2019-2020年山东省A(H1N1)pdm09亚型流感病毒抗原性及血凝素基因特征分析北大核心CSCD

目的了解山东省2019-2020年分离株A(H1N1)pdm09亚型流感病毒的抗原性及血凝素(hemagglutinin, HA)基因变异情况。方法采用单向红细胞凝集抑制试验对2019-2020监测年分离的15株A(H1N1)pdm09亚型流感病毒进行抗原性分析;PCR扩增病毒的HA基因并测序,进行进化分析,以及糖基化位点、抗原变异位点、受体结合位点的变异分析。结果 2019-2020年流行株A(H1N1)pdm09亚型流感病毒均为疫苗株A/Brisbane/02/2018的类似株。HA基因均属于6B.1分支,毒株HA基因核苷酸序列与当季疫苗株A/Brisbane/02/2018的同源性为98.5%~99.0%,与最新疫苗株A/Guangdong-Maonan/SWL1536/2019的核苷酸同源性为99.3%~99.8%。所有毒株与当季疫苗株相比均出现了T185I位点变异。结论 2019-2020监测年度山东省流行的A(H1N1)pdm09亚型流感病毒与WHO推荐的疫苗组分匹配性良好,疫苗株推荐存在滞后性。HA基因与其编码的氨基酸持续变异,因此应加强对病毒变异的监测,为疫苗筛选及流感防控措施的制定提供参考。     

2019-2020年山东省A(H1N1)pdm09亚型流感病毒抗原性及血凝素基因特征分析

目的了解山东省2019-2020年分离株A(H1N1)pdm09亚型流感病毒的抗原性及血凝素(hemagglutinin, HA)基因变异情况。方法采用单向红细胞凝集抑制试验对2019-2020监测年分离的15株A(H1N1)pdm09亚型流感病毒进行抗原性分析;PCR扩增病毒的HA基因并测序,进行进化分析,以及糖基化位点、抗原变异位点、受体结合位点的变异分析。结果 2019-2020年流行株A(H1N1)pdm09亚型流感病毒均为疫苗株A/Brisbane/02/2018的类似株。HA基因均属于6B.1分支,毒株HA基因核苷酸序列与当季疫苗株A/Brisbane/02/2018的同源性为98.5%～99.0%,与最新疫苗株A/Guangdong-Maonan/SWL1536/2019的核苷酸同源性为99.3%～99.8%。所有毒株与当季疫苗株相比均出现了T185I位点变异。结论 2019-2020监测年度山东省流行的A(H1N1)pdm09亚型流感病毒与WHO推荐的疫苗组分匹配性良好,疫苗株推荐存在滞后性。HA基因与其编码的氨基酸持续变异,因此应加强对病毒变异的监测,为疫苗筛选及流感防控措施的制定提供参考。     

2017-2019年呼和浩特市甲型A(H1N1)pdm09流感病毒基因特征分析

目的 了解呼和浩特市甲型A(H1N1)pdm09流感病毒遗传进化特征及抗原位点、糖基化位点和耐药位点的变异情况,为流感防控提供科学依据。方法 采集2017-2019年呼和浩特市3家哨点医院的流感样病例(ILI)咽拭子标本进行核酸检测,阳性样本通过MDCK细胞和鸡胚进行病毒分离。随机抽取15株甲型A(H1N1)pdm09流感毒株进行基因测序分析。采用MEGA7.0.14软件、DNASTAR7.0.1软件和NetNGlyc1.0软件进行基因进化树分析、核苷酸同源性分析和糖基化位点分析。结果 呼和浩特市2017-2019年15株甲型A(H1N1)pdm09分离株与疫苗株A/Brisbane/02/2018和A/Michigan/45/2015共同属于6B.1分支。各年度分离株与疫苗株A/Brisbane/02/2018的组间遗传距离分别为0.004、0.011和0.013。相较于疫苗株A/California/07/2009和A/Michigan/45/2015,2017年起甲型A(H1N1)pdm09病毒抗原位点变异较大,但与疫苗株A/Brisbane/02/2018相比并未发生抗原漂移现...     

