2005～2011年青岛地区流感病毒监测与分子流行病学研究
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摘要
流行性感冒病毒属正粘病毒科,流感病毒属,是引起各种鸟类、人、猪、马等哺乳动物发生上呼吸道感染的主要病原。根据其核蛋白(nuclear protein, NP)(?)口基质蛋白(matrix protein, MP)抗原性的不同,流感病毒可分为甲、乙、丙三型。流感每年在全球范围内可致5%-15%的人群感染,约50万人死亡。其中,甲型流感病毒感染范围最广,并可通过多种变异形式逃避宿主免疫从而造成暴发甚至全球性暴发流行,因此危害最大。甲型流感病毒据其两种主要表面抗原血凝素(hemagglutinin, HA)和神经氨酸酶(neuraminidase, NA)结构和基因特性的不同又可分成许多亚型。目前造成人类感染的主要为季节性H1N1、季节性H3N2和新甲型H1N1流感亚型。
基于半个多世纪以来的研究成果,现已发展出M2离子通道阻滞剂和神经氨酸酶活性抑制剂(neuraminidase inhibitor, NAI)两类抗病毒药物,而预防感染的三价流感病毒疫苗须每年两次更新候选疫苗株以应对病毒频繁的抗原变异。然而,甲型流感病毒迅速而又无法预期的动态进化仍可导致以下现象发生：(1)间或出现的重大抗原变异导致严重疾病及疫苗株-流行株失匹配；(2)可抵抗现有抗病毒药物的新变异株的增殖；(3)偶见致死性人感染甲型禽流感病毒病例。这些事件发生的进化机制尚未明确。而就在全世界流感监测及研究机构都聚焦于甲型禽流感病毒跨宿主致哺乳动物感染,并可能在人群导致新的流感暴发流行时,2009年4月一株“全新”的甲型H1N1流感病毒感染在墨西哥暴发,并迅速波及全球。
由于甲型流感病毒可对人类健康造成持续、而又无法准确预期的威胁,因此甲型流感病毒的进化一直是国内外学者的研究热点。甲型流感病毒基因具有多态性,很大程度上来源于共感染同一宿主细胞的两株遗传距离可能较远的病毒可以发生重配。上世纪最主要的几次大流行之一,1947年的流感大流行即与病毒的重配事件有关,而新世纪首次流感暴发更是来源于一株经过多次基因重配产生的抗原转变株。因此,对甲型流感病毒进行全基因组水平进化研究,全面考虑基因组重配事件及各基因节段的进化动态,比仅仅针对主要表面抗原HA的分子进化研究更为重要。
本研究于2005-2011年间采集青岛地区哨点医院流感样病例(influenza-like illness, ILI)鼻咽拭子标本开展流感监测,并采用细胞培养法分离流感毒株；2009年新甲型H1N1流感暴发期间开展紧急监测,采集4个目标人群鼻咽拭子标本进行核酸检测以监控病毒的流行形势。反转录聚合酶链反应(reverse-transcription polymerase chain reaction, RT-PCR)扩增甲型流感病毒的全基因组或部分基因组序列并进行序列测定。通过化学发光法检测流感病毒对NAI类抗病毒药物(oseltamivir,奥司他韦)的敏感性,并分析病毒M2蛋白序列判断病毒对离子通道阻滞剂,即烷胺类药物的敏感性。通过MEGA5.0软件对测得甲型流感病毒全基因组序列进行生物信息学分析,对青岛地区甲型流感病毒的进化规律、耐药株产生原因及分子变异特点进行初步研究；并对青岛地区季节性H3N2流感病毒与全球随机抽取的该亚型病毒HA序列进行进化分析以探讨青岛地区毒株的国际代表性。本研究共得出以下结论：
1.本研究于2005-2011年间在青岛地区共采集和监测2560份鼻咽拭子标本,检出245份流感病毒阳性样品,据此了解到近6年来青岛地区季节性H3N2、季节性H1N1、大流行H1N1和乙型流感流行概况：除2007-2008季以乙型为主外,甲型流感病毒是青岛地区流感的主要流行型别。2009新甲型H1N1流感大流行前,基本呈现季节性H1N1、季节性H3N2流感病毒此消彼长的交替流行；新甲型H1N1流感大流行后,季节性H1N1再未分离到,而呈现季节性H3N2、新甲型H1N1共流行态势；本研究同时测定了部分甲型流感病毒全基因组或部分基因组序列,所得数据对流感的防控工作具有重要的指导意义,并可为我国流感病毒的分子流行病学研究提供重要信息。
2.2009年新甲型H1N1流感大流行时开展紧急监测,及时跟踪和监视当时疫情状态,并对大流行应对决策制定和风险分析提供支持。对检出的48株来自输入性病例和本土病例的新甲型H1N1流感病毒进行分子流行病学研究,发现本土病例中存在一种HA S128P变异株；进而比对GenBank录入的新甲型H1N1流感病毒序列发现,该变异株于2009年在包括中国、俄国、蒙古及韩国在内的欧亚地区异常传播,在2010年后则随新甲型H1N1流感病毒活动消减而遭淘汰；突变株的“一过性”传播优势提示我们现有知识尚不足以预测流感病毒发展及进化轨迹,唯有继续加强流感病毒监测才可能及时发现突变株以减小病毒所致危害,同时病毒的基因变异、尤其全基因组变异及进化尚需更深入的研究。此外,本研究发现流感大流行期间存在不同型别病毒间共感染情况,提示须警惕不同型别流感病毒发生重配的可能。新甲型H1N1流感大流行期间这一靶向重点人群的紧急监测策略,对于某地区针对未来可能发生的急性呼吸道传染性疾病流行建立早期预警系统具有重要指导意义。
3.病毒M2蛋白序列分析发现青岛地区2005-2011年间流行的甲型流感病毒中,除6株奥司他韦耐药季节性H1N1流感病毒外,均在M2基因发生耐药相关的S31N突变,即为烷胺类耐药毒株；NAI敏感性试验发现,目前青岛地区流行的季节性H3N2亚型、新甲型H1N1亚型流感病毒均无奥司他韦耐药性；季节性H1N1流感病毒自2008-2009季出现28.6%比例的耐药株,至2009-2010季耐药株比例升至50%。但2010年以来再未分离到季节性H1N1亚型流感病毒,故NAI类药物对目前流行的流感病毒尚敏感。以上数据表明NAI类药物应作为目前流感临床及预防性用药的首选。此外,我们同时发现了NAI和烷胺类药物双重耐药季节性H1N1流感毒株,该双重耐药株的出现应使我们提高警惕,因其可使现有治疗方案完全失效。
4.季节性H1N1流感病毒序列分析表明：病毒抗原变异与病毒流行直接相关,2008-2009季病毒较2005-2006流行季病毒发生多个基因多处位点的突变,使得新生病毒在时隔两个流行季后再次暴发。2009-2010流行季仅在2009年8月底、9月初分离到4株季节性H1N1亚型病毒,病毒较上一流行季毒株未有新增突变发生,故未引起持续的流行,且2010-2011季再无该亚型病毒分离。NAI耐药株除具有NA蛋白H275Y突变外,尚在多个基因节段具有“搭车”突变,这些突变虽不足以提供病毒对NAI药物的耐受性,但它们很可能弥补了NA蛋白H275Y这一突变对病毒生存及传播能力的影响,促进了耐药突变株的生存及广泛传播。
