Oseltamivir-resistant pandemic A(H1N1) 2009 influenza viruses detected through enhanced surveillance in the Netherlands, 2009–2010

Enhanced surveillance of infections due to the pandemic A(H1N1) influenza virus, which included monitoring for antiviral resistance, was carried out in the Netherlands from late April 2009 through late May 2010. More than 1100 instances of infection with the pandemic A(H1N1) influenza virus from 2009 and 2010 [A(H1N1) 2009] distributed across this period were analyzed. Of these, 19 cases of oseltamivir-resistant virus harboring the H275Y mutation in the neuraminidase (NA) were detected. The mean 50% inhibitory concentration (IC₅₀) levels for oseltamivir- and zanamivir-susceptible A(H1N1) 2009 viruses were 1.4-fold and 2-fold, respectively, lower than for the seasonal A(H1N1) influenza viruses from 2007/2008; for oseltamivir-resistant A(H1N1) 2009 virus the IC₅₀ was 2.9-fold lower. Eighteen of the 19 patients with oseltamivir-resistant virus showed prolonged shedding of the virus and developed resistance while on oseltamivir therapy. Sixteen of these 18 patients had an immunodeficiency, of whom 11 had a hematologic disorder. The two other patients had another underlying disease. Six of the patients who had an underlying disease died; of these, five had received cytostatic or immunosuppressive therapy. No indications for onward transmission of resistant viruses were found. This study showed that the main association for the emergence of cases of oseltamivir-resistant A(H1N1) 2009 virus was receiving antiviral therapy and having drug-induced immunosuppression or an hematologic disorder. Except for a single case of a resistant virus not linked to oseltamivir therapy, the absence of detection of resistant variants in community specimens and in specimens from contacts of cases with resistant virus suggested that the spread of resistant A(H1N1) 2009 virus was limited. Containment may have been the cumulative result of impaired NA function, successful isolation of the patients, and prophylactic measures to limit exposure.     

Genotypic and phenotypic resistance of pandemic A/H1N1 influenza viruses circulating in Germany

In response to the rapid global spread of an antigenically novel A/H1N1 influenza virus in 2009, the World Heath Organization (WHO) recommended surveillance and monitoring for antiviral resistance of influenza viruses. We designed and evaluated pyrosequencing (PSQ)-based genotypic assays for high-throughput analysis of the susceptibility of pandemic A/H1N1 influenza viruses to neuraminidase (NA) inhibitors. A total of 1570 samples circulating in Germany between April 2009 and April 2010 were tested for determination of molecular markers of resistance to the NA inhibitors oseltamivir and zanamivir, and 635 of them were evaluated by phenotypic fluorescence-based assay with MUNANA substrate. Eight (0.5%) viruses were resistant to oseltamivir due to the H274Y NA substitution (N2 numbering). Six of these oseltamivir-resistant cases were treatment-related; four of them were selected in immunocompromised patients, two in patients suffered from chronic diseases. The two remaining oseltamivir-resistant viruses seem to have evolved in the absence of drug treatment and were isolated from immunocompetent healthy patients. All tested A/H1N1 pandemic viruses were sensitive to zanamivir. In addition, analysis of 1011 pandemic A/H1N1 virus samples by a PSQ-based assay according to the WHO protocol revealed the presence of mutation S31N in the M2 protein that conferred resistance to M2 ion channel inhibitors. Our data demonstrate a low incidence of oseltamivir-resistant pandemic A/H1N1 influenza variants isolated under drug selection pressure as well as community-acquired or naturally evolving viruses.     

