性率最高,为25.0%(21／84),其次是成年组(25岁～),血清抗体阳性率为10.0%(8／80),5-25岁年龄组血清抗体阳性率较低为2.5%(2／80)。随着甲型H1N1在上海地区流行,人群甲型H1N1抗体水平增强,2009年10月中旬,5-15岁年龄组血清抗体阳性率为26.5%(53／200)。表明在疫苗接种前,人群已经开始对甲型H1N1流感病毒产生免疫保护。
3.选择一定条件甲型流感病毒进行HA、NA、PB2、M等全基因测序,并与WHO疫苗推荐株序列比较,分析甲型流感病毒基因进化。2005～2009年,季节性H1N1亚型HA基因进化树呈同一主干发展,相同年份序列相近,成簇分布；不同年份之间侧枝较长,表明序列变异较大。与A/NewCaledonia/20/1999 (H1N1)(2000～2007年的疫苗推荐株)、A/SolomonIslands/3/2006(H1N1) (2007～2008年的疫苗推荐株)相比,A/Brisbane/59/2007(H1N1) (2008～2010年的疫苗推荐株)的侧枝长度最短,表明A/Brisbane/59/2007(H1N1)对同期上海地区人群流感流行的保护性较好。但2008年夏秋季分离株及部分2009年冬春季分离株单独形成一个分枝,与疫苗推荐株A/Brisbane/59/2007 (H1N1)距离相对较远,HA基因序列变异较大。基因推断的氨基酸序列比较显示,变异位点涉及3个抗原决定区域,特有的变异位点多达十余个。
2004～2009年,A／H3N2的HA基因进化树呈一个主干向上发展,侧枝很短的特征。进化树的主干很长,基本按照毒株出现的时间先后发展；但侧枝较短。相同年份毒株序列相近聚集成簇,不同年份的新旧毒株序列之间相互穿插,比较季节性H1N1流感病毒遗传继承关系更为明显。A/Brisbane/10/2007(H3N2)作为2008～2010的疫苗推荐株,距离2008～2009流感流行株主干侧枝长度较短,表明其与大部分同期流行株序列相近。基因推导氨基酸序列比较显示,变异位点位于三至四个抗原决定区域。
2009年甲型H1N1的HA、NA、PB2基因进化树都呈现与季节性H1N1、A／H3N2 HA进化树相似的模式,不同区域、不同月份分离株互有穿插,呈现一定的变异继承关系。12月份以来的一些分离株侧枝较长,与主枝干距离较远,有一定的变异。HA基因推断的氨基酸序列比较显示,一些氨基酸位点已发生变异,大部分位于HA1片段上,但都不位于抗原决定区域,也没有增减潜在的糖基化位点。分离株NA基因推断的氨基酸序列中,第274位氨基酸均为H,未检测到耐奥司他韦的变异(Y),对病毒聚合酶PB2序列分析,PB2位点第627位和701位氨基酸分别是E和D,是禽源流感病毒PB2蛋白的特征。
4.时间序列分析流感病毒抗原变异状况,采用金黄地鼠作为动物模型,选取2005～2009年上海地区散发和聚集性事件中的流行株,制备免疫血清,并进行交叉HAI实验,研究甲型流感病毒HA抗原的变异情况。结果显示,上海地区H3甲型流感病毒在05年以后到现在流行株抗原进化相对比较缓慢,而甲型流感H1亚型08年下半年流行株较之以前的疫苗株和流行株均发生了抗原变异。甲型H1N1流感病毒HA抗原与人季节性H1N1流感病毒抗原有很大的差异。
Influenza is a highly contagious acute respiratory disease caused by influenza virus. As the wide host range and high genetic variation, influenza A virus causes global seasonal epidemics with different scales every year and pandemic per 10 to 15 years.250000 to 500000 people were killed worldwide each year, which brought great challenges to the public health. Vaccination on susceptible people is an important measure to prevent influenza, but flu vaccine will soon lose the protection for people because of frequency virus mutation. Therefore, we study the flu virus variation, immune status against influenza virus in the population, seasonal epidemics and subtype distribution of influenza and time and area series analysis on evolutionary characteristics of influenza virus genes via field epidemiology, laboratory virology and molecular biology, as the following four aspects:
1. Influenza activity was surveilled during 2009 to 2010 in Shanghai to study seasonal epidemics and subtype distributions. Seasonal H1N1 and A/H3N2 co-circulated in winter and spring of 2009 (from 1st to 25th week) with seasonal H1N1 being dominated, and the peak occurred from January to February, while influenza B viruses were isolated in the end. Swine origin influenza viruses (A-H1N1) began to co-circulated with seasonal A/H3N2 in August, and then became dominating since October (the 40th week) while seasonal influenza B virus and H1N1 were isolated occasionally. the peak of summer and autumn of 2009 (from 26th to 52nd week) was August to September, and the influenza activity was stronger and lasted longer with positive isolates, mostly A-H1N1 isolates, in the 52nd week, This is because influenza virus A-H1N1 was not associated with seasonal H1N1 in gene and antigen and immunity of population early could not effectively protect from it. From the 1st to 10th week in 2010, though A-H1N1 still co-circulated with influenza B virus, A-H1N1 activity was weaker relatively, and the number of influenza B virus gradually increased until the 5th week of 2010 and finally became dominating. It might relate to the epidemic and vaccination of A-H1N1 in population.
