2017-2019年度上海市松江区A(H1N1)pdm09流感病毒HA和NA基因特征分析

目的 分析上海市松江区2017-2019监测年度A(H1N1)pdm09流感病毒HA和NA基因特征.方法 对松江区分离的毒株随机挑选46株A(H1N1)pdm09流感病毒毒株进行HA和NA基因序列进化分析和氨基酸位点变异分析,采用神经氨酸酶抑制实验开展奥司他韦和扎那米韦敏感性监测.结果 A(H1N1)pdm09流感病毒...     

2017-2020年流感流行季节河北省甲型H1N1流感病毒耐药性及耐药基因分析

  目的  分析河北省2017 — 2020年流感流行季节甲型H1N1流感病毒对神经氨酸酶抑制剂类药物（NAI）的耐药情况,为流感的预防控制提供依据。  方法  选取2017 — 2020年流感流行季节37株甲型H1N1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那米韦的药物敏感性。 选取38株甲型H1N1流感病毒提取核酸后对神经氨酸酶NA基因进行PCR扩增,测序后对耐药位点进行分析。  结果  选取的“A/河北新华/SWL1106/2017”对奥司他韦敏感性降低（IC₅₀=30.98 nmol/L）,其余36株甲型H1N1流感病毒全部对奥司他韦和扎那米韦敏感,对奥司他韦的半数抑制浓度（IC₅₀）平均数为0.46 （0.07～1.14 ）nmol/L;对扎那米韦的IC₅₀平均数为0.27 （0.07～0.85 ）nmol/L。 序列分析发现1株发生了H275Y突变,为奥司他韦耐药株。  结论  2017—2020年河北省流行的绝大部分甲型H1N1流感病毒对NAI类药物敏感。     

浙江省近年甲型H1N1流感病毒神经氨酸酶基因序列分析

目的 分析浙江省2009 2013年初甲型H1N1流感病毒神经氨酸酶基因(NA)序列进化特征。 方法 提取浙江省2009 2013年初29株甲型H1N1流感病毒基因组RNA,反转录－聚合酶链反应(RT-PCR)扩增NA基因,测序并拼接出ORF。以浙江省不同年份与地域15份代表性毒株NA序列和GenBank数据库中选取的22株2009 2012年国内外甲型H1N1流感病毒NA基因序列,采用MEGA 5.1软件对其进行序列比对并构建种系发生树。 结果 扩增并测序获得29株甲型H1N1流感病毒的NA基因ORF的全长序列,与国内外甲型H1N1流感病毒NA序列比对后显示序列的同源性较高,浙江省毒株与北美早期甲型H1N1流感病毒典型代表株A/California/04/2009(H1N1)的同源性为98.20％~99.50%,其中采集于2012年末和2013年初的4份病毒NA与A/California/04/2009(H1N1)的同源性为98.63%~99.14%。在1株采集于2010年1月的甲型H1N1流感病毒NA的基因序列中发现可导致奥司他韦耐药的H275Y突变基因型。 结论 虽然浙江省后期的甲型H1N1流感病毒神经氨酸酶基因累积了更多的变异,但所有毒株之间基因同源性仍然较高,所有毒株的神经氨酸酶基因同源性达到98.20％及以上,序列分析结果证实1份毒株携带奥司他韦耐药基因型突变。     

奥司他韦和扎那米韦治疗甲型H1N1流感病毒H274Y突变株的疗效:2007—2008年和2008—2009年流感季节在日本进行的多中心研究

甲型H1N1流感病毒出现H274Y突变株并在全球范围流行。然而,神经氨酸酶抑制剂奥司他韦和扎那米韦的临床疗效尚未得到充分的重新评估。本研究对2007—2008年和2008—2009年流感季节应用神经氨酸酶抑制剂治疗的流感病毒感染病例进行分析。     

中草药抗流感病毒的实验研究状况

流感病毒是引起急性呼吸道感染的重要病原体.病毒抗原易变异引起流行,甚至引起世界大流行[1].在抗病毒治疗的药物选择方面,初步药物试验提示,甲型H1N1流感病毒对奥司他韦和扎那米韦敏感,对化学药物金刚烷胺和金刚乙胺耐药田.     