Design, synthesis, and in vitro biological evaluation of 1H-1,2,3-triazole-4-carboxamide derivatives as new anti-influenza A agents targeting virus nucleoprotein

The influenza virus nucleoprotein (NP) is an emerging target for anti-influenza drug development. Nucleozin (1) and its closely related derivatives had been identified as NP inhibitors displaying anti-influenza activity. Utilizing 1 as a lead molecule, we successfully designed and synthesized a series of 1H-1,2,3-triazole-4-carboxamide derivatives as new anti-influenza A agents. One of the most potent compounds, 3b, inhibited the replication of various H3N2 and H1N1 influenza A virus strains with IC(50) values ranging from 0.5 to 4.6 μM. Compound 3b also strongly inhibited the replication of H5N1 (RG14), amantidine-resistant A/WSN/33 (H1N1), and oseltamivir-resistant A/WSN/1933 (H1N1, 274Y) virus strains with IC(50) values in sub-μM ranges. Further computational studies and mechanism investigation suggested that 3b might directly target influenza virus A nucleoprotein to inhibit its nuclear accumulation.     

Protective efficacy of an H1N1 cold-adapted live vaccine against the 2009 pandemic H1N1, seasonal H1N1, and H5N1 influenza viruses in mice

Vaccination is a key strategy for preventing influenza virus infections. Here, we generated a reassortant virus (SC/AAca) containing the hemagglutinin and neuraminidase genes from a 2009 pandemic influenza virus A/Sichuan/1/2009 (H1N1) (SC/09) and six internal genes from the cold-adapted virus A/Ann Arbor/6/60 (H2N2) (AAca). The SC/AAca reassortant induced a sound humoral immune response and complete protection against homologous SC/09 virus challenge in mice after intranasal administration of an at least 10(6) 50% egg infectious dose (EID(50)) of SC/AAca. SC/AAca inoculation also induced significant CD4+ and CD8+ T cell responses and provided solid protection against heterologous H1N1 and H5N1 virus challenge. Our results suggest that this 2009 H1N1 live vaccine will provide protection against both 2009 pandemic and seasonal H1N1 virus infection and might reduce the severity of H5N1 virus infection in humans. The induction of cross-reactive virus-specific T cell responses may be an effective approach to develop universal influenza vaccines.     

上海地区在校中学生接种甲型H1N1流感疫苗前后抗体水平监测分析

 目的  了解上海地区在校中学生接种甲型H1N1流感疫苗前后的抗体水平;观察甲型H1N1流感疫苗对该人群的免疫保护作用。 方法    应用常规微量血凝抑制试验（micro-hemagglutination inhibition test, HIT）对上海地区在校中学生分3个时间段进行甲型H1N1流感病毒抗体的血清学监测,3个时间段的抗体阳性率比较采用Pearson χ2 检验进行分析。 结果  2009年甲型H1N1流感流行前上海地区在校中学生的血清抗体阳性率仅为1.3%。经过一段时间流行后,血清抗体阳性率升至8.5%。接种甲型H1N1流感疫苗后,血清抗体阳性率升至87.3%。经Pearson χ² 检验分析,3个时间段在校中学生的血清甲型H1N1流感抗体阳性率差异有统计学意义（χ²=243.7,P<0.05）。 结论  在甲型H1N1流感流行前,上海地区在校中学生对其几乎没有免疫力,接种甲型H1N1流感疫苗能为该人群提供较好的免疫保护作用。     

H1N1 influenza A virus neuraminidase modulates infectivity in mice

In the 2years since the onset of the H1N1 2009 pandemic virus (H1N1pdm09), sporadic cases of oseltamivir-resistant viruses have been reported. We investigated the impact of oseltamivir-resistant neuraminidase from H1N1 Brisbane-like (seasonal) and H1N1pdm09 viruses on viral pathogenicity in mice. Reassortant viruses with the neuraminidase from seasonal H1N1 virus were obtained by co-infection of a H1N1pdm09 virus and an oseltamivir-resistant H1N1 Brisbane-like virus. Oseltamivir-resistant H1N1pdm09 viruses were also isolated from patients. After biochemical characterization, the pathogenicity of these viruses was assessed in a murine model. We confirmed a higher infectivity, in mice, of the H1N1pdm09 virus compared to seasonal viruses. Surprisingly, the oseltamivir-resistant H1N1pdm09 virus was more infectious than its sensitive counterpart. Moreover, the association of H1N1pdm09 hemagglutinin and an oseltamivir-resistant neuraminidase improved the infectivity of reassortant viruses in mice, regardless of the NA origin: seasonal (Brisbane-like) or pandemic strain. This study highlights the need to closely monitor the emergence of oseltamivir-resistant viruses.     

