甲型H1N1流行性感冒病毒血凝素基因变异状况及序列分析

目的 了解2009年度甲型H1N1流行性感冒(流感)病毒的检测情况和血凝素(HA)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,通过实时(RT)-PCR进行病毒分型及甲型H1N1流感病毒检测,对阳性标本采用狗肾细胞(MDCK)进行病原分离,采用红细胞凝集试验测定病毒效价,用血凝抑制实验进行型别鉴定,通过RT-PCR扩增毒株HA1片段的基因并进行序列测定,利用生物信息学技术进行序列分析.结果 共检测咽拭子样本996份,其中核酸检测阳性病例包括甲型H1N1 337份,季节性H1N1亚型1份,季节性H3N2亚型67份,B型12份,流感核酸检测阳性率为41.87％,其中甲型流感核酸检测阳性率为33.84％.分离出甲型H1N1病毒36株,选择18株.测序成功的10株甲型H1N1流感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 2009年度分离到的流感病毒株中以甲型H1N1为绝对的优势毒株,毒株的血凝素基因与世界卫生组织(WHO)提供的疫苗株相比有变异,与疫苗株相比,抗原决定簇B区有改变,但关键位点第222位没有变化.     

马鞍山市2016-2017年流感病原学监测结果分析北大核心CSCD

目的分析2016-2017马鞍山市流感病毒病原学监测结果,为流感防制提供科学依据。方法收集、分析马鞍山市2016-2017年流感样病例(influenza-like illness,ILI)监测数据,同时采集部分ILI咽拭子标本,采用RT-PCR法检测流感病毒核酸,对病原学检测数据进行统计分析。结果 2016年共检测ILI咽拭子标本2 772份,流感病毒核酸检测阳性383例,总阳性率为13.82%。其中新甲型H1N1亚型59例(占15.40%),H3N2亚型111例(占28.98%),B-Yamagata亚型11例(占2.87%),B-Victoria亚型202例(占52.74%);2017年共检测ILI咽拭子标本2 170份,流感病毒核酸检测阳性614例,总阳性率为28.29%,其中新甲型H1N1亚型38例(占52.74%),H3N2亚型309例(占50.33%),B-Yamagata亚型178例(占28.99%),B-Victoria亚型89例(占14.50%)。不同月份阳性检出率比较差异有统计学意义(年X_(2016)~2=450.876,X_(2017)~2=240.002,P均<0.05)。核酸检测阳性率性别差异无统计学意义(X_(2016)~2=0.171,X_(2017)~2=0.174,P均>0.05)。不同年龄组间阳性率比较差异有统计学(X_(2016)~2=160.030,X_(2017)~2=132.901,P均<0.05)。2016、2017年MDCK细胞培养法分离流感病毒221株和98株,分离率分别为57.70%和15.96%;2016、2017年鸡胚培养法分离流感病毒26株和8株,分离率分别为6.79%和1.30%。两种方法比较有统计学差异(X^2=74.815,P<0.05)。结论 2016年马鞍山市流感呈现冬春季两个发病高峰,主要流行株为B-Victoria型;2017年出现夏、冬、春季3个发病高峰,主要流行株为H3N2型。流行株发病高峰的变化可为当地流感防控提供参考,流感监测应进一步加强。     