中草药抗流感病毒的实验研究状况

流感病毒是引起急性呼吸道感染的重要病原体。病毒抗原易变异引起流行．甚至引起世界大流行。在抗病毒治疗的药物选择方面．初步药物试验提示．甲型H1N1流感病毒对奥司他韦和扎那米韦敏感．对化学药物金刚烷胺和金刚乙胺耐药。     

2010年广州市甲型H1N1流感病毒分离株NA基因变异分析

目的比较2010年从广州市分离到的甲型H1N1流感病毒神经氨酸酶(NA)基因与2009年中国大陆甲型H1N1流感病毒NA基因的变异情况,为甲型H1N1流感的监测和防控提供参考资料。方法收集2010年广州市有发热和呼吸道症状患者的咽拭子标本,用甲型H1N1流感病毒特异性引物进行聚合酶链反应(PCR)检测,扩增分离到的甲型H1N1流感病毒NA基因片段,测序后与2009年的H1N1毒株进行比对和进化分析,并用生物信息学方法对耐药位点和糖基化位点进行分析。结果共收集1 194份咽拭子标本,检测到甲型流感病毒阳性327份,其中H1N1流感病毒6株,与2009年分离的甲型H1N1流感病毒相比,有16个位点发生了有义突变,3个位点和NA活性相关,其中222位氨基酸的变异位于NA活性位点上。结论成功扩增了2010年广州市6株甲型H1N1流感病毒株NA基因并测序,未发现H275Y耐药位点的变异。3毒株在NA活性位点222位、228位和425位等氨基酸位点处发生了变异,需继续加强监测。     

2009至2016年中国甲型H1N1流感病毒神经氨酸酶基因进化分析及流行趋势预测

摘要:目的：分析2009至2016年中国甲型H1N1流感病毒神经氨酸酶基因（NA）进化特征,预测其流行趋势,为流感疫苗评价提供依据。 方法：从全球共享禽流感数据倡议组织（GISAID）数据库及美国国家生物技术中心（NCBI）数据库下载甲型H1N1流感病毒NA基因序列1 141条。利用邻接法进行系统进化分析和遗传变异分析,利用Bayesian skyline Plot预测流行趋势。 结果：2009至2016年中国甲型H1N1毒株NA基因与参考毒株的同源性逐年降低。遗传进化分析显示同一年份的毒株在系统进化树上基本呈现集中分布。除2012年,其余各年份的毒株均通过不同模型得到正向压力选择位点。动力学分析显示2017年甲型H1N1可能达到一个流行高峰。 结论：甲型H1N1流感病毒NA基因所编码的氨基酸逐年变异,需进一步加强流感监测。     

广东人类禽流感H5N1亚型血凝素和神经氨酸酶基因同源性分析

目的采用生物信息学技术,寻找广东地区人类禽流感H5N1亚型基因同源毒株。方法检测广东人禽流感H5N1亚型HA和NA基因核苷酸序列,从GenBank下载国内各种动物和人类相应基因序列,采用Mega5.03和Lasergene7.1软件进行核苷酸同源性和氨基酸变异分析。结果在总共174个HA基因和173个NA基因序列中,发现广东毒株A/Guangdong/01/2006的HA和NA基因与湖南鸡和安徽鸭流感毒株同源性较高;广东毒株A/Guangdong/02/2006的HA和NA基因与香港鸟禽类流感毒株同源性较高。同源性较高的毒株基因之间,氨基酸位点存在差异,但尚未显示流行病学和临床意义。结论广东A/Guangdong/01/2006毒株与湖南鸡和安徽鸭流感毒株具有共同的最近祖先,广东A/Guangdong/02/2006毒株与香港鸟禽类流感毒株具有共同的最近祖先。     