5.季节性H1N1、H3N2流感病毒全基因组进化分析发现,两种病毒均存在某一流行季内基因来源不同的两系毒株并存及基因组重配现象,而且2009-2010季的季节性H1N1流感病毒、2010-2011季的季节性H3N2流感病毒基因组全部涉及基因组重配事件,提示重配乃病毒进化的主要方式之一；某一季的优势株常常并非由上一季优势株直接进化产生,而是来自外源基因库的引入；季节性H1N1流感病毒耐药性的产生不仅仅需要NA蛋白H275Y变异和多个基因多处位点的附加变异,还涉及到全基因组的重配事件；青岛地区季节性H1N1流感病毒的进化早于国际疫苗株,则青岛地区毒株有条件作为疫苗候选株；青岛地区2006-2008年季节性H3N2流感病毒两个表面抗原HA和NA基因均与2008-2009季的疫苗推荐株A/Brisbane/10/2007(H3N2)遗传相近,而与当季疫苗株A/Wisconsin/67/2005(H3N2)遗传距离较远,提示可能存在疫苗失匹配现象。
6.对青岛地区季节性H3N2流感病毒与全球随机抽取的该亚型病毒HA序列进行进化分析发现,青岛地区流行的毒株具有国际代表性,因此青岛地区毒株可作为全球疫苗推荐株,青岛可作为国际流感监测哨点,在本地区开展流感监测对及时掌握全国乃至全球流感的流行和变异情况具有重要意义。
总之,流感的监测网络,尤其暴发流行期间针对目标人群的紧急监测对于急性呼吸道传染性疾病的防控极为重要。病毒的分子变异与每个流行季的病毒流行情况直接相关。NAI类药物应作为目前流感临床及预防性用药的首选,但同时应警惕NAI和烷胺类药物双重耐药流感毒株的出现。基因组重配频繁发生,为流感病毒进化的主要方式之一,流感病毒NAI耐药毒株的出现及广泛传播即不仅仅需要NA蛋白H275Y变异,还涉及到全基因组的重配事件及多个基因多处位点的附加变异。青岛地区流行的毒株具有国际代表性,因而可作为全球疫苗推荐株。尽管由于甲型流感病毒永久存在于野生水禽中致使人类难以彻底消除该病原,但对于甲型流感病毒进化动态的深入了解可以提高我们对它的监测能力,并有可能预计其进化轨迹进而选择合适流感疫苗株。本研究对甲型流感病毒的进化研究具有科学意义,为更有效的流感疫苗研制和流感的防控策略提供了科学依据。
Influenza viruses belong to the Orthomyxoviridae family. They are the major respiratory pathogens that can cause amount of morbidity and mortality worldwide. Three types of influenza viruses, influenza A, B, and C, could be identified based on the different antigenic characters of matrix protein (M) and nuclear protein (NP). Influenza viruses infect5%～15%of the global population annually, resulting in～500,000deaths.Among these, influenza A virus are characterized by high mutation rates with consequent antigenic changes, thus cause epidemics even pandemics through immune escape. Influenza A virus could be further divided into different subtypes, based on antigenic differences in the two main surface glycoproteins:the hemagglutinin (HA) and neuraminidase (NA). Currently16HA and9NA subtypes of influenza A virus have been identified, while three subtypes, seasonal A/H1N1, seasonal A/H3N2, and2009pandemic influenza A virus establishes transmission on humans at present.
Based on the knowledge obtained through studies carried over half a century, two classes of antiviral agents that block the M2ion channel and neuraminidase activity have been developed to treat infection, and a trivalent influenza vaccine is updated twice annually to account for frequent antigenic changes. However, the rapid and unpredictable evolutionary dynamics of the influenza A virus result in (a) sporadic extreme antigenic changes associated with severe disease and vaccine-epidemic mismatches,(b) the proliferation of new variants that are resistant to antiviral agents, and (c) occasional lethal infection of humans with avian influenza A viruses. The evolutionary basis for these events is still not well understood. While all the institutes are focusing on the capacity of avian influenza A viruses to cause pandemics in humans, during April2009a novel A(H1N1) influenza virus emerged in Mexico and since then has spread worldwide.