Evaluation of the antiviral drug susceptibility of influenza viruses in Italy from 2004/05 to 2009/10 epidemics and from the recent 2009 pandemic

Antiviral monitoring of influenza viruses circulating in Italy has been carried out since 2007 by the National Influenza Centre (NIC), using both phenotypic and sequence-based assays. Here, we report results of the susceptibility evaluation to neuraminidase (NA) inhibitors (NAIs, zanamivir and oseltamivir) and adamantanes of nearly 300 influenza type A and B seasonal viruses isolated in Italy during six recent seasons, together with over 30 pandemic (H1N1) 2009 virus strains. The present work is the first such study conducted in Italy, aimed to develop national data on antiviral drug profile and to establish a nationwide surveillance programme on antiviral susceptibility. Sequencing of the NA gene was undertaken either to confirm the phenotypic findings or to identify any NA change, in potentially resistant viruses (outliers), which might be associated with reduced susceptibility to NAIs. The 50% inhibitory concentration values (IC(50)s) showed slightly different sensitivities of the seasonal Italian isolates to the two NAI drugs, depending on the specific NA subtype. We found mean zanamivir IC(50)s of 0.74, 1.33 and 7 nM, and oseltamivir IC(50)s of 0.67, 2.34 and 30.1 nM for the N2, N1 and B NAs, respectively. The pandemic (H1N1) 2009 viruses showed IC(50)values overall comparable to the seasonal N1 viruses from previous years, showing mean zanamivir IC(50)s of 1.02 nM and mean oseltamivir IC(50)s of 2.82 nM. Oseltamivir resistance was found in a total of 19 seasonal N1viruses of 2007/2008 and 2008/2009, and in three pandemic (H1N1) 2009 strains. A gradual increase of resistance to adamantanes was observed among the N2 viruses isolated in recent seasons; no resistant viruses were found among the seasonal N1 strains, whereas all the pandemic (H1N1) 2009 isolates analysed were resistant to the M2 blockers.     

表没食子儿茶素没食子酸酯抗甲型流感病毒的作用研究

目的初步探讨表没食子儿茶素没食子酸酯(EGCG)抗甲型流感病毒的活性及其作用机制。方法采用H5N1假病毒检测体系,观察EGCG对A/Thailand/Kan353/2004H5N1毒株假病毒的抑制作用;采用神经氨酸酶抑制实验进一步分析其作用机制;采用H1N1 FM_1病毒检测体系,根据药物与病毒、细胞的不同作用,通过两种给药方式,评估EGCG对甲型流感病毒FM_1的抑制作用。结果 EGCG能特异性的抑制H5N1假病毒的进入,IC50为145.10μmol.L-1;EGCG对神经氨酸酶具有抑制作用,IC50为417.20μmol.L-1,EGCG对预防和治疗给药方式均有一定的抑制作用,其IC50分别为6.88μmol.L-1和14.83μmol.L-1,治疗指数分别为58.02和26.92。结论 EGCG在体外具有明显的抗甲型流感病毒作用,其作用机制包括抑制病毒的进入和神经氨酸酶活性。     

A single intramuscular injection of neuraminidase inhibitor peramivir demonstrates antiviral activity against novel pandemic A/California/04/2009 (H1N1) influenza virus infection in mice

