摘要
第一部分呼吸道病毒高通量悬浮芯片检测方法的建立
目的建立常见呼吸道病毒的悬浮芯片检测方法,实现呼吸道病毒的快速、高通量检测。
方法
(1)建立两组悬浮芯片检测方法,1组为流感病毒悬浮芯片检测方法,用于流感病毒型/亚型检测,包括季节性H1、季节性H3、新现的新甲型H1N1、高致病性禽流感H5及B型流感病毒；2组为常见呼吸道病毒悬浮芯片检测方法,检测15种常见呼吸道病毒,其中包括A、B型流感病毒(FluA、FluB)及新现的呼吸道病毒人博卡病毒(HBoV)、人偏肺病毒(HMPV)、WU多瘤病毒(WUPyV)、NL63型冠状病毒(Cov NL63)和HKU1型冠状病毒(Cov HKU1)。
(2)引物和探针设计：流感病毒悬浮芯片检测方法(1组)引物和探针参照WHO推荐序列合成。常见呼吸道病毒悬浮芯片检测方法(2组)病毒的引物和探针设计如下：从GenBank下载不同呼吸道病毒的基因序列,用ClustalX进行序列比对后,找到每种病毒的基因保守序列,用Primier5.0和Oligo6.0软件在保守区设计引物和探针,尽量使不同病毒引物Tm值接近,引物扩增的目的片段大小在100bp-300bp之间。同时采用Oligo6.0软件对设计好的引物进行引物二级结构及互相之间形成引物二聚体的能力进行评价,根据评价结果将15对呼吸道病毒引物分为两个pool,用于多重PCR扩增。对探针序列进行blast比对,评价其特异性。
(3)探针与微球耦联：按照微球供应商提供的方法进行探针与微球的耦联,并用倍比稀释的与探针互补的validation oligo进行耦联效果评价。1组用与季节性H1、H3、新甲型H1N1及B型流感病毒探针互补的validation oligo进行了耦联效果评价,2组用与OC43型冠状病毒(Cov OC43)、HMPV、FluA及FluB探针互补的validation oligo进行了耦联效果评价
(4)多重PCR扩增及杂交条件优化：1组为5重PCR扩增,2组分为两个pool,1个为7重PCR扩增,另1个为8重PCR扩增。通过不同退火温度及引物浓度组合,优化反应条件及引物浓度。优化多重PCR反应条件后,在不同温度下将PCR产物与耦联有探针的微球进行杂交,用Bio-Plex 200检测仪进行检测,优化杂交温度。标本荧光强度值(MFI)=阴性对照标本5倍判断为阳性。
(5)用本实验室保存的不同型/亚型的流感病毒毒株(H5亚型除外)按TCID50(半数组织细胞感染量)倍比稀释后进行1组悬浮芯片检测,评价敏感性和特异性。对本实验室保存的14种常见呼吸道病毒(副流感病毒2型(PIV2)除外)阳性标本及灭活H5亚型流感病毒培养液进行RT-PCR/PCR扩增,PCR产物纯化、定量后,10倍系列稀释核酸进行2组悬浮芯片检测(H5亚型纯化产物稀释液进行1组悬浮芯片检测),评价所建立方法的敏感性和特异性。
(6)临床标本验证实验：按照以上建立的方法,对2009年1月采集的8份及2010年2月采集的100份流感样病例(ILI)的咽拭子标本进行1组悬浮芯片检测,并与WHO推荐的Real-time PCR方法检测结果进行比较。对2008年9月-2009年12月采集的88份下呼吸道感染患者的鼻咽吸取物(NPA)标本进行2组悬浮芯片检测,并与单引物PCR(检测HBoV及WUPyV)和多重PCR试剂盒(Seegeen,韩国)检测结果进行比较。
结果
(1)单引物PCR验证引物：用设计的不同呼吸道病毒特异的引物对已知呼吸道病毒阳性的标本进行单引物PCR扩增,均扩增出目的条带,说明设计的引物可用于后续多重PCR方法建立。
(2)探针与微球耦联：不同稀释度vallidation olligo与微球杂交后MFI值均与vallidation olligo的稀释倍数成正比,最高稀释度的vallidation olligo与微球杂交后MFI值也在700以上,说明探针与微球耦联成功。
(3)多重PCR扩增条件及杂交温度优化：用不同型/亚型流感病毒和已知病毒阳性的标本核酸进行梯度多重PCR扩增,1组和2组多重PCR的最佳退火温度分别为60℃和62℃。通过不同引物浓度组合比较,确定了多重PCR的最佳引物浓度。1组和2组悬浮芯片检测方法的最佳杂交温度分别为54℃和50℃。
