摘要
急性呼吸道感染(Acute respiratory tract infection, ARTI)是世界范围内人类最常见疾病原因之一,可以引起以发热为主的流感样症状疾病(Influenza like illness,ILI),是导致儿童死亡的第二大病因。儿童中高达90%的肺炎是由病毒引起。由于传播途径的特殊性,呼吸系统病毒感染易在人群中传播。自2001年以来,一系列新发现病原体或者重组病原体与急性呼吸系统感染相关。因此,开展以ILI为主的症状监测研究将为明确我国呼吸系统病毒性病原谱构成、病原体变异规律乃至为新病原体发现提供依据。
本研究在我国北京、天津、重庆、山东、陕西、河南等地区开展了呼吸系统病毒性病原体监测,包括甲型、乙型、丙型(Influenza A、B、C,Flu-A、-B、-C)流感病毒、副流感病毒I-IV型(Parainfluenza I~IV,PIV-I~PIV-IV)、腺病毒(Adenovirus,ADV)、冠状病毒229E和OC43(Coronavirus-229E,CoV-229E;Coronavirus-OC43,CoV-OC43)、呼吸道合胞病毒(Respiratory syncytial viruses -A、-B,RSV-A、-B)、偏肺病毒(Metapneumovirus,MPV)以及博卡病毒(Bocavirus,BoV)等,分析了呼吸道病毒性病原体感染的流行病学特征,探索了流感病毒和冠状病毒变异规律。
主要研究结果:
1.我国部分地区常见呼吸系统病毒性病原体监测研究
我国北京、天津、重庆、陕西、河南等地区采用哨点医院监测和实验研究相结合的方法进行常见呼吸系统病毒性病原体监测研究,研究纳入ILI病例3865人次,月平均为160人次,ILI病例从每年9月份开始上升,持续到次年的2-3月份。2009年4月-2011年3月间共检出至少有一种呼吸道系统病毒阳性病例1463人次,阳性检出率为37.85%(1463/3865)。呼吸系统病毒性病原体最为常见的是Flu(检出率为21.57%,834/3865),其余依次为PIV(6.65%,257/3865)、ADV(6.57%,245/3865)、RSV(5.46%,211/3865)、BoV(5.02%,194/3865)、MPV(2.25%,87/3865)和CoV(1.81%,70/3865)。呼吸系统病毒病原体复合感染为8.82%(341/3865),占所有感染病例的比例为23.30%(341/1463)。复合感染病例中,75.07%(256/341)为两种病毒复合感染,19.94%(68/341)为三种病毒感染、4.99%(17/341)为四种及以上病毒复合感染。最常见复合感染是Flu和ADV、ADV和PIV、BoV和RSV。最易发生复合感染的病原体是MPV和BoV,复合感染比例分别占MPV和BoV阳性检出标本80.46%(70/87)和65.46%(127/194)。
呼吸系统病毒性感染有明显年龄分布特征,无性别分布差异。儿童ILI病例呼吸系统病毒检出率(62.08%,650/1047)显著高于成人(29.05%,700/2410)(P<0.01)。儿童感染中以0-5岁儿童检出率最高(69.62%,550/790),且有随年龄增加而降低趋势(P<0.01)。儿童复合感染率(26.36%,276/1047)显著高于成人(2.45%,59/2410)(P<0.01)。呼吸系统病毒性感染四季均有发生,不同病原体具有不同季节流行趋势。一年中各病原体发病高峰分别是:PIV、ADV和MPV在9月份,RSV在10月份,BoV在11月中旬,Flu在12月份,CoV在1月底。
ILI病例最为常见症状为咳嗽(72%),其余症状依次为咳痰(39%)、咽喉痛(35%)、流涕(30%)、头痛(22%)、乏力(18%)、肌肉酸痛(14%)、腹泻(10%)、呼吸困难(6%)、胸痛(5%)及腹痛(3%)等。呼吸系统病毒感染较常见的症状为咳嗽、咳痰、流涕等;而阴性者更加常见的症状为流涕、咽喉痛、咳嗽等。
单因素分析结果显示年龄、疾病既往史、就诊前的初步治疗或服药情况以及从发病到就诊的时间间隔等因素与病毒检出相关。其中,年龄增加可降低呼吸系统病毒性感染检出(OR=0.97,95%CI 0.97-0.98)。具有以往疾病史、就诊前进行了初步治疗或服药以及发病后就诊的时间间隔长等因素可能降低呼吸道病毒检出(P<0.01)。调整其它因素后,仅年龄和发病后的就诊时间间隔与呼吸系统病毒检出相关。与呼吸系统病毒检出的因素在不同感染数量(单纯感染和复合感染)、不同人群(成人和儿童)以及不同病原体中的分布有所不同。2.我国猪流感的血清流行病学和遗传特征分析研究
通过文献汇总对我国猪流感的血清流行病学状况进行分析,利用流感基因数据库对我国猪群流感进行遗传特征研究。结果显示我国猪群流感血清型有H1、H3、H5、H7和H9亚型,以H1亚型和H3亚型为主。H1亚型血清抗体水平(32.55%,3402/10451)高于H3亚型(28.60%,2900/10139)(P<0.01)。H1血清型阳性率在不同的地区有显著差别(P<0.01),其中华南地区最高、华中地区最低。H3型不同地区阳性率总体无显著差别。不同猪群养殖密度猪群流感抗体水平不同。高密度猪群养殖区H1血清抗体水平明显高于中、低密度养殖区(P=0.02和P=0.04)。
基因序列分析显示我国报道猪流感基因序列与2009年暴发的甲型H1N1流感(pH1N1)片段之间为中度相似,尤其与我国华东和华南地区的猪流感基因片段相似度更高。
3.我国部分地区2009年4月-2011年3月间流感病原学监测