New and emerging influenza virus strains, such as the pandemic influenza A (H1N1) virus require constant vigilance for antiviral drug sensitivity and resistance. Efficacy of intramuscularly (IM) administered neuraminidase (NA) inhibitor, peramivir, was evaluated in mice infected with recently isolated pandemic A/California/04/2009 (H1N1, swine origin, mouse adapted) influenza virus. A single IM injection of peramivir (four dose groups), given 1h prior to inoculation, significantly reduced weight loss (p < 0.001) and mortality (p < 0.05) in mice infected with LD90 dose of pandemic A/California/04/2009 (H1N1) influenza virus compared to vehicle group. There was 20% survival in the vehicle-treated group, whereas in the peramivir-treated groups, survival increased in a dose-dependent manner with 60, 60, 90 and 100% survivors for the 1, 3, 10, and 30 mg/kg doses, respectively. Weight loss on day 4 in the vehicle-treated group was 3.4 gm, and in the peramivir-treated groups was 2.1, 1.5, 1.8 and 1.8 g for the 1, 3, 10 and 30 mg/kg dose groups, respectively. In the treatment model, peramivir given 24h after infection as a single IM injection at 50mg/kg dose, showed significant protection against lethality and weight loss. There was 13% survival in the vehicle-treated group while in the peramivir-treated group at 24, 48, and 72 h post infection, survival was 100, 40, and 50%, respectively. Survival in the oseltamivir groups (10 mg/kg/d twice a day, orally for 5 days) was 90, 30 and 20% at 24, 48 and 72 h, respectively. These data demonstrate efficacy of parenterally administered peramivir against the recently isolated pandemic influenza virus in murine infection models.     

Oseltamivir-resistant influenza A(H1N1) 2009 virus in Greece during the post-pandemic 2010-2011 season

Information on the drug susceptibility of influenza epidemic strains is important for antiviral resistance monitoring. In Greece, the 2009-2010 pandemic waves were very mild and seroprevalence rates remained low after this influenza season, resulting in exclusive detection of the pandemic strain during the 2010-2011 influenza season. In the present study during the post-pandemic 2010-2011 season, 50 consecutive influenza A(H1N1) 2009 virus-positive samples from patients hospitalised in Greek hospitals were analysed for resistance to the neuraminidase inhibitor oseltamivir. All patients were hospitalised with severe influenza complications and had previously received oseltamivir. Influenza A(H1N1) 2009 virus detection and testing for oseltamivir resistance were performed with real-time PCR amplification assays. The H275Y substitution associated with resistance to oseltamivir was identified in two immunocompetent patients who received oseltamivir treatment for 3 days and 5 days, respectively. In both cases, patients were discharged in good condition despite development of resistance to antiviral treatment.     

某区甲型H1N1流感病毒分离鉴定及同源性的分析

目的检测并分离甲型H1N1流感病毒,对开封地区首次分离到的病毒株进行全基因组序列测定及同源性分析,为研究流感病毒的流行及变异规律提供科学依据。方法采用Real-time RT-PCR方法检测,筛选确定出甲型H1N1流感病毒阳性标本;利用狗肾传代细胞分离得到甲型H1N1流感病毒株A/Kaifeng/01/2009(H1N1);测定并分析其全基因组序列;利用序列比对进行了同源性分析。结果从1828份流感样病例中检出甲型H1N1流感病毒阳性标本286份,阳性率15.6%。在开封地区首次获得甲型H1N1流感病毒株及全基因组序列。基因组序列分析表明:该毒株与2009年大流行株高度同源,为同一进化分支。与以往流行的猪流感病毒株对比发现,HA基因有12个碱基发生了点突变。结论 MDCK细胞对甲型H1N1流感病毒具有较高敏感性;开封地区首例甲型H1N1流感病例分离病毒株与北美流行株高度同源;相对于以往古典型猪流感代表株出现了HA蛋白抗原性漂移;为今后进一步开展甲型H1N1流感病毒分子生物学研究奠定基础。     

贵州省新型甲型HIN1(2009)流感病毒HA-1基因分析

目的 建立并完善贵州省新型甲型H1N1流感病毒基因分析检测技术平台,了解贵州省新型甲型H1N1(2009)流感病毒HA1片断与WHO公布的流行株有否发生变异.方法 收集16株新型甲型H1N1流感病毒毒株,提取病毒核酸RNA,通过RT-PCR扩增HA1片断,用电泳观察PCR产物目的条带大小,经过纯化、测序,用Biodeit、MEGA4相关软件进行序列比对,同源性及差异性分析,构建系统发育树.结果 贵州省(2009)代表株与中俄代表株A/Habarovak/01/2009 (H1N1)和墨西哥第一株甲型H1N1流感A/Mexio City/001/2009(H1N1)同属一个谱系,亲缘关系较近,同源性在93.2％～95.8％之间.结论 贵州省(2009)代表株与WHO公布的流行株在核苷酸水平上有一定程度的变异.     

