2017 - 2018年山东省甲型H1N1流感病毒耐药性分析

目的 调查山东省2017 - 2018流感监测年度甲型H1N1流感病毒对神经氨酸酶抑制剂（NAI）的耐药情况,分析其神经氨酸酶（NA）基因特征。方法 选取山东省2017 - 2018流感监测年度分离的20株甲型H1N1流感病毒进行生物学耐药实验,检测病毒对奥司他韦和扎那米韦的药物敏感性。提取病毒核酸后对NA基因进行测序,利用生物信息学软件分析NA基因上与耐药有关的氨基酸位点及其基因变异情况。结果 选取的20株甲型H1N1流感病毒全部对奥司他韦和扎那米韦敏感,对奥司他韦的IC50平均数为0.37 nM（0.15～0.89 nM）,对扎那米韦的IC50平均数为0.28 nM（0.14～0.78 nM）。NA基因测序结果也没有发现导致耐药的突变位点。结论 山东省的甲型H1N1流感病毒对NAI敏感,临床上可继续使用此类药物对流感患者进行治疗。     

2017-2018年河南省流感病毒对神经氨酸酶抑制剂药物的耐药性研究

  目的  分析河南省2017-2018年流感病毒对神经氨酸酶抑制剂（neuraminidase inhibitor, NAI）药物的耐药性, 为流感抗病毒药物的使用提供依据。  方法  病毒来自2017-2018年河南省流感监测网络送检的毒株标本, 经复核鉴定后, 挑选H1pdm09亚型15株、H3N2亚型6株、B型15株共36株毒株使用荧光发光法（fluorescence-basedassay, FL）检测其对奥司他韦和扎那米韦的耐药性。药物敏感参考株为A/California/12/2012（H1pdm09）-275H、A/北京海淀/1942/2014（H3N2）-119E和B/Rochester/02/2001-198D;药物耐药参考株为A/Texas/23/2012（H1pdm9）-H275Y、A/Texas/12/2007（H3N2）-E119V和B/Rochester/02/2001-D198N。  结果  药物敏感参考株为A/California/12/2012（H1pdm09）-275H、A/北京海淀/1942/2014（H3N2）-119E和B/Rochester/02/2001-198D对奥司他韦的半数抑制浓度（half maximal inhibitory concentration, IC₅₀）值分别为0.29 nmol/L（nM）、0.10 nM和12.71 nM, 对扎那米韦的IC₅₀值分别为0.2 nM、0.49 nM和0.33 nM。2017-2018年河南省H1pdm09和H3N2亚型流感病毒对奥司他韦的IC₅₀值范围分别为（0.28~1.37 nM）和（0.08~0.17 nM）, 对扎那米韦的IC₅₀值范围为（0.15~0.53 nM）和（0.12~0.22 nM）, 均在相对应型别参考株IC₅₀值10倍以内; B型对奥司他韦的IC₅₀值范围为（11.83~24.59 nM）, 对扎那米韦的IC₅₀值范围为（0.48~1.25 nM）, 均在参考株的IC₅₀值的5倍以内。  结论  2017-2018年河南省检测的流感毒株都对神经氨酸酶抑制剂药物敏感。     

2018-2019年流感流行季节河北省流感病毒对神经氨酸酶抑制剂药物的耐药性研究

目的  分析河北省2018-2019年流感流行季节流感病毒对神经氨酸酶抑制剂类(neuraminidase inhibitor, NAI)药物的耐药情况, 为流感的预防控制提供依据。 方法  选取H1N1(pdm09)亚型毒株25株、H3N2亚型毒株11株共36株流感病毒毒株进行生物学耐药实验, 使用荧光发光法检测病毒对奥司他韦和扎纳米韦的药物敏感性。 结果  2018-2019年河北省H1N1(pdm09)和H3N2亚型流感病毒全部对奥司他韦和扎那米韦敏感, 对奥司他韦的半数抑制浓度(half maximal inhibitory concentration, IC₅₀)平均数分别为0.50 nM(0.07~1.14 nM)和0.25 nM(0.09~0.69 nM), 对扎那米韦的IC₅₀平均数分别为0.29 nM(0.09~0.85 nM)和0.87 nM(0.17~1.81 nM), 均在相对应型别参考株IC₅₀值10倍以内。 结论  2018-2019年河北省检测的流感毒株都对NAI类药物敏感。     