马鞍山市2016-2017年流感病原学监测结果分析北大核心CSCD

目的分析2016-2017马鞍山市流感病毒病原学监测结果,为流感防制提供科学依据。方法收集、分析马鞍山市2016-2017年流感样病例(influenza-like illness,ILI)监测数据,同时采集部分ILI咽拭子标本,采用RT-PCR法检测流感病毒核酸,对病原学检测数据进行统计分析。结果 2016年共检测ILI咽拭子标本2 772份,流感病毒核酸检测阳性383例,总阳性率为13.82%。其中新甲型H1N1亚型59例(占15.40%),H3N2亚型111例(占28.98%),B-Yamagata亚型11例(占2.87%),B-Victoria亚型202例(占52.74%);2017年共检测ILI咽拭子标本2 170份,流感病毒核酸检测阳性614例,总阳性率为28.29%,其中新甲型H1N1亚型38例(占52.74%),H3N2亚型309例(占50.33%),B-Yamagata亚型178例(占28.99%),B-Victoria亚型89例(占14.50%)。不同月份阳性检出率比较差异有统计学意义(年X_(2016)~2=450.876,X_(2017)~2=240.002,P均<0.05)。核酸检测阳性率性别差异无统计学意义(X_(2016)~2=0.171,X_(2017)~2=0.174,P均>0.05)。不同年龄组间阳性率比较差异有统计学(X_(2016)~2=160.030,X_(2017)~2=132.901,P均<0.05)。2016、2017年MDCK细胞培养法分离流感病毒221株和98株,分离率分别为57.70%和15.96%;2016、2017年鸡胚培养法分离流感病毒26株和8株,分离率分别为6.79%和1.30%。两种方法比较有统计学差异(X^2=74.815,P<0.05)。结论 2016年马鞍山市流感呈现冬春季两个发病高峰,主要流行株为B-Victoria型;2017年出现夏、冬、春季3个发病高峰,主要流行株为H3N2型。流行株发病高峰的变化可为当地流感防控提供参考,流感监测应进一步加强。     

2010～2011年马鞍山市流感疫情监测分析

目的了解马鞍山市流感流行动态,探索流行规律,为流感的防制提供科学依据。方法收集监测医院流感样病例(ILI)数据,采集部分ILI咽拭子标本,采用实时荧光定量RT-PCR技术检测流感病毒核酸。结果马鞍山市2010～2011年共报告ILI 13 151例,占门诊就诊人数的4.79%。采咽拭子3 185份,核酸检测阳性486份,阳性率15.26%,其中A型H1N1亚型47份、A型H3N2亚型119份、B型320份。结论 2010～2011年马鞍山市流感呈现冬春季及夏秋季两个发病高峰,流感病毒以B型为主;流感的监测应进一步加强。     

2013—2022年阿克苏地区儿童流感样病例监测结果分析

目的 分析新疆阿克苏地区儿童流行性感冒(简称流感)病原学特征,为当地流感防控提供科学依据。方法收集2013—2022年阿克苏地区流感监测哨点医院流感样病例(ILI)信息,采集咽试子标本,利用实时荧光RT-PCR法检测流感病毒,统计分析儿童流感样病例监测结果。结果 2013—2022年共检测儿童流感样病例标本4 582份,其中阳性464份、阳性率10.13%。不同年度甲乙流感病毒构成差异有统计学意义(χ²=559.066,P<0.05)、甲型流感不同亚型构成差异有统计学意义(χ²=424.779,P<0.05)、乙型流感不同系别构成差异有统计学意义(χ²=350.000,P<0.05);儿流行高峰从12月—次年3月,12月阳性率最高27.78%;6—9月阳性率最低0.00%。7～10岁儿童阳性率最高14.95%,1岁以下儿童阳性率最低3.73%;儿童流感阳性率无性别差异(χ²=3.718,P>0.05)。MDCK细胞分离培养出流感病毒93株。结论阿克苏地区出现儿童甲乙型流感病毒...     

犬肾传代细胞分离流行性感冒病毒致细胞病变的敏感性

目的 了解犬肾传代细胞系(MDCK)细胞对甲型流行性感冒(流感)患者鼻咽拭标本培养1代、2代、3代致细胞病变的敏感性.方法 甲型流感患者鼻咽拭标本在MDCK细胞盲传3代培养,倒置显微镜观察每代细胞致细胞病变效应产生情况.结果 经胶体金快速检测为甲型流感的279份流感样患者鼻咽拭标本,MDCK培养第1代产生致细胞病变为184份,占65.9%,第2代产生致细胞病变为255份,占91.4%,第3代产生致细胞病变为269份,占96.4%.279份标本的细胞培养液经多重RT-PCR检测鉴定为甲型流感病毒的有271份.结论 MDCK分离培养甲型流感患者鼻咽拭标本经盲传3代后,基本可达到95%以上的阳性分离率.     