Inhibition of adenovirus infection and adenain by green tea catechins

Green tea catechins have been reported to inhibit proteases involved in cancer metastasis and infection by influenza virus and HIV. To date there are no effective anti-adenoviral therapies. Consequently, we studied the effect of green tea catechins, and particularly the predominant component, epigallocatechin-3-gallate (EGCG), on adenovirus infection and the viral protease adenain, in cell culture. Adding EGCG (100 microM) to the medium of infected cells reduced virus yield by two orders of magnitude, giving and IC(50) of 25 microM and a therapeutic index of 22 in Hep2 cells. The agent was the most effective when added to the cells during the transition from the early to the late phase of viral infection suggesting that EGCG inhibits one or more late steps in virus infection. One of these steps appears to be virus assembly because the titer of infectious virus and the production of physical particles was much more affected than the synthesis of virus proteins. Another step might be the maturation cleavages carried out by adenain. Of the four catechins tested on adenain, EGCG was the most inhibitory with an IC(50) of 109 microM, compared with an IC(50) of 714 microM for PCMB, a standard cysteine protease inhibitor. EGCG and different green teas inactivated purified adenovirions with IC(50) of 250 and 245-3095, respectively. We conclude that the anti-adenoviral activity of EGCG manifests itself through several mechanisms, both outside and inside the cell, but at effective drug concentrations well above that reported in the serum of green tea drinkers.     

In vitro and in vivo efficacy of fluorodeoxycytidine analogs against highly pathogenic avian influenza H5N1, seasonal, and pandemic H1N1 virus infections

Various fluorodeoxyribonucleosides were evaluated for their antiviral activities against influenza virus infections in vitro and in vivo. Among the most potent inhibitors was 2′-deoxy-2′-fluorocytidine (2′-FdC). It inhibited various strains of low and highly pathogenic avian influenza H5N1 viruses, pandemic H1N1 viruses, an oseltamivir-resistant pandemic H1N1 virus, and seasonal influenza viruses (H3N2, H1N1, influenza B) in MDCK cells, with the 90% inhibitory concentrations ranging from 0.13 to 4.6 μM, as determined by a virus yield reduction assay. 2′-FdC was then tested for efficacy in BALB/c mice infected with a lethal dose of highly pathogenic influenza A/Vietnam/1203/2004 H5N1 virus. 2′FdC (60 mg/kg/d) administered intraperitoneally (i.p.) twice a day beginning 24 h after virus exposure significantly promoted survival (80% survival) of infected mice (p = 0.0001). Equally efficacious were the treatment regimens in which mice were treated with 2′-FdC at 30 or 60 mg/kg/day (bid X 8) beginning 24 h before virus exposure. At these doses, 70-80% of the mice were protected from death due to virus infection (p = 0.0005, p = 0.0001; respectively). The lungs harvested from treated mice at day four of the infection displayed little surface pathology or histopathology, lung weights were lower, and the 60 mg/kg dose reduced lung virus titers, although not significantly compared to the placebo controls. All doses were well tolerated in uninfected mice. 2′-FdC could also be administered as late as 72 h post virus exposure and still significantly protect 60% mice from the lethal effects of the H5N1 virus infection (p = 0.019). Other fluorodeoxyribonucleosides tested in the H5N1 mouse model, 2′-deoxy-5-fluorocytidine and 2′-deoxy-2′,2′-difluorocytidine, were very toxic at higher doses and not inhibitory at lower doses. Finally, 2′-FdC, which was active in the H5N1 mouse model, was also active in a pandemic H1N1 influenza A infection model in mice. When given at 30 mg/kg/d (bid X 5) beginning 24 h before virus exposure), 2′-FdC also significantly enhanced survival of H1N1-infected mice (50%, p = 0.038) similar to the results obtained in the H5N1 infection model using a similar dosing regimen (50%, p < 0.05). Given the demonstrated in vitro and in vivo inhibition of avian influenza virus replication, 2′FdC may qualify as a lead compound for the development of agents treating influenza virus infections.     