As the influenza A virus poses a continual, but unpredictable threat to human health, the evolution of the influenza A virus is of particular interest. The extensive genetic diversity is largely produced by the genomic reassortment between two genetically distinct viruses that co-infect a host cell. A particularly notable reassortment event was associated with one of the most important influenza epidemics of the twentieth century, the major epidemic of1947. These findings confirm the importance of studying influenza A virus evolution at a whole-genome level, taking into consideration genomic reassortment and the critical evolutionary dynamics of genes other than the main surface antigen HA.
In the present study, pharyngeal swab samples were collected from influenza-like illness (ILI) patients treated at three Qingdao sentinel hospitals during2005～2011for routine influenza surveillance, while an emergency surveillance targeting four defined groups were carried out during the2009influenza pandemic. Whole-or part-genome viral sequences were amplified by reverse-transcription polymerase chain reaction and determined. A chemical luminal method was applied to determine the virus sensitivity to oseltamivir, a kind of NAI antiviral drug, and the M2amino acid sequences were analyzed to determine the virus sensitivity to ion channel blockers, adamantanes. MEGA5.0software was explored to perform bioinformatic analysis on the genome sequences, thus constitute a primary study on the evolution disciplines, drug-resistant mechanism, and characters of molecular changes of influenza A virus in Qingdao. Furthermore, to investigate if the Qingdao influenza virus have an international representation, a phylogenetic analysis was performed on the HA sequences of seasonal H3N2virus from Qingdao and those randomly selected from the whole world. Six major conclusions were drawn as following:
1. A total of2560pharyngeal swab samples were collected in Qingdao during2005～2011,245of which were tested as influenza virus positive. The general situation of influenza prevalence in Qingdao during the recent6years was thus illuminated:except for the2007-2008epidemic season, which presented an influenza B virus prevalence, influenza A virus was generally predominant in Qingdao over the recent years. Seasonal HlNl and H3N2influenza virus alternated with each other before the2009pandemic, while the co-circulation of seasonal H3N2and the pandemic H1N1virus was established after the pandemic, as seasonal H1N1virus haven't been isolated since then. Whole-or part-genome sequences of influenza A virus were meanwhile determined in the present study, and the obtained data should be of great significance for the prevention and control of influenza, as well as provide important information for the molecular epidemiology of influenza virus in China.