在常见病毒性病原体监测时,恰逢pH1N1流感爆发,为此本研究在北京、天津、重庆、山东、陕西、河南专门进行了流感监测。纳入流感监测ILI病例数为6143人次,月平均为255人次。2009-2010流感流行期间ILI病例从每年8月份开始上升,持续到次年的3月份,2010-2011流感流行期间ILI病例进入11月份开始明显增加,持续到次年1月左右。
流感阳性检出率为26.78%(1645/6143),其中pH1N1占31.98%(526/1645)、季节性H3N2(sH3N2)占41.76%(687/1645)、季节性H1N1(sH1N1)占1.88%(31/1645)、A型未分型占3.22%(53/1645)、B型占21.15%(348/1645)。2009-2010流感流行趋势与2010-2011流感流行不同。首先从发病幅度来看,2009-2010年的流感幅度总体高于2010-2011年度,前者最高检出率为52.84%,而后者最高检出率为24.12%。从病原构成来看,2009-2010年流感病流行株有sH3N2、pH1N1、B型及sH1N1亚型;2010-2011年流感流行主要流行株有sH3N2、pH1N1、B型,sH1N1流行株非常少见。流感内部不同亚型的出现的时间顺序来看,2009-2010年首先出现流感A型sH1N1亚型,随后为sH3N2亚型,再后来为pH1N1流感,最后为流感B型;2010-2011年度首先出现sH3N2亚型,随后为pH1N1亚型,最后为B型。与2009-2010年流感季节相比,2010-2011年度sH1N1检出极低,pH1N1流行幅度也有较大降低,sH3N2亚型流行幅度无显著改变,B型流感的流行幅度却有升高的趋势。未见达菲(H274Y)耐药性株出现,但均为金刚烷胺耐药(S31N)。
不同(亚)型流感的性别分布不同,成人A型流感的男性构成高于B型流感(P<0.01)。不同(亚)型流感年龄分布不同,pH1N1检出率最高为10-14岁人群(16.04%,47/293)、sH3N2最高为30-39岁人群(14.93%,63/422)、sH1N1最高为70岁以上人群(2.63%,2/76)、B型最高为5-9岁人群(12.30%,61/496)。
流感感染后呼吸道症状依次为咳嗽(76%)、咽喉痛(47%)、咳痰(38%)、流涕(35%)、头痛(26%)、乏力(18%)、肌肉酸痛(12%)、腹泻(8%)、胸痛(5%)、呼吸困难(4%)及腹痛(2%)。成人和儿童中症状分布稍有不同。成人以咳嗽、咽喉痛、流涕、头痛等症状较常见,儿童以咳嗽、咳痰、腹泻、流涕等症状较常见。
因素分析结果显示年龄、治疗史与流感检出相关。不同流感(亚)型,这两个因素所起作用不同。发病后不进行前期治疗或处理,pH1N1检出率增加2.5倍,sH3N2增加1.7倍。B型流感不进行就诊前的治疗或处理却可减少该型病毒检出。同时,成人随年龄增加而增加sH3N2和sH1N1检出,儿童却会增加pH1N1检出。
4.季节性流感H3N2亚型在甲型H1N1流行期间的进化分析
季节性H3N2亚型流感在2009年4月-2010年7月之间的变异进化分析结果显示,sH3N2各个片段非同义替代碱基与同义替代碱基(dN/dS)比值<1,提示流感病毒没有正向选择压力存在。其中,PB1-F2基因片段、HA基因、NA基因、M2基因、NS基因变异度较高。
研究期间内sH3N2流感基因组不同基因片段的进化率不一致,NA基因变异率最高(4.30×10-3/碱基/年),最稳定的基因片段为NP和PB1。时间进化显示sH3N2流感株来源于2007年,但不同片段来源时间稍有区别。其中,PB1片段的来源时间最长,其起始时间约为2007年3月;NA基因能来源时间约为2007年11月。
重组分析结果显示,sH3N2流感8片段中仅PB1基因存在较高重组突变比(P=0.02),可能存在重组。重组发生在该基因40-821bp位置区域。未见其他基因片段重组现象。
5.我国部分地区冠状病毒的分子流行病学研究冠状病毒感染分子流行病学研究结果显示,冠状病毒感染以CoV-229E和CoV-OC43为主。Spike蛋白基因序列分析结果显示CoV-229E与我国2009年的分离株在同一个进化大枝上,但与2009年澳大利亚、美国、德国等报道的冠状病毒株更接近;CoV-OC43与2003年比利时和2006年法国报道株在一个大进化分支上。CoV-229E和CoV-OC43的dN/dS比值均小于0.5,但CoV-OC43的该指标更高。研究结果提示两种冠状病毒变异是在无选择压力下突变所致。同时,CoV-229E在1681位点(A561S)、2692位点(Y898H)出现两个有意义多态性位点;CoV-OC43在3073位点(D1024H)、3085位点(E1028Q)及3202位点(S1064A)出现三个有意义多态性位点。
CoV-229E的Spike蛋白信号肽区长度为16aa残基,CoV-OC43的Spike蛋白信号肽区长度为17aa残基。CoV-229E和CoV-OC43的Spike蛋白受体结合域(Receptor binding domain, RBD)均能够成功进行蛋白同源模建,CoV-229E模建蛋白能够与人血管紧张素转换酶-2(Angiotensin-convertion enzyme-2,ACE-2)结合,而CoV-OC43模建蛋白不能ACE-2与结合,这种差别可能是两种不同冠状病毒感染频率不同的原因之一。研究结论:
通过2009年4月-2011年3月间在我国部分地区开展的呼吸道病原体监测,获得以下研究结论:
1.我国部分地区的常见呼吸系统病毒性病原体依次为流感病毒、副流感病毒、腺病毒、呼吸道合胞病毒、博卡病毒、偏肺病毒和冠状病毒。呼吸系统病毒病原体感染常存在复合感染,以两种病原体复合感染为主,最常见复合感染是流感病毒和腺病毒、腺病毒和副流感病毒、博卡病毒和呼吸道合胞病毒。复合感染率最高的病原体是偏肺和博卡病毒。呼吸系统病毒性感染有明显的年龄分布特征,而无性别分布差异。不同呼吸系统病毒性感染季节分布不一致,各病原体发病高峰分别是:副流感病毒、腺病毒和偏肺病毒在9月份、呼吸道合胞病毒在10月份、博卡病毒在11月中旬、流感病毒在12月份、冠状病毒在1月底。呼吸系统病毒感染最常见症状为咳嗽、咳痰、流涕。影响呼吸道病毒检出的因素是年龄和发病后的就诊时间间隔。
2.1999-2007年间我国猪流感的血清型主要为H1和H3型,其中H1型血清分布有地区差别,并与猪群养殖密度有关。基因序列分析显示我国报道猪流感基因序列与2009年爆发的甲型H1N1流感(pH1N1)片段之间为中度相似,尤其与我国华东和华南地区的猪流感基因片段相似度更高。
3.2009年4月-2011年3月间我国部分地区流感病毒主要有季节性H3N2、甲型H1N1和B型流感。2009年的甲型H1N1暴发流行对流感流行趋势产生明显影响。2009-2010年与2010-2011年的流感流行趋势具有不同特征,流感流行强度、流感流行持续时间及不同亚型出现时间顺序均发生改变。流感不同亚型导致不同年龄人群临床症状不同。年龄和就诊前治疗史与不同流感亚型检出相关。
4.甲型H1N1流感流行期间,季节性流感H3N2亚型变异是随机、无选择压力存在下发生,但NA片段进化速度最快。该期间的流感病毒可能来源于2007年的相应流行株,来源时间为2007年3月-2007年11月间。季节性流感H3N2亚型PB1基因片段可能存在重组。
5.我国部分地区冠状病毒的主要流行株为CoV-229E和CoV-OC43,该两种冠状病毒变异是在无选择压力下突变所致。基因序列分析结果显示CoV-229E在1681位点(A561S)、2692位点(Y898H)出现两个有意义多态性位点。CoV-OC43在3073位点(D1024H)、3085位点(E1028Q)及3202位点(S1064A)出现三个有意义多态性位点。CoV-229E模建蛋白能够与人ACE-2结合,而CoV-OC43模建蛋白不能结合,这种差别可能是两种不同冠状病毒感染频率不同的原因之一。
Acute respiratory tract infections (ARTI) are among the most common infectious diseases of human worldwide, causing influenza like illnesses. ARTI is the secondary cause of mortality in children and nearly 90% of pneumonias among this population are caused by viral pathogens. Respiratory viral infections are easy to transmit in population because of their special route for transmission. A series of newly discovered or recombination pathogens had been confirmed to be related with respiratory infections since 2001. Influenza-like illness (ILI) definitions have been used worldwide for influenza surveillance. However, ILI may be difficult to distinguish influenza infection based on the symptoms alone from symptomatic infections caused by other respiratory viruses. Therefore, ILI is usually used for surveillance of respiratory infections. According to ILI surveillance, the spectrum of viral pathogens in respiratory infections could be described, and the evolutionary pattern of virus may be further highlight and even clues for novel pathogens discovery would be offered.
In this thesis, the most common respiratory viral pathogens, influenza A, B C(Flu-A, -B, -C), parainfluenza viruses 1-4 (PIV-1~IV), adenovirus(ADV), coronavirus-229E, -OC43 (CoV-229E, CoV-OC43), respiratory syncytial viruses-A, -B (RSV-A, -B), human metapneumovirus (MPV) and human bocavirus(BoV), were included in the surveillance for respiratory infectious diseases. The regions were Beijing, Tianjin, Chongqing, Shandong, Shanxi and Henan. In addition, clinical characteristics of viral infections, evolutionary dynamics of influenza and coronavirus were further studied based on the surveillance.