In vitro and in vivo effects of a long-acting anti-influenza agent CS-8958 (laninamivir octanoate, Inavir) against pandemic (H1N1) 2009 influenza viruses

Laninamivir is a novel neuraminidase inhibitor of influenza viruses and it has been reported that its prodrug, CS-8958 shows a long-lasting characteristics. Using viruses isolated in Nagasaki of pandemic (H1N1) 2009 influenza virus which cause pandemic in 2009, it was shown that laninamivir has a strong inhibitory activities against their neuraminidases and virus replication in cultured cells, and strong binding stability to the virus NA. Furthermore, a single intranasal administration of CS-8958 showed a superior reduction of virus load in lungs in mouse infection model. These suggest that CS-8958 will work as a long-acting neuraminidase inhibitor to an infection with pandemic (H1N1) 2009 influenza viruses as well.     

Activity of the neuraminidase inhibitor A-315675 against oseltamivir-resistant influenza neuraminidases of N1 and N2 subtypes

Clinical use of the neuraminidase inhibitor (NAI) oseltamivir has been associated with the emergence of viral resistance resulting from subtype-specific neuraminidase (NA) mutations. In this study, we evaluated the impact of the most frequent oseltamivir-resistant NA mutations including E119V, H274Y, R292K and N294S on the susceptibility profile to a novel NAI (A-315675) using recombinant NA proteins of N1 and N2 subtypes and also selected oseltamivir-resistant influenza H1N1 and H3N2 viruses. In the N1 subtype, recombinant NA proteins containing mutations H274Y and N294S previously associated with resistance to oseltamivir (754- and 197-fold increases in IC(50) values, respectively, compared to WT) remained susceptible to A-315675 (2.5- and 2-fold increases in IC(50) values(,) respectively). In the N2 subtype, NA proteins harboring mutations E119V and R292K conferring high levels of resistance to oseltamivir (1016- and >10,000-fold increases in IC(50) values, respectively) had IC(50) values that increased by only 1.5- and 13-fold, respectively, against A-315675. Similar susceptibility patterns to A-315675 were obtained when testing recombinant H1N1 mutant viruses (H274Y and N294S) and clinical H3N2 mutants (E119V). The V116A and I117V mutations, previously associated with oseltamivir resistance in H5N1 viruses, were susceptible to oseltamivir when tested in the H1N1 background suggesting a strain-specific impact of these mutations. These results confirm the potent inhibitory effect of A-315675 against oseltamivir-resistant influenza viruses of the N1 and N2 subtypes and support the clinical development of its bioavailable prodrug A-322278.     

Avian influenza: a review.

PURPOSE: A review of the avian influenza A/H5N1 virus, including human cases, viral transmission, clinical features, vaccines and antivirals, surveillance plans, infection control, and emergency response plans, is presented. SUMMARY: The World Health Organization (WHO) considers the avian influenza A/H5N1 virus a public health risk with pandemic potential. The next human influenza pandemic, if caused by the avian influenza A/H5N1 virus, is estimated to have a potential mortality rate of more than a hundred million. Outbreaks in poultry have been associated with human transmission. WHO has documented 258 confirmed human infections with a mortality rate greater than 50%. Bird-to-human transmission of the avian influenza virus is likely by the oral-fecal route. The most effective defense against an influenza pandemic would be a directed vaccine to elicit a specific immune response toward the strain or strains of the influenza virus. However, until there is an influenza pandemic, there is no evidence that vaccines or antivirals used in the treatment or prevention of such an outbreak would decrease morbidity or mortality. Surveillance of the bird and human populations for the highly pathogenic H5N1 is being conducted. Infection-control measures and an emergency response plan are discussed. CONCLUSION: Avian influenza virus A/H5N1 is a public health threat that has the potential to cause serious illness and death in humans. Understanding its pathology, transmission, clinical features, and pharmacologic treatments and preparing for the prevention and management of its outbreak will help avoid its potentially devastating consequences.     