Structural basis for oseltamivir resistance of influenza viruses

Oseltamivir, one of the two anti-neuraminidase drugs, is currently the most widely used drug against influenza. Resistance to the drug has occurred infrequently among different viruses in response to drug treatment, including A H5N1 viruses, but most notably has emerged among recently circulating A H1N1 viruses and has spread throughout the population in the absence of drug use. Crystal structures of enzyme–drug complexes, together with enzymatic properties, of mutants of H5N1 neuraminidase have provided explanations for high level oseltamivir resistance due to the common H275Y mutation, with retention of zanamivir susceptibility, and intermediate level resistance due to the N295S mutation. Complementation of enhanced NA activity due to a D344N mutation by the H275Y mutation suggests an explanation for the recent emergence and predominance of oseltamivir-resistant influenza A H1N1 viruses.     

Monitoring and characterization of oseltamivir-resistant pandemic (H1N1) 2009 virus, Japan, 2009-2010

To monitor and characterize oseltamivir-resistant (OR) pandemic (H1N1) 2009 virus with the H275Y mutation, we analyzed 4,307 clinical specimens from Japan by neuraminidase (NA) sequencing or inhibition assay; 61 OR pandemic (H1N1) 2009 viruses were detected. NA inhibition assay and M2 sequencing indicated that OR pandemic (H1N1) 2009 virus was resistant to M2 inhibitors, but sensitive to zanamivir. Full-genome sequencing showed OR and oseltamivir-sensitive (OS) viruses had high sequence similarity, indicating that domestic OR virus was derived from OS pandemic (H1N1) 2009 virus. Hemagglutination inhibition test demonstrated that OR and OS pandemic (H1N1) 2009 viruses were antigenically similar to the A/California/7/2009 vaccine strain. Of 61 case-patients with OR viruses, 45 received oseltamivir as treatment, and 10 received it as prophylaxis, which suggests that most cases emerged sporadically from OS pandemic (H1N1) 2009, due to selective pressure. No evidence of sustained spread of OR pandemic (H1N1) 2009 was found in Japan; however, 2 suspected incidents of human-to-human transmission were reported.     

Oseltamivir-resistant pandemic (H1N1) 2009 virus, Mexico

During May 2009-April 2010, we analyzed 692 samples of pandemic (H1N1) 2009 virus from patients in Mexico. We detected the H275Y substitution of the neuraminidase gene in a specimen from an infant with pandemic (H1N1) 2009 who was treated with oseltamivir. This virus was susceptible to zanamivir and resistant to adamantanes and oseltamivir.     

Antiviral Susceptibility of Highly Pathogenic Avian Influenza A(H5N1) Viruses Isolated from Poultry,Vietnam, 2009–2011

We assessed drug susceptibilities of 125 avian influenza A(H5N1) viruses isolated from poultry in Vietnam during 2009–2011. Of 25 clade 1.1 viruses, all possessed a marker of resistance to M2 blockers amantadine and rimantadine; 24 were inhibited by neuraminidase inhibitors. One clade 1.1 virus contained the R430W neuraminidase gene and reduced inhibition by oseltamivir, zanamivir, and laninamivir 12-, 73-, and 29-fold, respectively. Three of 30 clade 2.3.4 viruses contained a I223T mutation and showed 7-fold reduced inhibition by oseltamivir. One of 70 clade 2.3.2.1 viruses had the H275Y marker of oseltamivir resistance and exhibited highly reduced inhibition by oseltamivir and peramivir; antiviral agents DAS181 and favipiravir inhibited H275Y mutant virus replication in MDCK-SIAT1 cells. Replicative fitness of the H275Y mutant virus was comparable to that of wildtype virus. These findings highlight the role of drug susceptibility monitoring of H5N1 subtype viruses circulating among birds to inform antiviral stockpiling decisions for pandemic preparedness.     