The main results:
1. Surveillance of respiratory viral pathogens in some regions of China During a two-year period of surveillance in six sentinel hospitals from Apr 2009 to Mar 2010, 3865 ILI cases (person-time) with monthly of 160 were included in this study. Surveillance data showed that ILI cases increased from September, lasting for 7 months, to March next year. The least detected positive rate for viral pathogen was 37.85% (1463/3865) and the most common virus was influenza with positive rate of 21.57%(834/3865). Following influenza were PIV(6.65%,257/3865), ADV(6.57%, 245/3865), RSV(5.46%, 211/3865), BoV(5.02%, 194/3865), MPV(2.25%, 87/3865) and CoV(1.81%,70/3865). Co-infections with more than two viruses, which accounted for 23.30%(341/1463) of all positive samples, were also observed (positive rate 8.82%,341/3865). Among co-infections, 75.07% (256/341) were two-virus, 19.94%(68/341) were triple-virus and 4.99%(17/341) were more than four viruses. Co-infections of Flu and ADV, ADV and PIV, BoV and RSV were the most common. MPV (80.46%, 70/87) and BoV (65.46%, 127/194) were more frequently detected with co-infections.
Epidemiological results showed that the viral positive rates were higher in children(62.08%, 650/1047) than in the adults(29.05%, 700/2410)(P<0.00). Among children, the group of less than 5-years old was more frequently detected with viruses infection (69.62%, 550/790) and there was a remarkable decrease trend with age in this group(P<0.00). In addition, co-infection rates were much higher in children (26.36%, 276/1041) than that of the adults (2.45%, 59/2410)(P<0.01).
Though there were yearly occurrences of respiratory viral infections, the seasonality was virus-dependent. For example, PIV, ADV and MPV reached their climax in September, RSV in October, BoV in the middle of November, Flu in December and CoV in the end of January.
For ILI cases, the symptoms were cough(72%), expectoration(39%), sore throat(35%), runny nose(30%), headache(22%), hypodynamia(18%), muscular soreness(14%), diarrhea(10%), dyspnea (6%), chest pain (5%) and bellyache(3%). However, in viral positive cases, cough, expectoration, and runny nose were more common compared with negative cases among which runny nose, sore throat and cough were more frequently seen.