(4)敏感性：1组悬浮芯片检测方法的敏感性为季节性H1N1亚型5TCID50/mL、季节性H3N2亚型0.05 TCID50/mL、新甲型H1N1 0.05 TCID50/mL、B型5TCID50/mL及H5亚型10-8ng/μL; 2组悬浮芯片检测方法的敏感性为HMPV 10-8ng/μL、HBoV 10-9ng/μL、WUPyV 10-8ng/μL、Adv(腺病毒)10-8ng/μL、Cov OC43 10-8ng/μL、Cov 229E (229E型冠状病毒)10-7ng/μL、Cov NL63 10-8ng/μL、Cov HKU1 10-8ng/μL、PIV1 (副流感病毒1型)10-9ng/μL、PIV3(副流感病毒3型)10-9ng/μL、HRV(鼻病毒)10-7ng/μL、RSV(呼吸道合胞病毒)10-8ng/μL、FluA 10-9ng/μL、FluB 10-8ng/μL。
(5)特异性：用1组和2组悬浮芯片方法检测不同型/亚型流感病毒或不同种类呼吸道病毒,病毒之间均无交叉反应。
(6)108份咽拭子标本进行1组悬浮芯片检测,结果与Real-time PCR检测结果完全一致,符合率100%。88份NPA标本进行2组悬浮芯片检测,与多重PCR试剂盒(Seegeen)及单引物PCR结果进行比较,2组悬浮芯片方法检测不同呼吸道病毒的灵敏度为50.0%M00.0%,特异度为96.4%-100.0%,阳性预期值为33.3%-100.0%,阴性预期值为94.8%-100.0%,符合率为93.2%-100%。
结论本文成功建立了包括新现呼吸道病毒(新甲型H1N1流感病毒、HMPV、HBoV、WUPyV、Cov NL63及Cov HKU1)在内的流感病毒和常见呼吸道病毒悬浮芯片检测方法,并对实验条件进行了优化,确定了最佳实验方案。该方法的敏感性、特异性均较好,检测时限为6h,同时可检测15种常见呼吸道病毒及5种型/亚型流感病毒。
第二部分新现呼吸道病毒的分子特征分析
目的研究新现呼吸道病毒新甲型H1N1流感病毒、HMPV、HBoV及WUPyV的感染状况及病毒相关基因特性。
方法
(1)用病毒分离或Real——time PCR法,对2009年4月-2011年3月采集的IL1咽拭子标本和重症呼吸道感染患者的NPA、鼻咽拭子及气管盥洗液等呼吸道标本进行包括新现甲型H1N1在内的流感病毒检测。并对分离自上述病例中重症/死亡病例的14株新甲型H1N1流感病毒毒株进行HA基因序列测定,其中6株进行NA基因序列测定,3株进行全基因组序列测定,分析序列特征,绘制种系发生树。
(2)采集2006年和2008年春季、2008年和2009年秋冬季的急性呼吸道感染住院儿童的310份NPA标本,进行巢式RT-PCR扩增HMPVN和F基因片段,RT-PCR扩增G基因,并对扩增阳性产物进行序列分析,绘制种系发生树。同时收集所有阳性患者的临床资料。并采用PCR和多重PCR对HMPVN基因阳性标本进行其他呼吸道病毒检测。HMPV检测阳性标本同时用Vero-E6和LLC-MK2细胞进行病毒分离。
(3)采集2008年春季、2008年秋冬季和2009年冬春季的急性下呼吸道感染住院儿童的238份NPA标本,采用PCR检测HBoV NP-1基因片段,并对VP1/VP2基因进行PCR扩增和序列分析,绘制种系发生树。同时采用PCR和多重PCR检测其它呼吸道病毒。
(4)采集2008年春季、2008年秋冬季和2009年冬春季的急性下呼吸道感染住院儿童的NPA标本238份,2009年冬季的急性上呼吸道感染的咽拭子标本68份以及2009年2月-11月对照组咽拭子标本43份,进行WUPyV VP2基因片段PCR扩增,同时对扩增阳性标本采用PCR和多重PCR检测其他呼吸道病毒。
结果
(1)流感病毒