Oseltamivir-Resistant Influenza Viruses A (H1N1) during 2007�C2009 Influenza Seasons, Japan

To monitor oseltamivir-resistant influenza viruses A (H1N1) (ORVs) with H275Y in neuraminidase (NA) in Japan during 2 influenza seasons, we analyzed 3,216 clinical samples by NA sequencing and/or NA inhibition assay. The total frequency of ORVs was 2.6% (45/1,734) during the 2007–08 season and 99.7% (1,477/1,482) during the 2008–09 season, indicating a marked increase in ORVs in Japan during 1 influenza season. The NA gene of ORVs in the 2007–08 season fell into 2 distinct lineages by D354G substitution, whereas that of ORVs in the 2008–09 season fell into 1 lineage. NA inhibition assay and M2 sequencing showed that almost all the ORVs were sensitive to zanamivir and amantadine. The hemagglutination inhibition test showed that ORVs were antigenetically similar to the 2008–09 vaccine strain A/Brisbane/59/2007. Our data indicate that the current vaccine or zanamivir and amantadine are effective against recent ORVs, but continuous surveillance remains necessary.     

流感病毒神经酰胺酶功能及其抑制剂的研究进展

神经酰胺酶(NA)是流感病毒的一个表面糖蛋白,在病毒感染宿主细胞及其传播过程中起着重要作用。NA活性中心的多个带正电荷的氨基酸残基高度保守。NA结构的阐明有利于特异性的NA抑制剂的研发。当前广泛应用的2种NA抑制剂——扎那米韦和奥司米韦能有效地抗击流感病毒,但针对NA及血凝素(HA)保守序列突变的耐药株也不断出现。     

2008—2009年南宁市人季节性H1N1流感病毒神经氨酸酶潜在的抗原位点和抑制剂耐药位点分析

目的了解2008—2009年南宁市人季节性H1N1流感病毒神经氨酸酶(NA)基因的遗传进化特征,探讨NA基因潜在的抗原位点和活性位点(抑制剂耐药性位点)变异规律。方法提取2008—2009年20株H1N1流感病毒RNA,采用RT-PCR扩增病毒NA基因后进行序列测定,通过CTLPred软件预测NA基因上潜在的抗原位点,并对NA分子进化和其重要功能位点的遗传变异进行分析。结果将20株H1N1流感病毒毒株与北半球疫苗推荐株A/Solomon/3/2006和A/Bris-bane/59/2007构建进化树,NA进化树共分成3个类群,10株2008年H1N1流感病毒毒株聚集成分支Ⅱ,10株2009年毒株与疫苗株A/Brisbane/59/2007聚集成分支Ⅰ,A/Solomon/3/2006则独立形成1个分支(Ⅲ)。2008年毒株抗原位点替换频率较高,相对于A/Solomon/3/2006氨基酸替换分布在不同的3个区域的3个位点,分别是N64H、Y100H、L367I;2009年度的毒株抗原决定簇位点相对保守。在已知NA的酶活性位点中,2009年所有毒株均在275位点发生了H>Y的突变,而这种变异在2008年的毒株中未有出现。结论 A/Solomon/3/2006落后于2008年毒株,A/Brisbane/59/2007对应的疫苗在2009年能起到较好的保护作用;大量H275Y耐药株的出现提示在流感病毒监测中应密切关注其耐药位点的变异。     

Incidence of antiviral drug resistance markers among human influenza A viruses in the Eastern Mediterranean Region, 2005–2016