3种方法对发热伴血小板减少综合征检测结果比较

目的 采用实时荧光PCR法、酶联免疫吸附法及细胞培养分离病毒株的方法,对2013年临床疑似发热伴血小板减少综合征患者急性期血进行检测比较,寻求快速、准确的鉴定新布尼亚病毒感染的方法。方法 对180例临床疑似病例的急性期血液首先采用实时荧光PCR法检测,筛选出新布尼亚病毒核酸阳性的样本,同时将阳性样本采用酶联免疫吸附的方法检测抗体及采用VERO-E6细胞培养的方法分离病毒株。结果 180份样本经实时荧光PCR法检测,阳性率为42.78%(77/180 Ct值≤35曲线形态与阳性对照一致呈S型的),ELISA方法检测抗体阳性率为44.16%(34/77),将核酸检测阳性的77份样本全部感染VERO-E6细胞获得病毒株32株,阳性率为38.55%(32/77)。结论 实时荧光PCR法更适合于新布尼亚病毒感染者早期实验室快速诊断,发病在1w左右的病例不适合用ELISA的方法做确证实验。细胞培养分离病毒虽然是金标准,但耗时、费力,对早期诊断意义不大,但适于研究或疫苗研发。     

2021—2022年南通市流感监测结果分析

目的 了解2021—2022年度江苏省南通市流行性感冒(简称流感)流行特征及优势毒株监测情况,为流感防控提供科学依据。方法 收集2021—2022年南通市流感监测哨点医院上报的流感样病例(influenza-like illness,ILI)资料和病原学检测结果,采集ILI病例咽拭子标本,使用实时荧光定量PCR技术检测流感病毒核酸。采用描述流行病学方法分析数据。结果 两家哨点医院门急诊就诊病例共计836 164例,ILI病例78 965例、ILI%为9.44%;2021年和2022年ILI%分别为6.47%和13.38%。共采集咽拭子标本4 170份,流感病毒核酸检测阳性416份、阳性率9.98%;2021与2022年的阳性率分别为6.12%与13.85%,差异有统计学意义(χ2=69.213,P<0.05);不同年龄组阳性率差异有统计学意义(χ2=298.580,P<0.05)。不同性别阳性率差异无统计学意义(χ2=0.883,P>0.05)。呈冬春季和夏季双高峰分布。病毒流行株2021年为B型(Victoria),2022年为A型(H1N1、H3N2)。流感暴发疫情以中小学校为主、占91.78%。结论2021—2022年南通市流感病毒的流行优势株不同,阳性率和构成比均较高;流感仍然是重点防控传染病之一。     

严重急性呼吸综合征病原体的检测方法探讨

目的 在实验室建立SARS病原学检测方法。方法 采集临床诊断为SARS患者、疑似病人漱口液、用细胞培养分离,荧光定量PCR方法检测。病原体用间接免疫荧光鉴定。结果 漱口液、咽拭子液样本57份作病原体分离,阳性数5份,阳性率8.8％尸解标本7份,其中肺部组织3份、气管黏液、淋巴液、肺积液、心包液各1份。结果细胞分离到7株病原体。阳性率100％。荧光定量PCR方法检测临床确诊SARS患者标本57份,检出阳性结果38份,阳性率为66.7％。密切接触者标本14份,检出阳性结果7份,阳性率为50.0％。疑似患者标本59份中检出阳性12份,阳性率为20.3％。两种方法的比较,统计学上有显著性差异(P<0.001)。结论用荧光定量PCR方法检测SARS患者病原体敏感性高,可靠性和重复性好,优于细胞病毒分离的方法,可以作为SARS临床早期诊断和流行病学调查的重要指标之一。     

The diagnosis of influenza.