2016-2017年我国甲型流感病毒H3N2亚型HA和NA基因的分子特征分析

目的探讨2016-2017年我国甲型流感病毒H3N2亚型血凝素(hemagglutinin,HA)和神经氨酸酶(neuraminidase,NA)基因的分子特征,为甲型流感病毒H3N2亚型的防控提供科学依据。方法从全球共享禽流感数据库(GISAID)中获得我国2016~2017年分离的525株甲型流感病毒H3N2亚型病毒和13株H3N2亚型参考株的HA和NA基因序列。应用MEGA 6.0软件分析HA和NA的分子进化特征、氨基酸位点和耐药位点的变异情况。结果进化树分析显示13株参考株之间HA和NA氨基酸的同源性均为96.9%~99.1%。2016-2017年我国甲型流感病毒H3N2亚型的病毒株HA和NA氨基酸序列之间同源性均为96.3%~100%,HA和NA的优势亚分支均为3C.2a.1,相当一部分毒株分型不够明确。HA氨基酸变异分析显示9株发生N121E突变,103株发生N121K的突变且主要来源于2017年的香港毒株;182株发生G/R142K突变;A/Hong_Kong/3079/2017-HA和A/Guangdong-Chengguan/1383/2017-HA均发生A128I突变;NA蛋白仅3株(A/Hunan-Suxian/1980/2016-NA、A/Hunan-Yuhua/11799/2016-NA和A/Ningxia-Yuanzhou/1657/2016-NA)发现H275Y耐药位点突变。结论 2016-2017年我国甲型流感病毒H3N2亚型的HA和NA蛋白与参考株间存在一定的差异,优势亚型均为3C.2a.1;与病毒的毒力及药物相关的部分关键氨基酸位点发生变异。应密切关注H3N2病毒的分子特征,为其防控提供参考。     

In Vitro Generation of Neuraminidase Inhibitor Resistance in A(H5N1) Influenza Viruses

To identify mutations that can arise in highly pathogenic A(H5N1) viruses under neuraminidase inhibitor selective pressure, two antigenically different strains were serially passaged with increasing levels of either oseltamivir or zanamivir. Under oseltamivir pressure, both A(H5N1) viruses developed a H274Y neuraminidase mutation, although in one strain the mutation occurred in combination with an I222M neuraminidase mutation. The H274Y neuraminidase mutation reduced oseltamivir susceptibility significantly (900- to 2,500-fold compared to the wild type). However the dual H274Y/I222M neuraminidase mutation had an even greater impact on resistance, with oseltamivir susceptibility reduced significantly further (8,000-fold compared to the wild type). A similar affect on oseltamivir susceptibility was observed when the dual H274Y/I222M mutations were introduced, by reverse genetics, into a recombinant seasonal human A(H1N1) virus and also when an alternative I222 substitution (I222V) was generated in combination with H274Y in A(H5N1) and A(H1N1) viruses. These viruses remained fully susceptible to zanamivir but demonstrated reduced susceptibility to peramivir. Following passage of the A(H5N1) viruses in the presence of zanamivir, the strains developed a D198G neuraminidase mutation, which reduced susceptibility to both zanamivir and oseltamivir, and also an E119G neuraminidase mutation, which demonstrated significantly reduced zanamivir susceptibility (1,400-fold compared to the wild type). Mutations in hemagglutinin residues implicated in receptor binding were also detected in many of the resistant strains. This study identified the mutations that can arise in A(H5N1) under either oseltamivir or zanamivir selective pressure and the potential for dual neuraminidase mutations to result in dramatically reduced drug susceptibility.Large-scale outbreaks of highly pathogenic A(H5N1) avian influenza affecting poultry have occurred throughout many parts of Asia, North Africa, and the Middle East since 2003 (1). The virus, which now appears to be enzootic in many regions, has on occasion caused zoonotic infections in humans (1). Humans who acquire the infection develop severe pneumonia that can progress to acute respiratory distress syndrome with high risk of mortality. For the 6-year period 2003 to 2008, 395 confirmed A(H5N1) virus human infections were reported, and 250 were fatal (a case fatality rate of 63%) (http://www.who.int/csr/disease/avian_influenza/en/index.html). Human-to-human transmission of A(H5N1) virus appears to be rare and has been associated only with very close unprotected contact with severely ill patients (30). Of concern is the potential for the A(H5N1) virus to become easily transmissible between humans, which, because of the lack of prior immunity to this strain in humans, might result in a global influenza pandemic. Based on these theoretical concerns and the experiences of large-scale morbidity and mortality from previous influenza pandemics, many countries have prepared plans to address or mitigate such an occurrence, including the stockpiling of inactivated A(H5N1) influenza vaccines, as well as anti-influenza drugs. Because multiple vaccine doses may be necessary to achieve protection and some time would be required to generate a vaccine with an antigenically matched strain (1), antiviral drugs could play a critical role in the treatment or prophylaxis of influenza, particularly during the early stages of a pandemic. The oral neuraminidase (NA) inhibitor oseltamivir (Tamiflu) has been the most widely used anti-influenza drug for the treatment of A(H5N1) virus -infected patients and has been stockpiled for potential broad use. Results from uncontrolled clinical trials suggest that the use of oseltamivir may increase the survival rate of patients with A(H5N1) virus infection, particularly if administered early in the course of illness (1). However, oseltamivir-resistant A(H5N1) virus variants with an H274Y NA mutation have been isolated from treated patients and may be associated with clinical deterioration and fatal outcomes (9). Viruses with the H274Y NA mutations are susceptible to the NA inhibitor zanamivir, which has led to the inclusion of inhaled zanamivir, together with oseltamivir, in pandemic drug stockpiles. The volume of drug that might be used in the event of a pandemic would be significantly greater than has ever been used previously for treatment of seasonal influenza. There is concern that this may lead to a high frequency of drug resistance. While previous studies have identified a number of NA inhibitor resistance mutations that have arisen in seasonal influenza viruses under drug pressure, little is known about which NA inhibitor resistance mutations might arise in highly pathogenic A(H5N1) viruses. To investigate this question, two A(H5N1) strains from different phylogenetic clades were subjected to serial passage in Madin-Darby canine kidney (MDCK) cells in the presence of increasing levels of either oseltamivir or zanamivir, and the resultant viruses were analyzed functionally and genetically.     