Grippe A (H1N1) 2009. Revue générale

Influenza A(H1N1) 2009 is an acute contagious respiratory infection caused by influenza A virus subtype H1N1 appeared in 2009 and responsible for a pandemic. The new virus, different from the avian virus H5N1, is a variant containing genes from several known viruses from porcine, avian and human origin. Appeared originally in the northern hemisphere, the epidemic wave reached early most countries, and up to 24% of the population in metropolitan France. It is characterized by a low mortality estimated at 0.04 to 0.2%, and by a benign, or even asymptomatic presentation. However, more severe clinical expression has been observed in some subgroups, carrying or not risk factors (young, pregnant women). The preexisting immunity in a significant proportion of the population, the remarkable stability of the virus, determination of early antigenic characteristics of the virus, the development and rapid availability of suitable vaccines, the efficacy of antiviral drugs, and health care system contributed to the control the morbidity and mortality of the first pandemic wave. Other virological, clinical and epidemiological investigations, are needed to identify all potential risk factors for severity and determine the role of mutation and the diffusion of pandemic and seasonal viruses, which may alter the virulence and transmissibility of influenza A(H1N1)v 2009.     

Dynamics of antiviral-resistant influenza viruses in the Netherlands, 2005–2008

In the Netherlands, influenza specific antivirals are used for the therapy of influenza in nursing homes and hospitals, for prophylaxis in high risk groups and neuraminidase inhibitors are stockpiled as part of pandemic preparedness plans. To monitor the antiviral susceptibility profile, human influenza virus isolates derived from the Dutch influenza surveillance in 2005–2006 (n = 87), 2006–2007 (n = 58) and 2007–2008 (n = 128) were analyzed with phenotypic assays and sequencing. For adamantanes, a high proportion (>74%) of A(H3N2) viruses had the S31N mutation in M2 protein, while variation in the HA₁ region of adamantane-sensitive viruses suggested that adamantane-sensitive variants were reseeded into the Dutch population and re-emerged as drug-sensitive due to M-segment reassortment. For neuraminidase inhibitors oseltamivir and zanamivir, 98% of types A and B influenza viruses prior to 2007–2008 were sensitive for both, whereas 24% of the A(H1N1) viruses obtained in 2007–2008 were oseltamivir-resistant while retaining sensitivity to zanamivir and adamantanes. Furthermore, oseltamivir-resistant A(H1N1) or adamantane-resistant A(H3N2) virus infections were not associated with differences in clinical symptoms compared to infections with sensitive variants. Our data show the dynamic nature of emergence of drug-resistant influenza viruses, stressing the need for surveillance of resistance trends as part of influenza monitoring programs.     

黄芩苷联合帕拉米韦体内外抗甲型H1N1流感病毒作用

目的评价黄芩苷与帕拉米韦联合用药体内外抗甲型H1N1流感病毒作用。方法体外试验中,以甲型H1N1流感病毒感染MDCK细胞,黄芩苷与帕拉米韦联合用药,终点稀释法检测细胞上清液病毒滴度;体内试验中,以甲型H1N1流感病毒感染BALB/c小鼠,黄芩苷灌胃,帕拉米韦肌肉注射,两者联合给药,观察试验小鼠存活情况及体重变化。试验结果以MacSynergyⅡ软件分析两种药物体内外联合作用结果。结果细胞试验中,黄芩苷与帕拉米韦联用抗甲型H1N1流感病毒在95%置信区间内的协同值为3.2,表现为相加作用;小鼠试验中,黄芩苷与帕拉米韦联用,对提高感染流感病毒小鼠的存活率和抑制其体重下降表现为显著协同作用,协同值分别为69.0和105.2。结论黄芩苷与帕拉米韦联合抗流感作用比单独使用效果好,在临床上具有重要的应用价值。