2. An emergency surveillance targeting four defined groups were carried out during the2009influenza pandemic, providing banking for the city to establish its coping strategy and risk evaluation for the pandemic. A mutant with S128P substitution in the viral HA gene was detected in the domestic pandemic virus, as compared with those isolated from the imported cases. Further analysis on the pandemic virus sequences available on the GenBank revealed the abnormal transmission of the mutant in a defined Eurasian area including China, Russia, Mongolia, and South Korea in2009. However, the mutant was subsequently eliminated in the next year together with the weakening of the pandemic virus activity. The transitional transmission advantage of the mutant virus revealed our insufficient knowledge in predicting viral development and evolution. Therefore, intensified influenza surveillance should be applied for mutant searching as to possibly reduce viral damages, and further studies should be taken on the gene mutation, especially the whole-genome mutation of influenza virus. Moreover, co-circulation of pandemic virus and seasonal influenza virus was determined, which could facilitate reassortment between different subtypes. This emergency surveillance targeting defined risk groups should be of significance for the city to establish its early detection system for acute respiratory diseases in the future.
3. All the influenza A virus isolated in Qingdao during2005～2011was adamantine-resistant, as they all present the S31N substitution in M2,except for the six NAI-resistant seasonal A/H1N1influenza viruses in2008～2009. NAI sensitivity analysis showed that, seasonal H3N2, pandemic H1N1viruses were all oseltamivir-sensitive. The resistant viruses emerged in seasonal H1N1influenza A virus with a ratio of28.6%in Qingdao during the2008～2009season, and quickly reached50%during the2009～2010season. As the seasonal H1N1influenza viruses haven't been isolated since2010, NAI antivial agents are still potent to influenza virus prevalent at present, thus should be the preferred drugs for prophylactic or clinical application. Besides, the emerging of double-resistant virus to NAI and adamantine agents should arouse more attention, as the current treatments have no effects on it.
4. Sequence analysis revealed a direct relation between the viral antigenic changes and virus prevalence. Compared with those isolated in2005～2006season, multiple mutations in different viral genes was detected in virus of2008～2009season, which facilitate the virus breakout after a two-year interval. Only4seasonal H1N1influenza isolates were obtained during2009-2010season, all collected within late August and the beginning of September in2009. These viruses present no new mutations compared with those isolated in the last season thus didn't cause a sustaining prevalence, and didn't exist any longer in2010-2011. Besides the H275Y mutation in NA, the NAI-resistant virus also present multiple "hitch-hiking" substitutions, which could make up the compromised replication and transmission of the H275Y mutant, and facilitate the survival and wide transmission of the drug resistant virus.
5. Whole-genome evolution analysis of seasonal H1N1and H3N2influenza A virus revealed the co-circulation of multiple clades of the same subtype and the genome reassoitment events. All the seasonal H1N1influenza virus during2009～2010, and the H3N2virus during2010～2011are evolved with genome reassortment, indicating reassortment as one of the major evolution patterns of influenza virus. The viruses circulating in Qingdao in a given season tend to derive from newly imported genetic material rather than from isolates circulating in Qingdao in the previous season. The NAI-resistant seasonal H1N1virus emerged through a genomic reassortment in addition to the NA H275Y and other additional mutations. Influenza viruses in Qingdao could be served as vaccine candidates, as they evolved earlier than their counterparts from worldwide. On both the HA and NA phylogenies, Qingdao H3N2influenza virus during2006～2008were genetically close to A/Brisbane/10/2007isolate selected for the2008～2009vaccine, other than the2006～2007influenza vaccine strain A/Wisconsin/67/2005, indicating a vaccine mismatch.
6. Phylogenetic analysis performed on the HA sequences of Qingdao seasonal H3N2viruses and those randomly selected from the whole world revealed an international representation of the Qingdao isolates, which confirming the possibility of Qingdao influenza viruses as vaccine candidate. Furthermore, it's reasonable to set Qingdao as an international surveillance sentinel for influenza, which could be of great significance for the national, even worldwide monitoring of the influenza prevalence and changes.
Briefly, the influenza surveillance network, especially the emergency surveillance targeting defined groups during pandemic is of great significance for the coping method to acute respiratory diseases. Viral antigenic changes are directly associated to virus prevalence. NAI antiviral agents should be the preferred drugs for prophylactic or clinical application on influenza viruses. Genome reassortment occurs frequently as one of the major evolution patterns of influenza virus. The NAI-resistant influenza virus emerged through a genomic reassortment in addition to the NA H275Y and other additional mutations. Qingdao influenza viruses could be served as vaccine candidate owing to their international representation. Although influenza A virus can probably never be globally eradicated due to the permanent viral reservoir maintained in wild waterfowl, a greater understanding of the evolutionary dynamics of the influenza A virus will improve our capacity to effectively target global surveillance and potentially to predict evolutionary trajectories for appropriate selection of influenza vaccine strains. The present study could be of great scientific significance for further study on the evolution of influenza A virus, as well as provide foundation for the development of more effective influenza vaccine and strategy for prevention and controling of the virus.