BackgroundTwo classes of antiviral drugs are available for influenza antiviral therapy: the adamantanes and the neuraminidase inhibitors (NAIs). Due to the emergence of adamantane-resistant variants, the use of these drugs has been largely limited in the world. The NAIs became the drugs of choice for treatment of influenza A infections. However, amino acid substitutions in the NA protein might lead to reduced sensitivity to NAIs.MethodsThe frequency and distribution of matrix protein 2 (M2) and neuraminidase (NA) variants which confer resistance to antiviral drugs was investigated in the Eastern Mediterranean Region (EMR) between 2005 and 2016. A total of 314 M2 and 1209 NA protein sequences from influenza A/H1N1, A/H1N1pdm09, A/H3N2, and A/H5N1 available in the public database were analyzed.ResultsEighty-six percent of the influenza A viruses detected in the EMR were resistant to adamantanes, among which, H3 strains exhibited the highest (95.32%) level of adamantane resistance. Approximately 98.51% (265/269) of influenza A/H1N1 and H3N2 resistant viruses had the S31N substitution in their M2 sequences. The V27A mutation was the only resistance marker found in A/H5N1 viruses and was detected at a frequency of 7.40% among the investigated viruses. Other resistant mutations L26F, A30T, G34E, and L38F were not detected in any of the variants. We found that 2.81% (n = 34) of the detected NA sequences from influenza A viruses possessed at least one NAI-resistant mutation and the vast majority of resistant viruses 79.41% (27/34) bear the H274Y mutation. The frequency of NAI-resistant viruses was 3.29% (24/729) for the H1N1pdm09, 10.64% (5/47) for the seasonal H1N1, and 4.06% (5/123) for H5N1 viruses. None of the H3N2 viruses analyzed during the study period were resistant to NAIs.ConclusionOur study reveals the emergence and spread of antiviral drug resistant influenza A viruses in the EMR and emphasizes the importance of continuous surveillance to maintain the effective use of the current antivirals.     

Oseltamivir Resistance in Adult Oncology and Hematology Patients Infected with Pandemic (H1N1) 2009 Virus, Australia

We describe laboratory-confirmed influenza A pandemic (H1N1) 2009 in 17 hospitalized recipients of a hematopoietic stem cell transplant (HSCT) (8 allogeneic) and in 15 patients with malignancy treated at 6 Australian tertiary centers during winter 2009. Ten (31.3%) patients were admitted to intensive care, and 9 of them were HSCT recipients. All recipients of allogeneic HSCT with infection <100 days posttransplantation or severe graft-versus-host disease were admitted to an intensive care unit. In-hospital mortality rate was 21.9% (7/32). The H275Y neuraminidase mutation, which confers oseltamivir resistance developed in 4 of 7 patients with PCR positive for influenza after >4 days of oseltamivir therapy. Three of these 4 patients were critically ill. Oseltamivir resistance in 4 (13.3%) of 30 patients who were administered oseltamivir highlights the need for ongoing surveillance of such resistance and further research on optimal antiviral therapy in the immunocompromised.     

2012-2018年青岛市A型(H1N1)pdm09流感病毒奥司他韦耐药株基因特征

目的 了解2012-2018年青岛市人群A型(H1N1)pdm09流感病毒奥司他韦耐药株基因特征。方法 收集2012年4月-2018年3月间青岛市A(H1N1)pdm09毒株397份,逆转录聚合酶链反应(RT-PCR)扩增神经氨酸酶(Neuraminidase,NA)和血凝素(Hemagglutinin,HA)基因全长,序列测定后进行耐药位点和氨基酸变异及进化分析。结果 5株发生了H275Y突变,为奥司他韦耐药株;另有4株S247N突变,可能为奥司他韦耐药株。2012-2018年H275Y突变株检出率依次为2.8 %、2.0 %、0.0 %、1.1 %、0.0 %和0.7 %。NA和HA进化树显示,2012-2013年青岛H275Y突变株与美国耐药株A/Tennessee/03/2013更接近,2013-2014年青岛H275Y突变株与国内株和日本札幌耐药株更接近,这两个年度耐药株的毒株起源可能有所不同。突变株在酶活性位点、抗原决定簇、受体结合位点及其他功能位点(如HA位点D222、Q223和NA位点V241I、N369K和N386K)的转变与野生敏感株一致。结论 青岛市A型(H1N1)pdm09流感病毒奥司他韦耐药株明显增加且流行起源不同,但并未取得比野生株更强的流行能力。奥司他韦仍可作为流感预防和治疗的有效手段。     

Responses to live attenuated influenza vaccine in children vaccinated previously with Pandemrix (ASO3B adjuvanted pandemic A/H1N1pdm09)