Associated factors, which may affect the detection of viruses including age, disease histories, preclinical treatment of diseases and duration from onset to visit doctor, were studied and positive correlations were observed in univariate analysis. Opposite to age in which increase with decrease the detection rates(OR=0.97, 95% CI 0.97-0.98), disease histories, preclinical of diseases and duration from onset to visit doctor may lower the detection rates. However, only age and duration from onset to visit doctor displayed significantly affecting the detection rates when adjusted by other factors. And, the factors mentioned above may differ by infection type(single and coninfection), population (adults and children) and virus. 2. Seroprevalence and genetic characteristics of swine influenza viruses in Chinese swine population: a pooled data analysis.
A literature review and data pooling analysis were made on the antibodies prevalence against five influenza viruses subtypes in pigs in China for the last 10 years. The grand average of seropositive rates of subtypes H1, H3, H5, H7 and H9 were calculated as 32.55% (3402/10451), 28.60%(2900/10139), 1.20%(77/6392), 0%(0/1815) and 2.15% (86/3944), respectively. Moreever, there was a higher level of antibodies against H1 than against H3(P<0.01). There were large geographical variations in seroprevalence of subtypes -H1, with South and East China as the top regions with high seroprevalence in pigs. In addition, the region with high densities of pigs raising had higher seroprevalence than that of the regions with median and low densities (P=0.02, P=0.04).
BLAST analysis of genetic sequences revealed that genome segments with moderate homology to the 2009 pandemic influenza A (H1N1)(pH1N1) virus were present among swine influenza viruses isolated in China, especially in South and East China.
3. Influenza virologic surveillance during Apr 2009- Mar 2011 in some regions of China
In order to cope with ourbreaks of pH1N1, virologic surveillance for influenza was carried out during 2009-2011 in Beijing, Tianjin, Chonqging, Shandong, Henan and Shanxi. Based on respiratory viral surveillance, two more sentinel hospitals were added in this svrveillance and 6143 ILI cases (person-time) with monthly 255 cases (person-time) were included. ILI cases increased from August to March next year during 2009-2010 influenza season. However, in 2010-2011 season, ILI cases increased from November to January next year.
One thousand six hundred and forty five cases were diagnosed (26.78%,1645/6143) positive for influenza, in which pH1N1, seasonal H3N2(sH3N2), seasonal H1N1(sH1N1), untyped and influenza B accounted for 31.98%(526/1645), 41.76%(687/1645), 1.88%(31/1645), 3.22%(53/1645) and 21.15%(348/1645), respectively.
Different epidemic pattern was observed between 2009-2010 and 2010-2011 influenza season in this surveillance. Firstly, in 2009-2010 influenza season, the highest detected rate was 52.84%, obviously greater than that of the 2010-2011 season (24.12%). Secondly, in the 2009-2010 season, influenza viruses composed of sH3N2, pH1N1, influenza B and sH1N1. However, in 2010-2011 season, influenzas were mainly sH3N2, pH1N1, influenza B, and sH1N1 was less detected. Thirdly, in the 2009-2010 influenza season, the order of subtype scheduled as sH1N1, sH3N2, pH1N1 and influenza B and this pattern was changed with sH3N2, pH1N1 and influenza B in 2010-2011 season. Finally, no obvious changes of sH3N2 intensity in both seasons were observed though the epidemic strength of pH1N1 and sH1N1 lowered very much. In addition, no oseltamivir resistance mutations were observed according to drug resistance surveillance.
Epidemic data showed that the infections of the males were more frequently seen with influenza A than with influenza B. The age distribution was different among (sub)types of influenza, in which pH1N1 with the highest age specific rate in group of 10-14 years old, sH3N2 in 30-39 age group, sH1N1 in elders than 70 years and B in 5-9 age group.
For influenza cases, the symptoms were cough(76%), sore throat(47%), expectoration(38%), runny nose(35%), headache(26%), hypodynamia(18%), muscular soreness(12%), diarrhea(8%), chest pain (5%), dyspnea (4%) and bellyache (2%). Moreover, a different list of symptoms was observed among children and adults. For example, symptoms in adults were mainly cough, sore throat, runny nose and headache, however, in children the mainly symptoms were cough, expectoration, diarrhea and runny nose.
Factors analysis showed that age, preclinical treatments were associated with rates of influenza detection what was (sub)type dependent. Compared with the influenza negative cases, the risk for positive detection of pH1N1 and sH3N2 increased without preclinical treatments with odds ratio of 2.5 and 1.7, respectively. However, to influenza B, preclinical treatment decreased the detection rates. In adults, the detection rate was positively associated with age for seasonal influenza (sH1N1 and sH3N2), yet, in children, age increased the detection of novel influenza (pH1N1).