Comparison of efficacies of RWJ-270201, zanamivir, and oseltamivir against H5N1, H9N2, and other avian influenza viruses

The orally administered neuraminidase (NA) inhibitor RWJ-270201 was tested in parallel with zanamivir and oseltamivir against a panel of avian influenza viruses for inhibition of NA activity and replication in tissue culture. The agents were then tested for protection of mice against lethal H5N1 and H9N2 virus infection. In vitro, RWJ-270201 was highly effective against all nine NA subtypes. NA inhibition by RWJ-270201 (50% inhibitory concentration, 0.9 to 4.3 nM) was superior to that by zanamivir and oseltamivir carboxylate. RWJ-270201 inhibited the replication of avian influenza viruses of both Eurasian and American lineages in MDCK cells (50% effective concentration, 0.5 to 11.8 microM). Mice given 10 mg of RWJ-270201 per kg of body weight per day were completely protected against lethal challenge with influenza A/Hong Kong/156/97 (H5N1) and A/quail/Hong Kong/G1/97 (H9N2) viruses. Both RWJ-270201 and oseltamivir significantly reduced virus titers in mouse lungs at daily dosages of 1.0 and 10 mg/kg and prevented the spread of virus to the brain. When treatment began 48 h after exposure to H5N1 virus, 10 mg of RWJ-270201/kg/day protected 50% of mice from death. These results suggest that RWJ-270201 is at least as effective as either zanamivir or oseltamivir against avian influenza viruses and may be of potential clinical use for treatment of emerging influenza viruses that may be transmitted from birds to humans.     