BackgroundWe report a phase III/IV open-label study on the immunogenicity of a single dose of a Live Attenuated Influenza Vaccine (LAIV) (Fluenz™) in children naïve to, or in previous receipt of, AS03_B adjuvanted A/H1N1pdm09 influenza vaccine (Pandemrix™), to investigate whether early exposure to an adjuvanted subunit influenza vaccine impacts on subsequent response to quadrivalent LAIV (qLAIV).Method and findingsEligible children were enrolled to receive qLAIV and stratified according to previous Pandemrix™ vaccination. Functional antibody for the vaccine strains were analysed using Haemagglutination Inhibition (HAI); in addition antibodies to the A/H1N1pdm09 strain were measured by Neuraminidase Antibody Inhibition (NAI) and neutralisation assays. Fourfold titre increases by HAI were observed for 39% (95% confidence interval 33–46%) and 43% (37–51%) of subjects for the two influenza B vaccine strains and 8% (5–13%) for the A/H3N2 strain with no significant differences between the Pandemrix™ naïve or previously vaccinated groups in antibody tites pre- or post-vaccination or seroconversion rates. In both groups, the response to the qLAIV A/H1N1pdm09 component was barely detectable, overall HAI seroconversion rate 1.8% (0.5–4.7%). Previous receipt of Pandemrix™ was associated with significantly higher levels of A/H1N1pdm09 neutralising antibody, but decreased NAI titres pre-vaccination, with the differences maintained post-vaccination.ConclusionPrevious receipt of Pandemrix™ has had a significant impact on the influenza immune status of children several years later. Higher levels of neutralising antibody to A/H1N1pdm09 pre- and post-vaccination, but significantly lower levels of antibody to NA, were observed compared with Pandemrix™-naïve children, while responses to influenza B and A/H3N2 and antibody levels prior to vaccination were similar in both groups. This suggests that early vaccination with a powerful adjuvant maintains functional immunity for several years, which prevents natural infection. Alternatively, the AS03_B adjuvant may have re-directed the immune response, with focus towards viral HA and away from viral NA.     

Evolution of the susceptibility to antiviral drugs of A/H3N2 influenza viruses isolated in France from 2002 to 2005

In 2002, prior to the generalization of zanamivir and oseltamivir utilization in France, we had decided to test a large panel of influenza strains to establish the baseline sensitivity of these viruses to anti-neuraminidase drugs. Since 2002, routine monitoring of resistance relies mainly on the results of neuraminidase enzyme assay. The profile of sensitivity of A/H3N2 viruses has not changed during these three influenza seasons. Neuraminidase N2 was shown to be more sensitive to oseltamivir carboxylate than to zanamivir. Amongst 788 viruses tested, only 10 were found with outliers IC(50) values (seven in 2003, one in 2004, and two in 2005). After the sequencing of the neuraminidase segment of these viruses, seven mutations on the stalk or the head of the glycoprotein (A18S, L23F, C42R, R143V, E199K, S332K, R431N) were detected. Moreover during seasons 2003-2005, 12 additional viruses were characterized by a complete lack of neuraminidase activity. In these viruses, viral resistance to NA inhibitors can be related with the presence of a defective neuraminidase RNA segment as evaluated by a plaque reduction assay, these viruses having a reduced dependence on its neuraminidase activity.     

Molecular epidemiology and genetic characterization of influenza B virus in Lebanon during 2016–2018

BackgroundInfluenza B viruses are a major cause of serious acute respiratory infections in humans.MethodsNasopharyngeal swabs were collected from subjects with influenza-like illness during October 2016–June 2018 and screened for influenza A and B. The hemagglutinin (HA) and neuraminidase (NA) genes of the Lebanese influenza B specimens were sequenced and phylogenetically compared with the vaccine strains and specimens from the Eastern Mediterranean Region and Europe.ResultsInfluenza A and B viruses co-circulated between October and May and peaked between January and March. During the 2016–2017 season, A/H3N2 (33.4%) and B/Yamagata (29.7%) were the predominantly circulating viruses followed by B/Victoria and A/H1N1pdm09 viruses. During the 2017–2018 season, A/H3N2 (31.5%) and A/H1Npdm09 (29.3%) were most prevalent with co-circulation of B/Yamagata and to a lesser extent B/Victoria viruses. The B/Yamagata specimens belonged to clade-3 while the B/Victoria belonged to clade-1A. None of the analyzed specimens had a mutation known to confer resistance to NA inhibitors (NAIs).ConclusionMultiple subtypes of influenza co-circulate each year in Lebanon with a peak between January and March. The trivalent vaccine included a B/Victoria strain which mismatched the B/Yamagata lineage that predominated during the study period, highlighting the importance of quadrivalent vaccines.     