基于半个多世纪以来的研究成果,现已发展出M2离子通道阻滞剂和神经氨酸酶活性抑制剂(neuraminidase inhibitor, NAI)两类抗病毒药物,而预防感染的三价流感病毒疫苗须每年两次更新候选疫苗株以应对病毒频繁的抗原变异。然而,甲型流感病毒迅速而又无法预期的动态进化仍可导致以下现象发生：(1)间或出现的重大抗原变异导致严重疾病及疫苗株-流行株失匹配；(2)可抵抗现有抗病毒药物的新变异株的增殖；(3)偶见致死性人感染甲型禽流感病毒病例。这些事件发生的进化机制尚未明确。而就在全世界流感监测及研究机构都聚焦于甲型禽流感病毒跨宿主致哺乳动物感染,并可能在人群导致新的流感暴发流行时,2009年4月一株“全新”的甲型H1N1流感病毒感染在墨西哥暴发,并迅速波及全球。
由于甲型流感病毒可对人类健康造成持续、而又无法准确预期的威胁,因此甲型流感病毒的进化一直是国内外学者的研究热点。甲型流感病毒基因具有多态性,很大程度上来源于共感染同一宿主细胞的两株遗传距离可能较远的病毒可以发生重配。上世纪最主要的几次大流行之一,1947年的流感大流行即与病毒的重配事件有关,而新世纪首次流感暴发更是来源于一株经过多次基因重配产生的抗原转变株。因此,对甲型流感病毒进行全基因组水平进化研究,全面考虑基因组重配事件及各基因节段的进化动态,比仅仅针对主要表面抗原HA的分子进化研究更为重要。
本研究于2005-2011年间采集青岛地区哨点医院流感样病例(influenza-like illness, ILI)鼻咽拭子标本开展流感监测,并采用细胞培养法分离流感毒株；2009年新甲型H1N1流感暴发期间开展紧急监测,采集4个目标人群鼻咽拭子标本进行核酸检测以监控病毒的流行形势。反转录聚合酶链反应(reverse-transcription polymerase chain reaction, RT-PCR)扩增甲型流感病毒的全基因组或部分基因组序列并进行序列测定。通过化学发光法检测流感病毒对NAI类抗病毒药物(oseltamivir,奥司他韦)的敏感性,并分析病毒M2蛋白序列判断病毒对离子通道阻滞剂,即烷胺类药物的敏感性。通过MEGA5.0软件对测得甲型流感病毒全基因组序列进行生物信息学分析,对青岛地区甲型流感病毒的进化规律、耐药株产生原因及分子变异特点进行初步研究；并对青岛地区季节性H3N2流感病毒与全球随机抽取的该亚型病毒HA序列进行进化分析以探讨青岛地区毒株的国际代表性。本研究共得出以下结论：
1.本研究于2005-2011年间在青岛地区共采集和监测2560份鼻咽拭子标本,检出245份流感病毒阳性样品,据此了解到近6年来青岛地区季节性H3N2、季节性H1N1、大流行H1N1和乙型流感流行概况：除2007-2008季以乙型为主外,甲型流感病毒是青岛地区流感的主要流行型别。2009新甲型H1N1流感大流行前,基本呈现季节性H1N1、季节性H3N2流感病毒此消彼长的交替流行；新甲型H1N1流感大流行后,季节性H1N1再未分离到,而呈现季节性H3N2、新甲型H1N1共流行态势；本研究同时测定了部分甲型流感病毒全基因组或部分基因组序列,所得数据对流感的防控工作具有重要的指导意义,并可为我国流感病毒的分子流行病学研究提供重要信息。
2.2009年新甲型H1N1流感大流行时开展紧急监测,及时跟踪和监视当时疫情状态,并对大流行应对决策制定和风险分析提供支持。对检出的48株来自输入性病例和本土病例的新甲型H1N1流感病毒进行分子流行病学研究,发现本土病例中存在一种HA S128P变异株；进而比对GenBank录入的新甲型H1N1流感病毒序列发现,该变异株于2009年在包括中国、俄国、蒙古及韩国在内的欧亚地区异常传播,在2010年后则随新甲型H1N1流感病毒活动消减而遭淘汰；突变株的“一过性”传播优势提示我们现有知识尚不足以预测流感病毒发展及进化轨迹,唯有继续加强流感病毒监测才可能及时发现突变株以减小病毒所致危害,同时病毒的基因变异、尤其全基因组变异及进化尚需更深入的研究。此外,本研究发现流感大流行期间存在不同型别病毒间共感染情况,提示须警惕不同型别流感病毒发生重配的可能。新甲型H1N1流感大流行期间这一靶向重点人群的紧急监测策略,对于某地区针对未来可能发生的急性呼吸道传染性疾病流行建立早期预警系统具有重要指导意义。
3.病毒M2蛋白序列分析发现青岛地区2005-2011年间流行的甲型流感病毒中,除6株奥司他韦耐药季节性H1N1流感病毒外,均在M2基因发生耐药相关的S31N突变,即为烷胺类耐药毒株；NAI敏感性试验发现,目前青岛地区流行的季节性H3N2亚型、新甲型H1N1亚型流感病毒均无奥司他韦耐药性；季节性H1N1流感病毒自2008-2009季出现28.6%比例的耐药株,至2009-2010季耐药株比例升至50%。但2010年以来再未分离到季节性H1N1亚型流感病毒,故NAI类药物对目前流行的流感病毒尚敏感。以上数据表明NAI类药物应作为目前流感临床及预防性用药的首选。此外,我们同时发现了NAI和烷胺类药物双重耐药季节性H1N1流感毒株,该双重耐药株的出现应使我们提高警惕,因其可使现有治疗方案完全失效。