Neuraminidase inhibitor sensitivity and receptor-binding specificity of Cambodian clade 1 highly pathogenic H5N1 influenza virus

The evolution of the highly pathogenic H5N1 influenza virus produces genetic variations that can lead to changes in antiviral susceptibility and in receptor-binding specificity. In countries where the highly pathogenic H5N1 virus is endemic or causes regular epidemics, the surveillance of these changes is important for assessing the pandemic risk. In Cambodia between 2004 and 2010, there have been 26 outbreaks of highly pathogenic H5N1 influenza virus in poultry and 10 reported human cases, 8 of which were fatal. We have observed naturally occurring mutations in hemagglutinin (HA) and neuraminidase (NA) of Cambodian H5N1 viruses that were predicted to alter sensitivity to neuraminidase inhibitors (NAIs) and/or receptor-binding specificity. We tested H5N1 viruses isolated from poultry and humans between 2004 and 2010 for sensitivity to the NAIs oseltamivir (Tamiflu) and zanamivir (Relenza). All viruses were sensitive to both inhibitors; however, we identified a virus with a mildly decreased sensitivity to zanamivir and have predicted that a V149A mutation is responsible. We also identified a virus with a hemagglutinin A134V mutation, present in a subpopulation amplified directly from a human sample. Using reverse genetics, we verified that this mutation is adaptative for human α2,6-linked sialidase receptors. The importance of an ongoing surveillance of H5N1 antigenic variance and genetic drift that may alter receptor binding and sensitivities of H5N1 viruses to NAIs cannot be underestimated while avian influenza remains a pandemic threat.     

Characterization of Drug-Resistant Influenza Virus A(H1N1) and A(H3N2) Variants Selected In Vitro with Laninamivir

Neuraminidase inhibitors (NAIs) play a major role for managing influenza virus infections. The widespread oseltamivir resistance among 2007-2008 seasonal A(H1N1) viruses and community outbreaks of oseltamivir-resistant A(H1N1)pdm09 strains highlights the need for additional anti-influenza virus agents. Laninamivir is a novel long-lasting NAI that has demonstrated in vitro activity against influenza A and B viruses, and its prodrug (laninamivir octanoate) is in phase II clinical trials in the United States and other countries. Currently, little information is available on the mechanisms of resistance to laninamivir. In this study, we first performed neuraminidase (NA) inhibition assays to determine the activity of laninamivir against a set of influenza A viruses containing NA mutations conferring resistance to one or many other NAIs. We also generated drug-resistant A(H1N1) and A(H3N2) viruses under in vitro laninamivir pressure. Laninamivir demonstrated a profile of susceptibility that was similar to that of zanamivir. More specifically, it retained activity against oseltamivir-resistant H275Y and N295S A(H1N1) variants and the E119V A(H3N2) variant. In vitro, laninamivir pressure selected the E119A NA substitution in the A/Solomon Islands/3/2006 A(H1N1) background, whereas E119K and G147E NA changes along with a K133E hemagglutinin (HA) substitution were selected in the A/Quebec/144147/2009 A(H1N1)pdm09 strain. In the A/Brisbane/10/2007 A(H3N2) background, a large NA deletion accompanied by S138A/P194L HA substitutions was selected. This H3N2 variant had altered receptor-binding properties and was highly resistant to laninamivir in plaque reduction assays. Overall, we confirmed the similarity between zanamivir and laninamivir susceptibility profiles and demonstrated that both NA and HA changes can contribute to laninamivir resistance in vitro.     

Impact of neuraminidase mutations conferring influenza resistance to neuraminidase inhibitors in the N1 and N2 genetic backgrounds

Subtype-specific neuraminidase (NA) mutations conferring resistance to NA inhibitors (NAIs) have been reported during in vitro passages and in clinic. In this study, we evaluated the impact of various NA mutations (E119A/G/V, H274Y, R292K and N294S) on the susceptibility profiles to different NAIs (oseltamivir, zanamivir and peramivir) using recombinant NA proteins of influenza A/WSN/33 (H1N1) and A/Sydney/5/97-like (H3N2) viruses. In the Nl subtype, the E119V mutation conferred cross-resistance to oseltamivir, zanamivir and peramivir [1,727-2,144 and 5,050-fold increase in IC50 values compared with wild-type (WT)] whereas only oseltamivir-resistance (1,028-fold increase in IC50) was conferred by the same mutation in the N2 subtype. The N294S mutation conferred resistance to oseltamivir in both the NI and N2 subtypes (197- and 1,879-fold increase in IC50 values, respectively) whereas the H274Y mutation conferred resistance to oseltamivir (754-fold increase) and peramivir (260-fold increase) in the N1 subtype only. The virulence of reverse genetics-rescued A/WSN/33 viruses harbouring H274Y and N294S NA mutations was investigated in Balb/c mice. The WT and H274Y recombinants had identical LD50 values (103 PFUs) and generated similar viral lung titres, whereas a higher LD50 (10 PFUs) and a 1-log decrease in viral lung titres were obtained with the N294S mutant. This study shows that some NA mutations at framework residues may confer resistance to one or three NAIs depending on the viral subtype. It suggests that certain drug-resistant NA mutants may still be virulent although additional studies using clinical isolates are needed to confirm our results.     