2016-2020年福建省A(H1N1)pdm09流感病毒HA基因特征分析

目的 分析2016-2020年福建省A(H1N1)pdm09亚型流感病毒的血凝素(Hemagglutinin, HA)基因特征,了解与其他省份和全球范围内时间和空间上的分布差异。方法 选取2016-2020年福建省流感监测网络实验室分离A(H1N1)pdm09流感毒株基因序列测定。基因序列用MEGA、Net NGlyc 1.0 Server分析基因特征。结果 2016-2020年福建省A(H1N1)pdm09亚型流感病毒HA基因进化树分析与其他省份及国外大部分一致,但仍有少部分不同。HA分支由6B转化为6B.1并演化至6B.1A5A。HA基因变异主要在抗原性决定簇Sa、Ca、Sb。对于不同地区疫苗株匹配度也会有差异。结论 福建省A(H₁N₁)pdm09病毒流行株具有遗传多样性。     

2019—2020年山东省H3N2流感病毒抗原性及血凝素基因特征分析

目的 了解2019—2020年流感监测年度山东省分离的H3N2亚型流感病毒抗原性及血凝素(hemagglutinin,HA)基因遗传进化规律与氨基酸变异情况。方法 用免疫雪貂后的抗血清进行红细胞凝集抑制试验,对2019年4月至2020年3月山东省分离的40株H3N2亚型流感毒株进行抗原性分析,并对其中19株待检病毒的HA基因进行序列测定及分析。结果 抗原性分析结果显示检测的H3N2亚型流感病毒中仅仅35%(14/40)与疫苗株A/Kansas/14/2017抗原性类似。系统进化树显示,19株H3N2亚型流感病毒分离株血凝素基因在进化树上全部属于3C.2a分支,与处于3C.3a分支的疫苗株A/Kansas/14/2017亲缘关系较远;与疫苗株相比,所有待检毒株A抗原决定簇有3个位点,B抗原决定簇有2个位点发生改变;受体结合位点均发生T135K位和S137F位变异;19株分离株全部发生133位糖基化位点缺失。结论 抗原性分析及HA基因序列分析结果显示,WHO推荐的 2019—2020 年疫苗株保护效果有可能不理想。应继续密切关注 H3N2 亚型流感病毒的流行与基因变异情况,为流感病毒疫苗株推荐及防控提供科学依据。     

甲型H1N1与季节性H1N1流感病毒血凝素基因比较分析

目的分析泰安市2008～2009年度季节性流感与2009年度甲型H1N1流感病原学检测结果 ,比较季节性H1N1与甲型H1N1血凝素基因变异情况。方法选择国家级流感监测哨点医院以及暴发疫情的疫点,采集流感样病例的鼻咽拭子标本,通过RealtimePCR进行病毒检测,用MDCK细胞进行病毒分离,通过RT-PCR扩增血凝素HA1片段的基因并测序,利用生物信息学进行序列分析。结果 2008～2009年共检测鼻咽拭子标本283份,分离出流感病毒33株,分离阳性率为11.67%,其中季节性H1N1亚型31株。2009年5月1日～12月31日,检测鼻咽拭子标本996份,流感核酸检测阳性417份,阳性率为41.86%,其中甲型H1N1337份,季节性H1N1亚型1份。6株季节性H1N1病毒均在多个氨基酸位点上发生变异,与疫苗株A/Brisbane/59/2007(H1N1)比较,有11个位点发生了突变,其中5个位点位于抗原决定簇上;测序成功的6株甲型H1N1病毒在多个氨基酸位点发生变异,与疫苗株A/California/07/2009(H1N1)比较,有6个位点发生突变,其中1个位点位于抗原决定簇的B区。结论 2008～2009年度季节性H1N1为优势株,甲流暴发后,甲型H1N1成为绝对优势毒株。季节性H1N1分离株有多处氨基酸替换,抗原决定簇B区变异频繁;甲型H1N1病毒分离株的基因有变异,但关键位点第222位仍为D(天冬氨酸),与疫苗株相比抗原决定簇的关键位点变化不大。     