2009年4月-2011年3月共检测呼吸道标本7931份(其中ILI咽拭子标本6177份,重症呼吸道感染病例呼吸道标本754份),1567份(19.8%)检出流感病毒。2009年4月-2009年9月、2009年10月-2010年3月、2010年4月-2010年9月和2010年10月-2011年3月四个监测季流感病毒优势流行亚型分别为季节性H3(60.8%,185/304)、新甲型H1N1(76.8%,630/820)、季节性H3(77.6%,118/152)和新甲型H1N1(51.5%,150/291)。四个监测季中季节性H3亚型流感病毒HA基因变异较小,属抗原漂移。
(2)新甲型H1N1流感病毒：
1567份流感病毒阳性标本中,56.3%(882/1567)为新甲型H1N1流感病毒。25.5%(192/754)的重症呼吸道感染病例为新甲型H1N1流感病毒阳性。不同监测季新甲型H1N1流感病毒在所有流感病毒中的构成比如下：2009年4月-2009年9月为33.6%(102/304),2009年10月-2010年3月为76.8%(630/820),2010年4月-2010年9月为0.6%(1/152),2010年10月-2011年3月为51.5%(150/291)。新甲型H1N1流感病毒为2009年10月-2010年3月和2010年10月-2011年3月两个监测季的优势流行亚型。种系发生分析显示,新甲型HIN1流感病毒的HA、NA、M、NP和NS基因来源于猪流感病毒,PB2和PA基因来源于禽流感病毒,PB1基因来源于人流感病毒。天津地区新甲型H1N1流感病毒8个片段与WHO推荐的疫苗株A/California/07/2009(H 1N1)及中国的代表株A/Sichuan/1/2OO9(H1N1)相比,同源性很高,为98.2%-99.5%。HA基因氨基酸序列与疫苗株比较,未发现受体结合位点变异。NA基因氨基酸序列分析显示,天津地区新甲型H1N1流感病毒对神经氨酸酶抑制剂(如达菲)敏感。
(3) HMPV
310份NPA标本中共检出20份(6.5%)HMPV。HMPV阳性患儿的临床诊断均为支气管肺炎,其临床症状以咳嗽、喘息、呼吸急促、发烧等常见。HMPV阳性患儿的年龄中位数为15.0个月(16d-9岁),其中=2岁患儿占90.0%(18/20)。男性占60.0%(12/20)。17株HMPVN基因和18株HMPVF基因种系发生树分析显示,14株为A型(其中13株为A2b亚型),6株为B型。A、B型间临床特征未见明显差别。G基因变异最大,N和F基因片段相对保守。2例HMPV阳性患儿分别与腺病毒和鼻病毒混合感染。HMPV的细胞分离较困难,用Vero-6细胞从PCR阳性标本中成功分离到HMPV, Vero-E6分离HMPV优于LLC-MK2。
(4) HBoV
238份NPA标本中,17份(7.1%)为HBoV阳性。所有阳性患儿的年龄均=6y,男14例,女3例。6月-1y患儿感染率最高,为16.0%(8/50)。17例HBoV阳性患儿均为肺炎和喘息性支气管炎患者。14份(82.4%)合并其它呼吸道病毒感染。13株HBoVVP1/VP2基因扩增成功,VPl/VP2基因序列种系发生分析表明,13株HBoV均与瑞典分离的代表株ST2株(1b簇)聚在一个分支上,均属于1b簇。
(5) WUPyV
238份下呼吸道感染的NPA标本中,45份(18.9%)WUPyV VP2基因阳性。阳性患儿年龄中位数为9.0个月(6d-6y),=6个月检出率最高(37.8%,17/45),男28例,女17例。WUPyV阳性标本中,80%(36/45)与其他呼吸道病毒混合感染,其中RSV B占35.6%(16/45)。68份门诊上呼吸道感染儿童的咽拭子标本中,仅4.4%(3/68)为WUPyV VP2基因片段PCR扩增阳性,与上述下呼吸道感染的阳性率18.9%相比,差异有统计学意义(X2=8.4,P=0.0037)。43例对照组儿童的咽拭子标本经WUPyV VP2基因检测均为阴性。
结论
(1)流感病毒是冬春季重点监测的呼吸道病毒,2009年4月-2011年3月4个监测季的优势流行亚型分别为季节性H3、新甲型H1N1、季节性H3和新甲型H1N1。季节性H3亚型流感病毒HA基因变异属抗原漂移。