Our ability to establish a specific diagnosis of influenza infections has dramatically improved. Clinical signs and symptoms of influenza infection and epidemiological indicators of an influenza outbreak can be verified with a variety of rapid detection methods. Viral isolation and an acute change in serology, which characteristically took from 5 to 28 days, are now being supplemented with methods that detect influenza viral antigen directly on clinical specimens and/or influenza virus in tissue culture within 24 to 48 hours following inoculation. These rapid diagnostic techniques are easily adapted in clinical microbiology laboratories and will provide diagnostic information so that the clinician can prescribe specific antiviral therapy.     

Codon bias and frequency-dependent selection on the hemagglutinin epitopes of influenza A virus

Although the surface proteins of human influenza A virus evolve rapidly and continually produce antigenic variants, the internal viral genes acquire mutations very gradually. In this paper, we analyze the sequence evolution of three influenza A genes over the past two decades. We study codon usage as a discriminating signature of gene- and even residue-specific diversifying and purifying selection. Nonrandom codon choice can increase or decrease the effective local substitution rate. We demonstrate that the codons of hemagglutinin, particularly those in the antibody-combining regions, are significantly biased toward substitutional point mutations relative to the codons of other influenza virus genes. We discuss the evolutionary interpretation and implications of these biases for hemagglutinin's antigenic evolution. We also introduce information-theoretic methods that use sequence data to detect regions of recent positive selection and potential protein conformational changes.     

2019-2021年南通市活禽市场外环境禽流感病毒检测结果分析北大核心CSCD

目的对2019-2021年南通市活禽市场外环境标本禽流感病毒检测结果进行分析,了解禽流感病毒流行、分布特征,为南通市禽流感防控工作提供依据。方法选取2019-2021年南通市崇川区、通州区、海安市、如皋市活禽市场外环境标本,采用实时荧光反转录-聚合酶链反应(RT-PCR)方法对标本进行H5亚型、H7亚型、H9亚型流感病毒检测,采用描述性流行病学方法对检测结果进行统计分析。结果南通市全年均有禽流感阳性样本检出,其中以第4季度检出率较高。甲型禽流感病毒核酸阳性率为21.69%(141/650)。其中H5亚型阳性率为0.62%(4/650)、H7亚型阳性率为0.31%(2/141)、H9亚型阳性率为18.62%(121/650)。活禽市场的宰杀或摆放禽内案板表面擦拭标本和禽类内脏阳性率最高;各监测地区禽流感病毒核酸阳性率差异有统计学意义。结论南通市活禽市场外环境中禽流感病毒以H9亚型为主,应加强禽流感病毒的监测防控工作,加强对活禽市场的卫生监管。     

Potency determination of inactivated H7 influenza vaccines using monoclonal antibody‐based ELISA and biolayer interferometry assays

Background

The single radial immunodiffusion (SRID) assay, the accepted method for determining potency of inactivated influenza vaccines, measures an immunogenic form of the influenza hemagglutinin. Nevertheless, alternative methods for measuring vaccine potency have been explored to address some of the weaknesses of the SRID assay, including limited sensitivity and the requirement for large amounts of standardized reagents. Monoclonal antibody (mAb)‐based potency assays also have the ability to detect and measure relevant immunogenic forms of HA.

Objectives

The objective of this study was to continue evaluation of mAb‐based alternative methods for measuring the potency of inactivated influenza vaccines, focusing on A(H7N9) pandemic influenza vaccines.

Methods

Several murine mAbs that recognize different epitopes on the H7 hemagglutinin (HA) were identified and characterized. These mAbs were evaluated in both a mAb‐capture ELISA and a mAb‐based biolayer interferometry (BLI) assay.