2016-2018年广州市H6亚型禽流感病毒外环境分离株HA基因遗传特征分析

目的 对广州禽类市场外环境H6亚型禽流感病毒进行分离和测序,分析HA基因遗传进化特点。方法 通过接种鸡胚分离H6亚型禽流感病毒,采用RT-PCR方法扩增HA基因全长序列并测序,通过DNA Star7.1和MEGA 4.0软件,分析HA基因遗传特征。结果 2016-2018年广州禽类市场外环境分离到6株H6亚型禽流感病毒,HA基因核苷酸同源性在81.0%~99.1%之间。基因序列特征分析显示,所有分离株裂解位点序列相对保守,只有1个碱性氨基酸插入,呈现低致病性禽流感病毒分子特点。受体结合位点均为226Q和228G,为禽类呼吸道上皮受体结合位点。2016年分离株发现183-NNT糖基化位点的缺失。进化分析显示,广州早期分离株属于广东常见的ST2853-like分支,但2018年监测发现了ST339-like新流行分支病毒。结论 广州地区H6亚型禽流感病毒尚为禽类低致病性病毒,本研究毒株HA基因变异较大, 2018年毒株出现多遗传分支进化趋势,因此需要继续监测H6亚型禽流感病毒的流行和变异。     

甲型流感病毒株H1N1(09pdm)的分离及HA和NA基因分析北大核心CSCD

目的对成都某部人感染甲型流感病毒进行分离鉴定和基因突变分析。方法采集甲型流感患者咽拭子标本,通过MDCK细胞分离病毒毒株;采用免疫荧光法鉴定其感染细胞能力,采用基因分型特异性引物鉴定病毒亚型,PCR扩增血凝素基因(HA)和神经氨酸酶基因(NA)后测序,与NCBI数据库在线比对并利用MEGA软件构建系统发育进化树,分析突变位点。结果从甲型流感患者咽拭子标本中分离出1株流感病毒,经型特异性引物PCR鉴定为H1N1(09pdm)亚型,该毒株在37℃时对细胞致病力较强。免疫荧光检测到分离毒株感染细胞内甲型流感病毒核蛋白(NP)高表达,甲型流感病毒NP蛋白在细胞核和细胞质中均有大量分布。利用反转录PCR和测序获得该毒株HA和NA全长基因序列。在线比对及系统发育树分析显示,该毒株HA和NA序列与2017-2018流感季其他国家流行株同源性均>99%。对HA氨基酸突变位点进行分析,其序列的155位点存在组氨酸-酪氨酸(H-Y)点突变,该点突变也发生Influenza A/Hawaii/24/2018(H1N1)(MH245873)、Influenza A/North Carolina/19/2018(H1N1)(MH245873)和In-fluenza A/Missouri/51/2017(H1N1)(MH083792)毒株上。NA氨基酸序列与近期流行的毒株均相同。结论本起流感病毒株H1N1(09pdm)的HA、NA基因序列与同期其他国家流行株高度相似,但在HA氨基酸序列的155位点存在一个组氨酸-酪氨酸的点突变,其意义尚不清楚。     

2018-2019年克拉玛依市新甲型H1N1流感病毒NA基因分析

目的 分析新疆克拉玛依市新甲型H1N1流感病毒NA基因特征及变异情况.方法 采集2018年1月-2019年12月哨点医院流感样病例咽拭子,经MDCK细胞完成流感病毒分离后,随机抽取26株分离株测定其NA基因序列,并与疫苗株进行序列比对和分析.结果 与疫苗株A/Michigan/45/2015 NA基因核苷酸相比,2018年分离株与其同源性为98.56％～99.28％,2019年分离株与其同源性为98.35％～98.71％;26株分离株与同期国内其他地区代表株和疫苗株A/Michigan/45/2015在同一进化分支;分离株T180699较疫苗株A/Michigan/45/2015少1个糖基化位点(42～44位),分离株T190041发生了I223V位点变异.结论 相对于疫苗株A/Michigan/45/2015,2018-2019年克拉玛依市新甲型H1N1流感病毒NA基因抗原决定簇、糖基化位点和耐药相关位点已有改变;应继续加强流感监测,密切关注其基因变异情况.     