4.季节性H1N1流感病毒序列分析表明：病毒抗原变异与病毒流行直接相关,2008-2009季病毒较2005-2006流行季病毒发生多个基因多处位点的突变,使得新生病毒在时隔两个流行季后再次暴发。2009-2010流行季仅在2009年8月底、9月初分离到4株季节性H1N1亚型病毒,病毒较上一流行季毒株未有新增突变发生,故未引起持续的流行,且2010-2011季再无该亚型病毒分离。NAI耐药株除具有NA蛋白H275Y突变外,尚在多个基因节段具有“搭车”突变,这些突变虽不足以提供病毒对NAI药物的耐受性,但它们很可能弥补了NA蛋白H275Y这一突变对病毒生存及传播能力的影响,促进了耐药突变株的生存及广泛传播。
5.季节性H1N1、H3N2流感病毒全基因组进化分析发现,两种病毒均存在某一流行季内基因来源不同的两系毒株并存及基因组重配现象,而且2009-2010季的季节性H1N1流感病毒、2010-2011季的季节性H3N2流感病毒基因组全部涉及基因组重配事件,提示重配乃病毒进化的主要方式之一；某一季的优势株常常并非由上一季优势株直接进化产生,而是来自外源基因库的引入；季节性H1N1流感病毒耐药性的产生不仅仅需要NA蛋白H275Y变异和多个基因多处位点的附加变异,还涉及到全基因组的重配事件；青岛地区季节性H1N1流感病毒的进化早于国际疫苗株,则青岛地区毒株有条件作为疫苗候选株；青岛地区2006-2008年季节性H3N2流感病毒两个表面抗原HA和NA基因均与2008-2009季的疫苗推荐株A/Brisbane/10/2007(H3N2)遗传相近,而与当季疫苗株A/Wisconsin/67/2005(H3N2)遗传距离较远,提示可能存在疫苗失匹配现象。
6.对青岛地区季节性H3N2流感病毒与全球随机抽取的该亚型病毒HA序列进行进化分析发现,青岛地区流行的毒株具有国际代表性,因此青岛地区毒株可作为全球疫苗推荐株,青岛可作为国际流感监测哨点,在本地区开展流感监测对及时掌握全国乃至全球流感的流行和变异情况具有重要意义。
总之,流感的监测网络,尤其暴发流行期间针对目标人群的紧急监测对于急性呼吸道传染性疾病的防控极为重要。病毒的分子变异与每个流行季的病毒流行情况直接相关。NAI类药物应作为目前流感临床及预防性用药的首选,但同时应警惕NAI和烷胺类药物双重耐药流感毒株的出现。基因组重配频繁发生,为流感病毒进化的主要方式之一,流感病毒NAI耐药毒株的出现及广泛传播即不仅仅需要NA蛋白H275Y变异,还涉及到全基因组的重配事件及多个基因多处位点的附加变异。青岛地区流行的毒株具有国际代表性,因而可作为全球疫苗推荐株。尽管由于甲型流感病毒永久存在于野生水禽中致使人类难以彻底消除该病原,但对于甲型流感病毒进化动态的深入了解可以提高我们对它的监测能力,并有可能预计其进化轨迹进而选择合适流感疫苗株。本研究对甲型流感病毒的进化研究具有科学意义,为更有效的流感疫苗研制和流感的防控策略提供了科学依据。
Influenza viruses belong to the Orthomyxoviridae family. They are the major respiratory pathogens that can cause amount of morbidity and mortality worldwide. Three types of influenza viruses, influenza A, B, and C, could be identified based on the different antigenic characters of matrix protein (M) and nuclear protein (NP). Influenza viruses infect5%～15%of the global population annually, resulting in～500,000deaths.Among these, influenza A virus are characterized by high mutation rates with consequent antigenic changes, thus cause epidemics even pandemics through immune escape. Influenza A virus could be further divided into different subtypes, based on antigenic differences in the two main surface glycoproteins:the hemagglutinin (HA) and neuraminidase (NA). Currently16HA and9NA subtypes of influenza A virus have been identified, while three subtypes, seasonal A/H1N1, seasonal A/H3N2, and2009pandemic influenza A virus establishes transmission on humans at present.