(2)天津地区2009年6月出现新甲型H1N1流感病毒,2009年10月-2010年3月达流行高峰(76.8%),2010年4月-2010年9月急剧下降(0.6%),2010年10月-2011年3月又成为优势流行亚型(51.5%)。新甲型H1N1流感病毒可引起肺炎等重症呼吸道感染。新甲型H1N1流感病毒的HA、NA、M、NP和NS基因来源于猪流感病毒,PB2和PA基因来源于禽流感病毒,PB1基因来源于人流感病毒,其为三重重排病毒,与国外报道一致。目前的新甲型H1N1流感病毒基因与国外毒株相比,无显著变异。NA氨基酸序列分析表明,天津地区无达菲耐药株出现。
(3) HMPV、HBoV及WUPyV均在下呼吸道感染患者中检出率高,且都有混合感染。天津地区流行的HMPV以A2b为优势流行型,HBoV均属于1b簇。
Part I Development of high through-out liquid chip to detect common respiratory viruses
Objectives
Developing quick and high through-out liquid chip technique to detect common respiratory viruses in order to response better to emergency respiratory disease outbreaks.
Methods
(1) Two groups were developed. One includes seasonal H1N1, H3N2, novel H1N1, avain Influenza H5 and Influenza B. The other group aimed to test 15 respiratory viruses, including the emerging ones, human metapneumovirus (HMPV), human bocavirus (HBoV), WU polyomovirus (WUPyV), Coronavirus NL63(Cov NL63) and Coronavirus HKU1(Cov HKU1).
(2) Primers and probes design:5 sets of primer and probe were selected according to WHO recommedation for influenza virus testing group, and 15 sets of primer and probe were for respiratory virus. Gene sequences of various viruses were downloaded from GenBank, sequence alignment was proceeded by Clustal X to find conservative gene segments, and using Primier 5.0 and Oligo 6.0 softwares to design virus-specific primers and probes. Tm of different various viruses should be close to each other, and the size of PCR products shoule be in 100-300bp. Using Oligo6.0 to evaluate the primers secondary structure. Then the 15 sets of respiratory primers were divided into 2 pools to conduct multi-PCR. The specificity of probes was evaluated through blast alignment.