2017—2018年广州市H3N2流感病毒流行及血凝素基因分子特征分析

目的 对广州市H3N2流感病毒进行基因变异和进化分析,为H3N2流感科学防控提供科学依据。方法 对广州市2017年1月-2018年9月流感监测标本进行H3N2病毒检测和分离,并对HA基因进行测序,通过生物信息学软件分析病毒变异和进化特点。结果 所监测的8 535份标本中,检出H3N2流感阳性标本386份,阳性率为4.52%。对其中16株分离株HA基因测序结果显示,与同年度疫苗推荐株A/Hong Kong/4801/2014相比,2017年广州H3N2病毒A 区抗原位点变异频率较高,多发生于140位、144位和150位,其中有7株病毒发生新抗原漂移,变异毒株占比43.75%。受体结合位点变异主要发生在前壁,位于T131K位和T135K(N)位。糖基化位点变异同样多发生于A区抗原位点和受体结合位点前壁。2017年广州H3N2流感病毒均位于3C.2a分支,但分支内又进一步进化形成3个不同的小流行分支,包括3C.2a1分支,以及与2016年北京、2017年美国分离株亲缘相近的另外两个小分支。结论 2017年广州H3N2流感病毒HA蛋白重要氨基酸位点的变异表现遗传多态性,特别是在抗原性上可能发生了较大变异。在基因进化上表现为多样性和复杂性,呈现多分支进化特点。因此有必要密切监测流行毒株的进化趋势,以期及时对疫苗株的匹配性进行有效评估。     

福建省1例不明原因肺炎病例中甲型H1N1流感病毒的全基因组特征分析

目的了解1例不明原因肺炎病例中甲型H1N1流感病毒的生物学特性和变异情况,为临床治疗与疫情防控提供依据。方法利用MDCK细胞分离不明原因肺炎病例中的甲型H1N1流感病毒,分离的毒株经全基因组测序后分析其抗原性、致病性和耐药性等特征。结果从该病例的咽拭子标本中分离得到1株甲型H1N1流感病毒并命名为A/FujianGulou/SWL64/2016(H1N1),其8个节段的核苷酸和氨基酸序列与A/California/07/2009(H1N1)疫苗株的相似性分别为96.9%~98.9%和96.7%~99.5%。氨基酸序列分析显示HA蛋白共有18个位点发生突变,其中K163Q、S185T、S203T和D222N变异涉及到3个不同的抗原位点,提示病毒发生抗原漂移;与受体结合位点相关的D222N突变还提示病毒感染下呼吸道的能力增强。耐药性分析显示该病毒对金刚烷胺耐药,对达菲仍然敏感。结论本次研究的甲型H1N1流感病毒具有抗原漂移现象,且具备引发重症肺炎的分子特征,应进一步加强监测,为疫情防控奠定基础。     

Virological characterization of influenza H1N1pdm09 in Vietnam, 2010‐2013

Objectives

Influenza A/H1N1pdm09 virus was first detected in Vietnam on May 31, 2009, and continues to circulate in Vietnam as a seasonal influenza virus. This study has monitored genotypic and phenotypic changes in this group of viruses during 2010–2013 period.

Design and setting

We sequenced hemagglutinin (HA) and neuraminidase (NA) genes from representative influenza A/H1N1pdm09 and compared with vaccine strain A/California/07/09 and other contemporary isolates from neighboring countries. Hemagglutination inhibition (HI) and neuraminidase inhibition (NAI) assays also were performed on these isolates.

Sample

Representative influenza A/H1N1pdm09 isolates (n = 61) from ILI and SARI surveillances in northern Vietnam between 2010 and 2013.

Main outcome measures and results

The HA and NA phylogenies revealed six and seven groups, respectively. Five isolates (8·2%) had substitutions G155E and N156K in the HA, which were associated with reduced HI titers by antiserum raised against the vaccine virus A/California/07/2009. One isolate from 2011 and one isolate from 2013 had a predicted H275Y substitution in the neuraminidase molecule, which was associated with reduced susceptibility to oseltamivir in a NAI assay. We also identified a D222N change in the HA of a virus isolated from a fatal case in 2013.