Results

Results indicated that potency of inactivated A(H7N9) vaccines, including vaccine samples that were stressed by heat treatment, measured by either alternative method correlated well with potency determined by the traditional SRID potency assay.

Conclusions

The availability of multiple H7 mAbs, directed to different HA epitopes, provides needed redundancy in the potency analysis as A(H7N9) viruses continue to evolve antigenically and suggests the importance of having a broad, well‐characterized panel of mAbs available for development of vaccines against influenza strains with pandemic potential. In addition, the results highlight the potential of mAb‐based platform such as ELISA and BLI for development as alternative methods for determining the potency of inactivated influenza vaccines.     

114例疑似COVID-19患者呼吸道病毒感染分析

目的 分析114例COVID-19疑似患者呼吸道病毒感染情况, 探讨多种病毒同时检测在疫情防控中的价值。方法 利用Real Time RT-PCR技术检测发热门诊COVID-19疑似患者SARS-CoV-2核酸,利用基因芯片恒温扩增技术检测SARS-CoV-2核酸阴性患者其他常见的18种呼吸道病毒。结果 114例COVID-19疑似患者SARS-CoV-2核酸均为阴性,有21例感染了非其它呼吸道病毒,感染率达18.42%,21例患者中共检测出10种呼吸道病毒,包括冠状病毒NL63/229型、呼吸道合胞病毒、柯萨奇病毒A16型、乙型流感病毒、人副流感病毒1型、人副流感病毒3型、人偏肺病毒、甲型流感病毒、甲型流感病毒季节性H3亚型、肠道病毒/鼻病毒。其中乙型流感病毒感染患者最多,有6例,呼吸道合胞病毒有5例。有3例患者同时感染2种病毒:呼吸道合胞病毒与柯萨奇病毒A16型混合感染、冠状病毒NL63/229型与人副流感病毒1型混合感染、甲型流感病毒与甲型流感病毒季节性H3亚型混合感染。结论 在应对本次SARS-CoV-2疫情中,针对COVID-19疑似患者中,要注意鉴别SARS-CoV-2与其它呼吸道病毒,及时有效地排除疑似病例。     

Analytical detection of influenza A(H3N2)v and other A variant viruses from the USA by rapid influenza diagnostic tests

Please cite this paper as: Balish et al. (2012) Analytical detection of influenza A(H3N2)v and other A variant viruses from the USA by rapid influenza diagnostic tests. Influenza and Other Respiratory Viruses 7(4), 491–496. Background The performance of rapid influenza diagnostic tests (RIDTs) that detect influenza viral nucleoprotein (NP) antigen has been reported to be variable. Recent human infections with variant influenza A viruses that are circulating in pigs prompted the investigation of the analytical reactivity of RIDTs with these variant viruses. Objectives To determine analytical reactivity of seven FDA‐cleared RIDTs with influenza A variant viruses in comparison with the reactivity with recently circulating seasonal influenza A viruses. Methods Tenfold serial dilutions of cell culture–grown seasonal and variant influenza A viruses were prepared and tested in duplicate with seven RIDTs. Results All RIDTs evaluated in this study detected the seasonal influenza A(H3N2) virus, although detection limits varied among assays. All but one examined RIDT identified the influenza A(H1N1)pdm09 virus. However, only four of seven RIDTs detected all influenza A(H3N2)v, A(H1N2)v, and A(H1N1)v viruses. Reduced sensitivity of RIDTs to variant influenza viruses may be due to amino acid differences between the NP proteins of seasonal viruses and the NP proteins from viruses circulating in pigs. Conclusions Clinicians should be aware of the limitations of RIDTs to detect influenza A variant viruses. Specimens from patients with influenza‐like illness in whom H3N2v is suspected should be sent to public health laboratories for additional diagnostic testing.     