(3) Coupling of probes with beads:According to manufacturer's instruction to conduct the coupling, and evaluate the coupling effects with validation oligos which were complementary with probes. Influenza virus group was evaluated with seasonal HI, H3, novel A(H1N1), avain H5 subtype and B influenza virus probe coplmeatary validation oligos. Common respiratory virus group was evaluated withCov OC43, HMPV, Flu A and Flu B probe complementary validation oligos.
(4) Optimizing of multi-PCR and hybridization conditions:five pairs of primer- multiplex PCR were constructed for influenza virus.2 pools were developed for respiratory viruses,1 was seven pairs of primer-multiplex PCR, the other was 8 pairs of primer-multiplex PCR. Optimize the annealing temperature and concentration of primers through different combination. The hybridization temperature was optimized with mean fluorescence intensity (MFI) value of PCR products and beads hybridization. Specimens which MFI was 5 times or greater than negative control specimens were judged as positive.
(5) Assay sensitivity of influenza virus liquid chip was evaluated using serial dilution of tissue culture infectious dose (TCID50) strains which were stored in inhouse laboratory. For respiratory virus liquid chip, the sigle specific primer PCR products (except PIV2) were quantified and serial diluted to evaluate the sensitivity. Different respiratory viruses positive samples were used to evaluate specificity.
(6) Specimens detection to verify liquid chip method:108 throat swabs (8 were collected from Influenza Like Illness (ILI) cases in Janurary 2009,100 were collected in Feburary 2010) were tested with both influenza virus liquid chip technique and real-time PCR.88 NPAs (which were collected in September 2008 to December 2009) were tested by common respiratory virus liquid chip method, and the results were compared with conventional PCR (for HBoV and WUPyV testing) and commercial Multi-PCR kit (Seegeen, Korea).
Results
(1) Single pair primer-PCR to verify designed primers:all 15 pairs of primers were used to amplify specific respiratory viruses, anticipated products were obtained from these amplification. It indicated that these 15 primers can be used to develop the following multiplex PCR.
(2) Effects of probe and bead coupling:MFI value of validation oligo was in proportion to its concentration, the minimum MFI value was about 700. All these meaned that probe and bead coupling succeeded.
(3) Optimizing of multi-PCR and hybridization conditions:Optimized annealing temperatures of influenza virus group and respiratory virus groups (2 pools) multi-PCR were 60℃and 62℃respectivly. Opitimized hybridization temperatures of influenza virus and respiratory virus liquid chip methods were 54℃and 50℃respectivly.
(4)Sensitivity of influenza liquid chip assay was as follows:seasonal H1N1 5TCID50/L1, seasonal H3N20.05TCID50/mL, novel A(H1N1) 0.05TCID50/mL, and B 5TCID5o/ml and H510-8ng/μl. Sensitivity of respiratory virus liquid chip assay was as follows:HMPV10"8ng/μL, HBoV10-9ng/μl, WUPyV10-8ng/μl, AdvlO-8ng/ul, Cov OC4310-8ng/μl, Cov 229E10-7ng/μl, Cov NL6310-8ng/μl, Cov HKU110-8ng/μl, PIV110-9ng/μl, PIV3 10-9ng/μl, HRV 10-7ng/μl, RSV 10-8ng/μl, FluA 10-9ng/μl, FluB 10"8ng/μl.
(5) No interassay cross amplification of other influenza/respiratoryviruses was observed in both assays.
(6)The results of influenza virus liquid chip assay to detect 108 throat swabs were the same as that of Real-time PCR.88 NPA were detected using respiratory virus liquid chip assay. Compared with multiplex PCR (Seegeen, Korean) and single primer PCR, the sensitivity of 15 respiratory viruses liquid chip assay ranged from 50%-100%, specificity was 96.4%-100%, positive expected value was 33.3%～100%, negative expected value was 94.8%～100%. and accordance rate was 93.2%～100%.