Conclusions

Significant genotypic and phenotypic changes in A/ H1N1pdm09 influenza viruses were detected by the National Influenza Surveillance System (NISS) in Vietnam between 2010 and 2013 highlighting the value of this system to Vietnam and to the region. Sustained NISS and continued virological monitoring of seasonal influenza viruses are required for vaccine policy development in Vietnam. 3     

Large Scale Genome Analysis Shows that the Epitopes for Broadly Cross-Reactive Antibodies Are Predominant in the Pandemic 2009 Influenza Virus A H1N1 Strain

The past pandemic strain H1N1 (A (H1N1)pdm09) has now become a common component of current seasonal influenza viruses. It has changed the pre-existing immunity of the human population to succeeding infections. In the present study, a total of 14,210 distinct sequences downloaded from National Center for Biotechnology Information (NCBI) database were used for the analysis. The epitope compositions in A (H1N1)pdm09, classic seasonal strains, swine strains as well as highly virulent avian strain H5N1, identified with the aid of the Immune Epitope DataBase (IEDB), were compared at genomic level. The result showed that A (H1N1) pdm09 contains the 90% of B-cell epitopes for broadly cross-reactive antibodies (EBCA), which is in consonance with the recent reports on the experimental identification of new epitopes or antibodies for this virus and the binding tests with influenza virus protein HA of different subtypes. Our analysis supports that high proportional EBCA depends on the epitope pattern of A (H1N1)pdm09 virus. This study may be helpful for better understanding of A (H1N1)pdm09 and the production of new influenza vaccines.     

Antibody responses against influenza A(H1N1)pdm09 virus after sequential vaccination with pandemic and seasonal influenza vaccines in Finnish healthcare professionals

Background Influenza A(H1N1)pdm09 virus has been circulating in human population for three epidemic seasons. During this time, monovalent pandemic and trivalent seasonal influenza vaccination against this virus have been offered to Finnish healthcare professionals. It is, however, unclear how well vaccine‐induced antibodies recognize different strains of influenza A(H1N1)pdm09 circulating in the population and whether the booster vaccination with seasonal influenza vaccine would broaden the antibody cross‐reactivity. Objectives Influenza vaccine‐induced humoral immunity against several isolates of influenza A(H1N1)pdm09 virus was analyzed in healthcare professionals. Age‐dependent responses were also analyzed. Methods Influenza viruses were selected to represent viruses that circulated in Finland during two consecutive influenza epidemic seasons 2009–2010 and 2010–2011. Serum samples from vaccinated volunteers, age 20–64 years, were collected before and after vaccination with AS03‐adjuvanted pandemic and non‐adjuvanted trivalent seasonal influenza vaccine that was given 1 year later. Results Single dose of pandemic vaccine induced a good albeit variable antibody response. On day 21 after vaccination, depending on the virus strain, 14–75% of vaccinated had reached antibody titers (≥1:40) considered seroprotective. The booster vaccination 1 year later with a seasonal vaccine elevated the seroprotection rate to 57–98%. After primary immunization, younger individuals (20–48 years) had significantly higher antibody titers against all tested viruses than older persons (49–64 years) but this difference disappeared after the seasonal booster vaccination. Conclusions Even a few amino acid changes in influenza A HA may compromise the vaccine‐induced antibody recognition. Older adults (49 years and older) may benefit more from repeated influenza vaccinations.     

Whole‐genome analysis of influenza A(H1N1)pdm09 viruses isolated in Uganda from 2009 to 2011

We report a whole‐genome analysis of 19 influenza A(H1N1)pdm09 isolates from four Ugandan hospitals between 2009 and 2011. The isolates differed from the vaccine strain A/California/07/2009 by three amino acid substitutions P100S, S220T, and I338V in the hemagglutinin and by two amino acid substitutions V106I and N248D in the neuraminidase proteins with consistent mutations in all gene segments distinguishing isolates from the 2009/2010 to 2010/2011 seasons. Phylogenetic analysis showed low genetic evolution, with genetic distances of 0%–1.3% and 0.1%–1.6% for HA and NA genes, respectively. The amino acid substitutions did not lead to antigenic differences from the reference strains.     