Based on the knowledge obtained through studies carried over half a century, two classes of antiviral agents that block the M2ion channel and neuraminidase activity have been developed to treat infection, and a trivalent influenza vaccine is updated twice annually to account for frequent antigenic changes. However, the rapid and unpredictable evolutionary dynamics of the influenza A virus result in (a) sporadic extreme antigenic changes associated with severe disease and vaccine-epidemic mismatches,(b) the proliferation of new variants that are resistant to antiviral agents, and (c) occasional lethal infection of humans with avian influenza A viruses. The evolutionary basis for these events is still not well understood. While all the institutes are focusing on the capacity of avian influenza A viruses to cause pandemics in humans, during April2009a novel A(H1N1) influenza virus emerged in Mexico and since then has spread worldwide.
As the influenza A virus poses a continual, but unpredictable threat to human health, the evolution of the influenza A virus is of particular interest. The extensive genetic diversity is largely produced by the genomic reassortment between two genetically distinct viruses that co-infect a host cell. A particularly notable reassortment event was associated with one of the most important influenza epidemics of the twentieth century, the major epidemic of1947. These findings confirm the importance of studying influenza A virus evolution at a whole-genome level, taking into consideration genomic reassortment and the critical evolutionary dynamics of genes other than the main surface antigen HA.
In the present study, pharyngeal swab samples were collected from influenza-like illness (ILI) patients treated at three Qingdao sentinel hospitals during2005～2011for routine influenza surveillance, while an emergency surveillance targeting four defined groups were carried out during the2009influenza pandemic. Whole-or part-genome viral sequences were amplified by reverse-transcription polymerase chain reaction and determined. A chemical luminal method was applied to determine the virus sensitivity to oseltamivir, a kind of NAI antiviral drug, and the M2amino acid sequences were analyzed to determine the virus sensitivity to ion channel blockers, adamantanes. MEGA5.0software was explored to perform bioinformatic analysis on the genome sequences, thus constitute a primary study on the evolution disciplines, drug-resistant mechanism, and characters of molecular changes of influenza A virus in Qingdao. Furthermore, to investigate if the Qingdao influenza virus have an international representation, a phylogenetic analysis was performed on the HA sequences of seasonal H3N2virus from Qingdao and those randomly selected from the whole world. Six major conclusions were drawn as following:
1. A total of2560pharyngeal swab samples were collected in Qingdao during2005～2011,245of which were tested as influenza virus positive. The general situation of influenza prevalence in Qingdao during the recent6years was thus illuminated:except for the2007-2008epidemic season, which presented an influenza B virus prevalence, influenza A virus was generally predominant in Qingdao over the recent years. Seasonal HlNl and H3N2influenza virus alternated with each other before the2009pandemic, while the co-circulation of seasonal H3N2and the pandemic H1N1virus was established after the pandemic, as seasonal H1N1virus haven't been isolated since then. Whole-or part-genome sequences of influenza A virus were meanwhile determined in the present study, and the obtained data should be of great significance for the prevention and control of influenza, as well as provide important information for the molecular epidemiology of influenza virus in China.