Conclusion
Influenza/respiratory virus liquid chip assay were developed successfully. These assays can be used as a rapid, sensitive and specific method for major respiratory viruses detection and typing and subtyping influenza viruses. It only takes 6 hours to complete test and the respiratory virus liquid chip assay can test 15 prototype viruses at one time.
Part II Analysis molecular characters of emerging respiratory viruses
Objectives
To investigate the prevalence of emerging respiratory viruses, novel influenza A(H1N1) surveillance in all age population and HMPV, HBoV and WUPyV in infants and young children presented with acute respiratory tract infection, and and to identify the molecular characters.
Methods
(1) Samples collected from Influenza Like Illness (ILI) cases and severe respiratory tract infection in April 2009 to March 2011 were detected through virus isolation or Real-time PCR for influenza virus including novel Influenza A (H1N1). HA genes of 14 novel Influenza A (H1N1) strains isolated from severe/death cases and NA genes of 6 stains and complete genome of 3 strains from death cases were sequenced and analyzed.
(2) Nasopharyngeal aspirates (NPAs) were taken from 310 hospitalized pediatric patients in the spring in 2006 and 2008, and autume and winter in 2008 and 2009. And the N and F gene fragments of HMPV were detected by nested PCR, G gene by conventional PCR. Phylogenetic analysis of N, F and G genes was performed. The clinical materials of patients were collected and analyzed. All HMPV-positive samples were examined by multi-PCR for other respiratory viruses. PCR positive samples were used to isolate HMPV with Vero-E6 and LLC-MK2 cells.
(3)NPAs were taken from 238 hospitalized pediatric patients in spring, autume and winter in 2008, and spring and winter in 2009. And the NP1 gene fragments of HBoV were detected by PCR. VP1/VP2 genes were sequenced to analyze the variation characters and construct phylogenetic tree. All HBoV-positive samples were examined by PCR and multi-PCR for other 12 respiratory viruses.
(4)NPAs were taken from 238 hospitalized pediatric patients in spring, autume and winter in 2008, and spring and winter in 2009, throat swabs were collected from 68 children with upper respiratory tract infection (URTI) in winter in 2009, and 43 children without respiratory tract clinical manifestation in Feburary 2009 to November 2009(control group). WUPyV from all above samples were examined by PCR for VP2 gene segment. All WUPyV-positive NPAs were examined by PCR and multi-PCR for other respiratory viruses.
Results
(1) Influenza virus:
Among 7931 throat swab specimens from patients with ILI and severe respiratory tract infection in Tianjin area from April 2009 to March 2010,1567 (19.8%) were influenza viruses positive. Seasonal H3 (60.8%,185/304) was predomiant subtype detected from April 2009 to September 2009. From October 2009 to March 2010, novel influenza A(H1N1) (76.8%,630/820) became the most frequently detected subtype. From April 2010 to September 2010, Seasonal H3 (77.6%,118/152) was the predominant subtype. Novel influenza A(H1N1) (51.5%,150/291) became predomiant subtypes again from September 2010 to March 2011. Little variation of seasonal H3 subtype influenza viruses was observed in above 4 surveillance seasons, this variation belonged to antigen drift.
(2) Novel influenza A(H1N1):
Among 1567 influenza viruses,56.3%were novel influenza A(H1N1).25.5% (192/754) severe respiratory tract infections cases were novel influenza A(H1N1) positive. The proportion of novel influenza A(H1N1) in different surveillance seasons were as follows:33.6%(102/304) from April 2009 to September 2009, 76.8%(630/820) from April 2009 to March 2010,0.6%(1/152) fromOctober 2009 to March 2010,51.5%(150/291) from September 2010 to March 2011. Novel influenza A(H1N1) was the predominant subtype in two surveillance seasons (September 2009 to March 2010 and September 2010 to March 2011). Phylogenetic analysis showed that HA, NA, M, NP and NS genes of novel influenza A(H1N1) viruses gathered together with classical swine influenza A(H1N1). PB2 and PA genes came from avain influenza virus. PB1 gene came from human seasonal influenza virus. The high identities(98.2%-99.5%) of HA gene were observed between novel influenza A(H1N1) isolated in Tianjin and WHO recommended vaccine strain A/California/07/2009(H1N1) and China representative strain A/Sichuan/1/2009(H1N1). No variation of receptor binding sites was observed in HA protein. NA amino acid sequence analysis showed that novel A(H1N1) influenza viruses in Tianjin was sensitive with Neuraminidase Inhibitor (such as Oseltamivir).