Immunogenicity of influenza A(H1N1)pdm09 vaccine and the associated factors on lowered immune response in patients with hepatitis C

Background Patients with underlying disease represent a high‐risk group for influenza‐associated complications and hospitalization. However, few studies investigated the immunogenicity of influenza vaccine in patients with liver disease. Objective To examine immunogenicity of influenza A(H1N1)pdm09 vaccine in patients with liver disease and to explore the associated factors on lowered immune response. Patients/Methods A single subcutaneous dose of monovalent inactivated unadjuvanted split‐virus influenza A(H1N1)pdm09 vaccination was performed in 80 patients with chronic hepatitis C virus infection at Osaka City University Hospital in Japan. To measure the hemagglutination inhibition antibody titer, serum samples were collected before and 3 weeks after vaccination. Results No serious adverse events were observed. After vaccination, antibody titers ≥1:40 were observed in 56 patients (71%). The corresponding seroconversion proportion was 72%, and the mean fold rise was 10·3. Immune responses were robust regardless of severity of liver disease or existence of probable cirrhosis. However, patients with older age, lower body mass index, or receiving Stronger Neo‐Minophagen C tended to show lower antibody responses to A(H1N1)pdm09 vaccine. In addition, reduced immune responses were observed in patients who had received the 2009/10 seasonal vaccination prior to A(H1N1)pdm09 vaccination. Conclusions Single dose of A(H1N1)pdm09 vaccine achieved a sufficient level of immunity among patients with chronic hepatitis C. Antibody response may be affected by age, body mass index, Stronger Neo‐Minophagen C administration, and recent seasonal influenza vaccination.     

Genotypic Variants of Pandemic H1N1 Influenza A Viruses Isolated from Severe Acute Respiratory Infections in Ukraine during the 2015/16 Influenza Season

Human type A influenza viruses A(H1N1)pdm09 have caused seasonal epidemics of influenza since the 2009–2010 pandemic. A(H1N1)pdm09 viruses had a leading role in the severe epidemic season of 2015/16 in the Northern Hemisphere and caused a high incidence of acute respiratory infection (ARI) in Ukraine. Serious complications of influenza-associated severe ARI (SARI) were observed in the very young and individuals at increased risk, and 391 fatal cases occurred in the 2015/16 epidemic season. We analyzed the genetic changes in the genomes of A(H1N1)pdm09 influenza viruses isolated from SARI cases in Ukraine during the 2015/16 season. The viral hemagglutinin (HA) fell in H1 group 6B.1 for all but four isolates, with known mutations affecting glycosylation, the Sa antigenic site (S162N in all 6B.1 isolates), or virulence (D222G/N in two isolates). Other mutations occurred in antigenic site Ca (A141P and S236P), and a subgroup of four strains were in group 6B.2, with potential alterations to antigenicity in A(H1N1)pdm09 viruses circulating in 2015/16 in Ukraine. A cluster of Ukrainian isolates exhibited novel D2E and N48S mutations in the RNA binding domain, and E125D in the effector domain, of immune evasion nonstructural protein 1 (NS1). The diverse spectrum of amino-acid substitutions in HA, NS1, and other viral proteins including nucleoprotein (NP) and the polymerase complex suggested the concurrent circulation of multiple lineages of A(H1N1)pdm09 influenza viruses in the human population in Ukraine, a country with low vaccination coverage, complicating public health measures against influenza.     

Discordant antigenic drift of neuraminidase and hemagglutinin in H1N1 and H3N2 influenza viruses

Seasonal epidemics caused by influenza virus are driven by antigenic changes (drift) in viral surface glycoproteins that allow evasion from preexisting humoral immunity. Antigenic drift is a feature of not only the hemagglutinin (HA), but also of neuraminidase (NA). We have evaluated the antigenic evolution of each protein in H1N1 and H3N2 viruses used in vaccine formulations during the last 15 y by analysis of HA and NA inhibition titers and antigenic cartography. As previously shown for HA, genetic changes in NA did not always lead to an antigenic change. The noncontinuous pattern of NA drift did not correspond closely with HA drift in either subtype. Although NA drift was demonstrated using ferret sera, we show that these changes also impact recognition by NA-inhibiting antibodies in human sera. Remarkably, a single point mutation in the NA of A/Brisbane/59/2007 was primarily responsible for the lack of inhibition by polyclonal antibodies specific for earlier strains. These data underscore the importance of NA inhibition testing to define antigenic drift when there are sequence changes in NA.