2. An emergency surveillance targeting four defined groups were carried out during the2009influenza pandemic, providing banking for the city to establish its coping strategy and risk evaluation for the pandemic. A mutant with S128P substitution in the viral HA gene was detected in the domestic pandemic virus, as compared with those isolated from the imported cases. Further analysis on the pandemic virus sequences available on the GenBank revealed the abnormal transmission of the mutant in a defined Eurasian area including China, Russia, Mongolia, and South Korea in2009. However, the mutant was subsequently eliminated in the next year together with the weakening of the pandemic virus activity. The transitional transmission advantage of the mutant virus revealed our insufficient knowledge in predicting viral development and evolution. Therefore, intensified influenza surveillance should be applied for mutant searching as to possibly reduce viral damages, and further studies should be taken on the gene mutation, especially the whole-genome mutation of influenza virus. Moreover, co-circulation of pandemic virus and seasonal influenza virus was determined, which could facilitate reassortment between different subtypes. This emergency surveillance targeting defined risk groups should be of significance for the city to establish its early detection system for acute respiratory diseases in the future.
3. All the influenza A virus isolated in Qingdao during2005～2011was adamantine-resistant, as they all present the S31N substitution in M2,except for the six NAI-resistant seasonal A/H1N1influenza viruses in2008～2009. NAI sensitivity analysis showed that, seasonal H3N2, pandemic H1N1viruses were all oseltamivir-sensitive. The resistant viruses emerged in seasonal H1N1influenza A virus with a ratio of28.6%in Qingdao during the2008～2009season, and quickly reached50%during the2009～2010season. As the seasonal H1N1influenza viruses haven't been isolated since2010, NAI antivial agents are still potent to influenza virus prevalent at present, thus should be the preferred drugs for prophylactic or clinical application. Besides, the emerging of double-resistant virus to NAI and adamantine agents should arouse more attention, as the current treatments have no effects on it.
4. Sequence analysis revealed a direct relation between the viral antigenic changes and virus prevalence. Compared with those isolated in2005～2006season, multiple mutations in different viral genes was detected in virus of2008～2009season, which facilitate the virus breakout after a two-year interval. Only4seasonal H1N1influenza isolates were obtained during2009-2010season, all collected within late August and the beginning of September in2009. These viruses present no new mutations compared with those isolated in the last season thus didn't cause a sustaining prevalence, and didn't exist any longer in2010-2011. Besides the H275Y mutation in NA, the NAI-resistant virus also present multiple "hitch-hiking" substitutions, which could make up the compromised replication and transmission of the H275Y mutant, and facilitate the survival and wide transmission of the drug resistant virus.
5. Whole-genome evolution analysis of seasonal H1N1and H3N2influenza A virus revealed the co-circulation of multiple clades of the same subtype and the genome reassoitment events. All the seasonal H1N1influenza virus during2009～2010, and the H3N2virus during2010～2011are evolved with genome reassortment, indicating reassortment as one of the major evolution patterns of influenza virus. The viruses circulating in Qingdao in a given season tend to derive from newly imported genetic material rather than from isolates circulating in Qingdao in the previous season. The NAI-resistant seasonal H1N1virus emerged through a genomic reassortment in addition to the NA H275Y and other additional mutations. Influenza viruses in Qingdao could be served as vaccine candidates, as they evolved earlier than their counterparts from worldwide. On both the HA and NA phylogenies, Qingdao H3N2influenza virus during2006～2008were genetically close to A/Brisbane/10/2007isolate selected for the2008～2009vaccine, other than the2006～2007influenza vaccine strain A/Wisconsin/67/2005, indicating a vaccine mismatch.
6. Phylogenetic analysis performed on the HA sequences of Qingdao seasonal H3N2viruses and those randomly selected from the whole world revealed an international representation of the Qingdao isolates, which confirming the possibility of Qingdao influenza viruses as vaccine candidate. Furthermore, it's reasonable to set Qingdao as an international surveillance sentinel for influenza, which could be of great significance for the national, even worldwide monitoring of the influenza prevalence and changes.
Briefly, the influenza surveillance network, especially the emergency surveillance targeting defined groups during pandemic is of great significance for the coping method to acute respiratory diseases. Viral antigenic changes are directly associated to virus prevalence. NAI antiviral agents should be the preferred drugs for prophylactic or clinical application on influenza viruses. Genome reassortment occurs frequently as one of the major evolution patterns of influenza virus. The NAI-resistant influenza virus emerged through a genomic reassortment in addition to the NA H275Y and other additional mutations. Qingdao influenza viruses could be served as vaccine candidate owing to their international representation. Although influenza A virus can probably never be globally eradicated due to the permanent viral reservoir maintained in wild waterfowl, a greater understanding of the evolutionary dynamics of the influenza A virus will improve our capacity to effectively target global surveillance and potentially to predict evolutionary trajectories for appropriate selection of influenza vaccine strains. The present study could be of great scientific significance for further study on the evolution of influenza A virus, as well as provide foundation for the development of more effective influenza vaccine and strategy for prevention and controling of the virus.
引文
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