4. Evolutionary pattern of seasonal H3N2 during pandemic of H1N1 There was no positive selection pressure (dN/dS <1) for seasonal H3N2 during the pandemic of influenza A/H1N1, 2009. Higher dN/dS ratios for PB1-F2, HA, NA, M2 and NS genes existed, which imply higher evolutionary rate in these gene segments. Phylogenetic analysis showed that NA gene had the highest evolutionary rate with substitution rate of 4.30×10-3/nt/year. The most likely ancestor of influenza in this study may come from seasonal influenza strains in 2007. Among the eight segments, PB1 gene originated from strains in Mar 2007 and NA from strains in Nov 2007. Recombination existed in PB1 gene and the region between 40-821bp might from other influenza strains.
5. Molecular study of coronavirus in some regions of China Among coronavirus infections, CoV-229E and CoV-OC43 were the main strains. Phylogenetic analysis showed that CoV-229E was in the main branch of Chinese strain reported in 2009 but closer to strains reported by Australia, USA and Germany. However, CoV-OC43 was closely to strains reported by Belgium in 2003 and France in 2006. No positive selection pressure was observed in CoV-229E and CoV-OC43 when evaluated by dN/dS. Two polymorphism sites with nonsynonymous nucleotide substitutions located at 1681(A561S) and 2692(Y898H) in CoV-229E and three such sites in CoV-OC43 located at 3073(D1024H), 3085(E1028Q) and 3202(S1064A). Deduced amino acid sequence showed that there was a 16aa and a 17aa signal peptide in CoV-229E and CoV-OC43, respectively. Homology protein modeling of the receptor binding domain(RBD) was success for CoV-229E and CoV-OC43. Successfully docking of CoV-229E with angiotensin-convertion enzyme-2(ACE-2) was modeled, however, no docking of CoV-OC43 with ACE-2 was observed and this difference partial explained the difference of the infection caused by CoV-229E and CoV-OC43.
Conclusions:
1.The common respiratory viral pathogens spectrum were influenza virus, parainfluenza virus, adenovirus, respiratory syncytial viruses, human bocavirus, human metapneumovirus, and coronavirus. Co-infections with more than two viruses were frequently seen in respiratory viral infections and two-virus infection was the major of co-infections. Flu and ADV, ADV and PIV, BoV and RSV were the most common mix infections, and MPV and BoV had the high proportion of combining infection with other viruses. An obvious age specific distribution pattern existed in respiratory infections. The seasonalities of virus infection as following: PIV, ADV and MPV reached their climax in September, RSV in October, BoV in the middle of November, Flu in December and CoV in the end of January. Cough, expectoration, and runny nose were the most common symptoms. Age and duration from onset to visit doctor may affect the detection of respiratory viral infections.
2. H1 and H3 were the main serotypes in pig in China during 1999-2007 and a large geographical variation in seroprevalence of subtypes -H1 existed in South and East China, which related with densities of pig-raising. Genetic sequences blast revealed that swine influenza virus isolated in China were moderate homologous to the pH1N1, especially those isolated in South and East China.
3. Virological surveillance showed the major (sub)type of influenza were seasonal H3N2, pH1N1 and influenza B during Apr,2009 to Mar, 2011. The pandemic of H1N1 influenza in 2009 affected the epidemic influenza, in which the intensity, the duration of epidemic, and the peak of (sub)type were changed. The clinical symptoms of influenza were (sub)type dependent in population. Age and preclinical treatment may determine the detection of influenza.
4. Stochastic mutations without selection pressure may be the evolutionary manner of seasonal influenza H3N2 during pandemic of H1N1 in 2009. Among the segments, NA had the highest evolutionary rate. The most likely ancestor of influenza in this study may come from seasonal influenza strains in 2007 and PB1 gene might recombine whit other influenza strains.
5. The major strains of coronavirus were CoV-229E and CoV-OC43 and stochastic mutations without selection pressure was the source of mutations. Two polymorphism sites with nonsynonymous nucleotide substitutions located at 1681(A561S) and 2692(Y898H) in CoV-229E and three such sites in CoV-OC43 located at 3073(D1024H), 3085(E1028Q) and 3202(S1064A). Successfully docking of CoV-229E receptor binding domain with angiotensin-convertion enzyme-2(ACE-2) may partial explain the difference of the infection caused by CoV-229E and CoV-OC43.
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