Surveillance for neuraminidase inhibitor resistance among human influenza A and B viruses circulating worldwide from 2004 to 2008

The surveillance of seasonal influenza virus susceptibility to neuraminidase (NA) inhibitors was conducted using an NA inhibition assay. The 50% inhibitory concentration values (IC₅₀s) of 4,570 viruses collected globally from October 2004 to March 2008 were determined. Based on mean IC₅₀s, A(H3N2) viruses (0.44 nM) were more sensitive to oseltamivir than A(H1N1) viruses (0.91 nM). The opposite trend was observed with zanamivir: 1.06 nM for A(H1N1) and 2.54 nM for A(H3N2). Influenza B viruses exhibited the least susceptibility to oseltamivir (3.42 nM) and to zanamivir (3.87 nM). To identify potentially resistant viruses (outliers), a threshold of a mean IC₅₀ value + 3 standard deviations was defined for type/subtype and drug. Sequence analysis of outliers was performed to identify NA changes that might be associated with reduced susceptibility. Molecular markers of oseltamivir resistance were found in six A(H1N1) viruses (H274Y) and one A(H3N2) virus (E119V) collected between 2004 and 2007. Some outliers contained previously reported mutations (e.g., I222T in the B viruses), while other mutations [e.g., R371K and H274Y in B viruses and H274N in A(H3N2) viruses) were novel. The R371K B virus outlier exhibited high levels of resistance to both inhibitors (>100 nM). A substantial variance at residue D151 was observed among A(H3N2) zanamivir-resistant outliers. The clinical relevance of newly identified NA mutations is unknown. A rise in the incidence of oseltamivir resistance in A(H1N1) viruses carrying the H274Y mutation was detected in the United States and in other countries in the ongoing 2007 to 2008 season. As of March 2008, the frequency of resistance among A(H1N1) viruses in the United States was 8.6% (50/579 isolates). The recent increase in oseltamivir resistance among A(H1N1) viruses isolated from untreated patients raises public health concerns and necessitates close monitoring of resistance to NA inhibitors.     

Mechanism by which mutations at his274 alter sensitivity of influenza a virus n1 neuraminidase to oseltamivir carboxylate and zanamivir

Oseltamivir carboxylate is a potent and specific inhibitor of influenza neuraminidase (NA). An influenza A/H1N1 variant selected in vitro with reduced susceptibility to oseltamivir carboxylate contains a His274Tyr mutation. To understand the mechanism by which a His274Tyr mutation gives rise to drug resistance, we studied a series of NA variant proteins containing various substitutions at position 274. Replacement of His274 with larger side chain residues (Tyr or Phe) reduced the NA sensitivity to oseltamivir carboxylate. In contrast, replacement of His274 with smaller side chain residues (Gly, Asn, Ser, and Gln) resulted in enhanced or unchanged sensitivity to oseltamivir carboxylate. Previous studies have suggested that the slow-binding inhibition of NA by oseltamivir carboxylate is a result of the reorientation of Glu276. Loss of this slow-binding inhibition in the His274Tyr and His274Phe mutant NA but not in His274Asn, His274Gly, His274Ser, or His274Gln supports the conclusion that the conformational change of Glu276 is restricted in the His274Tyr and His274Phe mutant NA upon oseltamivir carboxylate binding. Interestingly, His274Asn, as well as His274Gly, His274Ser, and His274Gln, also displayed reduced sensitivity to zanamivir and its analogue, 4-amino-Neu5Ac2en. Substitution of His274 with Tyr in influenza A/Tokyo/3/67 (H3N2) recombinant NA did not affect the susceptibility to oseltamivir carboxylate. These data indicate that the volume occupied by the amino acid side chain at position 274 can influence the sensitivities of influenza N1 NA but not of N2 NA to both oseltamivir carboxylate and zanamivir.     