(3) HMPV
Of 310 pediatric patients,20(6.5%) were positive for HMPV. The median age of HMPV infected children was 15 months(from 16 days to 9 years old),90%(18/20) of the cases were under 2 months. Phylogenetic analysis of 17 N and 18 F gene fragments showed that 13 HMPV strains were A2b subtype. G gene was more variable than N and F genes. All the 20 HMPV-positive children were subjected to pneumonia. The common clinical manifestations of HMPV infected patients were cough, wheezing, shortness of breath and fever. No significant difference of clinical manifestations between type A and B were displayed. Two patients were coinfected with adenovirus and rhinovirus respectively. HMPV were successfully isolated from PCR positive specimens though it was so difficult, and Vero-E6 was better than LLC-MK2 to isolate HMPV.
(4)HBoV
Of 238 pediatric patients,17(7.1%) were positive for HBoV. All HBoV infected children was equal or less than 6 years old, the highest infection frequency age group was 6m-12m, infection rate was 16.0%(8/50). All HBoV-positive children were subjected to pneumonia and asthmatic bronchitis.82.4%(14/17) HBoV-positive patients were co-infected with other respiratory viruses.13 HBoV VP1/VP2 genes were successfully sequenced, phylogenetic analysis of VP1/VP2 genes showed that they were all belong to Ib cluster.
(5)WUPyV
Among 238 hospitalized pediatric patients,45(18.9%) were positive for WUPyV.The median age of WUPyV infected children was 9 months(from 6 days to 6y), the highest infection frequency age group was=6 months(37.8%,17/45). Among them,28 were male,17 were female.36 cases(80%) were coinfected with other respiratory viruses, the most commonly co-detected with RSV B (35.6%).3 of 68 (4.4%) throat swabs from URTI children were WUPyV positive, significantly lower than the infection rate (18.9%)of low respiratory tract illness(LRTI)(χ2= 8.4, P=0.0037). No WUPyV was detected in 43 samples collected from asymptomatic control patients.
Conclusions
(1) Influenza virus was the most important respiratory virus in spring and winter seasons. The predominant subtypes of the 4 surveillance seasons in April 2009 to March 2011 were as follows:seasonal H3, novel influenza A(H1N1), seasonal H3, and novel influenza A(H1N1). Seasonal H3 variation belonged to antigen drift.
(2) Novel Influenza A (H1N1) was first detected in Tianjin in June 2009, and it attained prevalence peak in October 2009 to March 2010(76.8%), then decreased dramatically in April 2010 to September 2010(0.6%), and became the predominant subtype in October 2010 to March 2011(51.5%). Novel Influenza A (H1N1) can lead to severe respiratory tract infection such as pneumonia. Consistent with other reports, novel influenza A (H1N1) viruse was a tri-rearrangment virus. HA, NA, M, NP and NS genes of novel influenza A (H1N1) viruses originated from swine influenza viruses. PB2 and PA originated from avain influenza viruses. PB1 originated from huamn influenza viruses. The gene variation of novel influenza A (H1N1) viruses was not so significant, no Oseltamivir-resistant strain was observed.
(3) HMPV, HBoV and WUPyV were detected as high frequency in LRTIs, and coinfection was common with them. A2b was the predominant subtype of HMPV, and all HBoV in Tianjin was clustered to Ib.
引文
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