2. HAI antibody levels against influenza A virus were detected from 2008 to 2009 in Shanghai population to understand the immune level due to past infection and to forecast the flu epidemic trends. The result showed the positive rate of antibodies to influenza virus A/Guangdong Luohu/219/06(H1N1) is 38.8%(134/345),to A/ Jiangxi Donghu/312/06(H3N2) is 42.3%(115/272)in the general population in 2008; the Shanghai general population have a certain immune barrier to A/Brisbane/59/2007 (H1N1) and A/Brisbane/10/2007 (H3N2) with 70.8%(638/901) and 43.5%(392/901) positive rate of HAI antibody respectively during 2009. In 2008, the positive rate of antibodies to influenza A virus subtype H5 is 0.3%(1/345) and 4%(8/198) in the general population and the contacted population respectively, and the positive rate of antibodies to influenza A virus subtype H9 is 2.6%(9/345) and 17.7%(35/198) respectively; during 2009 the positive rate of antibodies to H5 is 0.3%(1/332) and 4.2 %(15/356), to H9 is 2.4%(8/332) and 34.6%(123/356) in these two groups respectively. There is antibody to avian flu H5 and H9 in people, especially in the contacted population, and the increase of H5 and H9 positive rate indicated possible infected from avian, but no sign of human-human transmission. A-H1N1 influenza viruses outbroke and got pandemic in the world, we tested the antibody levels to A-H1N1 flu in serum collected from the Shanghai general population in January 2009. The positive rate of antibody of A-H1N1 among the whole population in Shanghai was 9.2%(37/404), revealed the whole population in Shanghai generally lacked the immunity against A-H1N1 and were highly sensitive to A-H1N1. Among various age groups the positive rate of antibody of the≥60 age group was the highest with 25%(21/84), then the 25-year group with 10%(8/80), the 5 to 25-year group was lower with 2.5%(2/80).With the epidemic of A-H1N1 in Shanghai, the antibody levels increased in population, until mid-October 2009, the positive rate of antibody of 5 to 15-year group was 26.5%(53/200), showed that people have begun to obtained the immunity against A-H1N1 before vaccination was applied widely.
3. We sequenced HA, NA, PB2 and M genes of some influenza A isolates and compared with the WHO reference vaccine strains subsequently, to analyze the genetic evolution of influenza A. During 2005 to 2009, HA genes of seasonal H1N1 were rooted in one trunk, and the sequences of the same year were clustered as similar to each other, but the branches between years were longer revealing notable variation. Compared to A/New Caledonia/20/1999 (H1N1) (recommend as vaccine from 2000 to 2007), A/Solomon slands/3/2006(H1N1) (recommend as vaccine from 2007 to 2008) and A/Brisbane/59/2007(H1N1) (recommend as vaccine from 2008 to 2010) had shorter branch, which indicated the two latter would have better protection to the population of Shanghai. However, all strains isolated in summer and autumn of 2008 and some in winter and spring of 2009 formed a separate branch, and were relatively far away from A/Brisbane/59/2007 (H1N1), which revealed that some large variations on HA gene. Amino acid sequence comparison showed that the variable sites involved in three antigenic regions, and the unique variable sites were up to a dozen.
The phylogenetic tree of A/H3N2 in 2004～2009 was characterized as one long trunk with very short brunches. The interlaced influenza viruses isolated from different years made the more remarkable successive connection compared to the seasonal H1N1. A/Brisbane/10/2007(H3N2) recommended as vaccine of 2008 to 2010 had shorter branch, which showed similar to most influenza viruses and better protection to the population of Shanghai. But mutations were found in some HA amino acid sites, and mostly involved in three or four antigenic regions.
The phylogenetic trees of HA, NA and PB2 gene of the A-H1N1 strains isolated in 2009 had the similar model with the HA phylogenetic trees of seasonal H1N1 and A/H3N2, the interlaced influenza viruses isolated from different regions and months had some successive connection. Several isolates had some variations with the longer lateral branches far from the trunk since December. Mutations were found in some HA amino acid site, mostly in the HA1 segment, but none of them was in the antigenic determinant region and there was no change in potential glycosylation sites. None changes were observed in the 274 NA amino acid residue which was related to the drug resistance to oseltamivir. PB2 protein analysis showed that the 627 and 701 amino acid residues were Glutamic acid (Glu, E) and Aspartic acid (Asp, D) respectively, which were same encoded amino acid with avian flu PB2 protein.
4. We also did time series analysis of the antigenic variation of influenza virus. We chose golden hamster as the animal model and selected influenza virus isolated from sentry hospitals and influenza outbreaks of Shanghai during 2005 to 2009 to get strain specific antiserum, and carried out cross HAI test to study the HA antigenic variation. The results showed that the antigenic evolution of A/H3N2 strains slowed down relatively since 2005, and seasonal H1N1 isolated in first half of 2008 had some antigenic variations than the previous isolated strains and the vaccine strain, while the HA antigen of A-H1N1 strains were very different from human seasonal H1N1.
引文
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