Comparison of performance between Fast Track Diagnostics Respiratory Kit and the CDC global reference laboratory for influenza rRT‐PCR panel for detection of influenza A and influenza B

BackgroundReliable diagnostics are a key to identifying influenza infections.ObjectivesOur objectives were to describe the detection of influenza among severe acute respiratory infection (SARI) cases, to compare test results from the Fast Track Diagnostics (FTD) Kit for influenza detection to the Centers for Disease Control (CDC) human influenza virus detection and characterization panel, and to assess seasonality of influenza in Burkina Faso.MethodsNasopharyngeal and oropharyngeal specimens from SARI cases (hospitalized patients with fever, cough, and onset in the previous 10 days) were tested using the FTD‐33 Kit and the CDC rRT‐PCR influenza assays. We assessed sensitivity and specificity of the FTD‐33 Kit for detecting influenza A, influenza B, and the influenza A(H1N1)pdm09 strain using the CDC human influenza rRT‐PCR panel as the gold standard.ResultsFrom December 2016 to February 2019, 1706 SARI cases were identified, 1511 specimens were tested, and 211 were positive for influenza A (14.0%) and 100 for influenza B (6.6%) by either assay. Higher influenza circulation occurred between November and April with varying peaks of influenza A and influenza B. Sensitivity of the FTD‐33 assay was 91.9% for influenza A, 95.7% for influenza B, and 93.8% for A(H1N1)pdm09 subtype. Specificity was over 99% for all three tests.ConclusionsOur study indicates that Burkina Faso has one peak of influenza each year which is similar to the Northern Hemisphere and differs from other countries in West Africa. We found high concordance of influenza results between the two assays indicating FTD‐33 can be used to reliably detect influenza among SARI cases.     

Search of amantadine-resistance in influenza A strains isolated in Santiago, Chile, 2001-2002

Amantadine has been used for prevention and treatment of influenza A infection. It blocks the proton through the M2 ion channel. Drug-resistant viruses appear quickly when this therapy is used. Single amino acids changes in the H2 protein can confer resistance, being the most frequent one in position 31. Different methods to detect resistant strains have been described. The objectives were to determine the existence of amantadine resistance of influenza A strains isolated in a virologic laboratory in Santiago, Chile, between 2001-2002, and to validate a new molecular method to detect resistant strains. A PCR restriction fragment length polymorphism analysis was employed for the detection of resistant viruses. In 31 processed strains no mutation in the position 31 was found. This result supports that amantadine resistance is very low or absent in Chile. This could be explained by a limited use of this drug in the study population. This method could be used as a monitoring system to survey resistant viruses.     

Rapid Detection and Differentiation of Swine-Origin Influenza A Virus (H1N1/2009) from Other Seasonal Influenza A Viruses

We previously developed a rapid and simple gold nanoparticle(NP)-based genomic microarray assay for identification of the avian H5N1 virus and its discrimination from other influenza A virus strains (H1N1, H3N2). In this study, we expanded the platform to detect the 2009 swine-origin influenza A virus (H1N1/2009). Multiple specific capture and intermediate oligonucleotides were designed for the matrix (M), hemagglutinin (HA), and neuraminidase (NA) genes of the H1N1/2009 virus. The H1N1/2009 microarrays were printed in the same format as those of the seasonal influenza H1N1 and H3N2 for the HA, NA, and M genes. Viral RNA was tested using capture-target-intermediate oligonucleotide hybridization and gold NP-mediated silver staining. The signal from the 4 capture-target-intermediates of the HA and NA genes was specific for H1N1/2009 virus and showed no cross hybridization with viral RNA from other influenza strains H1N1, H3N2, and H5N1. All of the 3 M gene captures showed strong affinity with H1N1/2009 viral RNA, with 2 out of the 3 M gene captures showing cross hybridization with the H1N1, H3N2, and H5N1 samples tested. The current assay was able to detect H1N1/2009 and distinguish it from other influenza A viruses. This new method may be useful for simultaneous detection and subtyping of influenza A viruses and can be rapidly modified to detect other emerging influenza strains in public health settings.