Impact of mutations at residue I223 of the neuraminidase protein on the resistance profile, replication level, and virulence of the 2009 pandemic influenza virus

Amino acid substitutions at residue I223 of the neuraminidase (NA) protein have been identified in 2009 pandemic influenza (pH1N1) variants with altered susceptibilities to NA inhibitors (NAIs). We used reverse genetics and site-directed mutagenesis to generate the recombinant A/Québec/144147/09 pH1N1 wild-type virus (WT) and five (I223R, I223V, H275Y, I223V-H275Y, and I223R-H275Y) NA mutants. A fluorimetry-based assay was used to determine 50% inhibitory concentrations (IC(50)s) of oseltamivir, zanamivir, and peramivir. Replicative capacity was analyzed by viral yield assays in ST6GalI-MDCK cells. Infectivity and transmission of the WT, H275Y, and I223V-H275Y recombinant viruses were evaluated in ferrets. As expected, the H275Y mutation conferred resistance to oseltamivir (982-fold) and peramivir (661-fold) compared to the drug-susceptible recombinant WT. The single I223R mutant was associated with reduced susceptibility to oseltamivir (53-fold), zanamivir (7-fold) and peramivir (10-fold), whereas the I223V virus had reduced susceptibility to oseltamivir (6-fold) only. Interestingly, enhanced levels of resistance to oseltamivir and peramivir and reduced susceptibility to zanamivir (1,647-, 17,347-, and 16-fold increases in IC(50)s, respectively) were observed for the I223R-H275Y recombinant, while the I223V-H275Y mutant exhibited 1,733-, 2,707-, and 2-fold increases in respective IC(50)s. The I223R and I223V changes were associated with equivalent or higher viral titers in vitro compared to the recombinant WT. Infectivity and transmissibility in ferrets were comparable between the recombinant WT and the H275Y or I223V-H275Y recombinants. In conclusion, amino acid changes at residue I223 may alter the NAI susceptibilities of pH1N1 variants without compromising fitness. Consequently, I223R and I223V mutations, alone or with H275Y, need to be thoroughly monitored.     

Changes in in vitro susceptibility of influenza A H3N2 viruses to a neuraminidase inhibitor drug during evolution in the human host

OBJECTIVES: Influenza A H3N2 viruses isolated recently have characteristic receptor binding properties that may decrease susceptibility to neuraminidase inhibitor drugs. A panel of clinical isolates and recombinant viruses generated by reverse genetics were characterized and tested for susceptibility to zanamivir. METHODS: Plaque reduction assays and neuraminidase enzyme inhibition assays were used to assess susceptibility to zanamivir. Receptor binding properties of the viruses were characterized by differential agglutination of red blood cells (RBCs) from different species. Sequence analysis of the haemagglutinin (HA) and neuraminidase (NA) genes was carried out. RESULTS: Characterization of a panel of H3N2 clinical isolates from 1968 to 2000 showed a gradual decrease in agglutination of chicken and guinea pig RBCs over time, although all isolates could agglutinate turkey RBCs equally. Sequence analysis of the HA and NA genes identified mutations in conserved residues of the HA1 receptor binding site, in particular Leu-226 --> Ile-226/Val-226, and modification of potential glycosylation site motifs. This may be indicative of changes in virus binding to sialic acid (SA) receptors in recent years. Although recent isolates had reduced susceptibility to zanamivir in MDCK cell based plaque reduction assays, no difference was found in an NA enzyme-inhibition assay. Assays with recombinant isogenic viruses showed that the recent HA, but not the NA, conferred reduced susceptibility to zanamivir. CONCLUSION: This study demonstrates that recent clinical isolates of influenza A H3N2 virus no longer agglutinate chicken RBCs, but despite significant receptor binding changes as a result of changes in HA, there was little variation in sensitivity of the NA to zanamivir.