甲型H1N1流感病毒神经氨酸酶基因进化分析

目的 探讨2009年山东省甲型H1N1流感病毒神经氨酸酶(neuram inidase,NA)基因的进化及NA基因编码蛋白抗原性、酶活性位点、糖基化位点变异情况.方法 对山东省分离的20株甲型H1N1流感病毒的NA基因序列,用MEGA 4.0软件进行基因进化分析和氨基酸序列分析.结果 山东省2009年9月-2010年1月分离的20株甲型H1N1流感病毒NA基因的同源性均>99.2%,其中有4株的同源性为100%;与疫苗株[A-California-07-2009(H1N1)]、国内推荐株[A-Sichuan-SWL1-2009(H1N1)]的同源性分别为99.1%~99.5%和99.1%~99.6%;有13个神经氨酸酶基因的氨基酸发生了替换,为21、23、25、42、43、60、76、94、106、122、248、307和351位;未发生神经氨酸酶蛋白275位H-Y的替换.结论 山东省分离的甲型H1N1流感病毒NA基因高度保守,潜在抗原位点氨基酸分布相同;所有毒株的酶活性中心位点以及糖基化位点均高度保守;所有测定样本均未发生对达菲类药物的耐药性突变.     

甲型H1N1流感病毒基因组序列分析及其特性研究

目的 分析甲型H1N1流感病毒的基因组序列特征,阐明该毒株的遗传变异及分子特性.方法 GenBank中获取流感病毒全序列,对各段基因与已知序列进行分析比较,绘制进化树,并分析和预测甲型毒株的致病性、药物敏感性和现有疫苗的预防保护作用.结果 甲型H1N1病毒的HA、PB2、PB1、PA、NP、NS基因与美国本土的猪流感病毒序列具有高度同源性,NA和M基因具有典型的欧亚株系猪流感病毒特征.该病毒具有人传人的分子基础,HA上HA1和HA2裂解位点序列为PSIQSR↓+GLFGAI,尚不具备高致病性流感病毒的特征.病毒对金刚烷胺类药物耐药,而对达菲和扎那米韦敏感.HA片段5个抗原决定区氨基酸序列与人用流感疫苗具有较大差异,推测现有疫苗对预防本次疫情基本无效.结论 甲型H1N1是一种北美和欧亚两种猪流感病毒的混合体,开发针对本病毒的流感疫苗有助于进一步控制疫情蔓延.     

H275Y mutant pandemic (H1N1) 2009 virus in immunocompromised patients

Most oseltamivir-resistant pandemic (H1N1) 2009 viruses have been isolated from immunocompromised patients. To describe the clinical features, treatment, outcomes, and virologic data associated with infection from pandemic (H1N1) 2009 virus with H275Y mutation in immunocompromised patients, we retrospectively identified 49 hematology-oncology patients infected with pandemic (H1N1) 2009 virus. Samples from 33 of those patients were tested for H275Y genotype by allele-specific real-time PCR. Of the 8 patients in whom H275Y mutations was identified, 1 had severe pneumonia; 3 had mild pneumonia with prolonged virus shedding; and 4 had upper respiratory tract infection, of whom 3 had prolonged virus shedding. All patients had received oseltamivir before the H275Y mutation was detected; 1 had received antiviral prophylaxis. Three patients excreted resistant virus for >60 days. Emergence of oseltamivir resistance is frequent in immunocompromised patients infected with pandemic (H1N1) 2009 virus and can be associated with a wide range of clinical disease and viral kinetics.