In vitro inhibitory effect of carrageenan oligosaccharide on influenza A H1N1 virus

Carrageenan polysaccharide has been reported to be able to inhibit the infection and replication of many different kinds of viruses. Here, we demonstrated that a 2 kDa κ-carrageenan oligosaccharide (CO-1) derived from the carrageenan polysaccharide, effectively inhibited influenza A (H1N1) virus replication in MDCK cells (selectivity index >25.0). Moreover, the 2 kDa CO-1 inhibited influenza A virus (IAV) replication better than that of 3 kDa and 5 kDa κ-carrageenan oligosaccharides (CO-2 and CO-3). IAV multiplication was suppressed by carrageenan oligosaccharide treatment in a dose-dependent manner. Carrageenan oligosaccharide CO-1 did not bind to the cell surface of MDCK cells but inactivated virus particles after pretreatment. Different to the actions of carrageenan polysaccharide, CO-1 could enter into MDCK cells and did not interfere with IAV adsorption. CO-1 also inhibited IAV mRNA and protein expression after its internalization into cells. Moreover, carrageenan oligosaccharide CO-1 had an antiviral effect on IAV replication subsequent to viral internalization but prior to virus release in one replication cycle. Therefore, inhibition of IAV intracellular replication by carrageenan oligosaccharide might be an alternative approach for anti-influenza A virus therapy.     

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.     

Use of plethysmography in assessing the efficacy of antivirals in a mouse model of pandemic influenza A virus

The recently emerged swine-origin H1N1 influenza A virus (IAV) caused a pandemic outbreak in 2009 with higher risk of severe disease among children and pregnant women in their third trimester (Van Kerkhove et al., 2011), and is continuing to be important seasonal IAV strain. Mice are commonly used in antiviral studies as models of influenza disease, which utilize morbidity and mortality to assess the efficacy of a test compound. Here, we investigated the utility of unrestrained plethysomography to quantify the lung function of IAV-infected BALB/c mice. Administration of a lethal dose (∼30X LD₅₀) of pandemic H1N1 IAV resulted in a rapid decline in breath volume, as determined by a significant (P < 0.001) decrease in the pressure associated with inspiration and expiration detected as early as 2 days after virus challenge. Severe disease was also accompanied by a significant (P < 0.05) increase in breath time on 8 dpi. Plethysmography parameters correlated with weight loss and other parameters of disease such as gross pathology and the weight of the lung. Breath time was reduced in surviving mice challenged with a sublethal dose of virus as compared with normal controls, and is a predictive indicator of outcome in these mice. In antiviral studies, the use of plethysmography resulted in the detection of a clear and rapid treatment response, which was similar to other non-invasive parameters, such as weight change. Oseltamivir and ribavirin significantly (P < 0.001) improved parameters of lung function, particularly mean breath volume, as early as 2 dpi and in a dose-dependent manner. Moreover, a combination of these two drugs further improved these parameters. Plethysmography provides a sensitive evaluation of lung function in IAV-infected mice in response to antiviral therapy.     

The proton translocation domain of cellular vacuolar ATPase provides a target for the treatment of influenza A virus infections

BACKGROUND AND PURPOSE

Cellular vacuolar ATPases (v-ATPase) play an important role in endosomal acidification, a critical step in influenza A virus (IAV) host cell infection. We investigated the antiviral activity of the v-ATPase inhibitor saliphenylhalamide (SaliPhe) and compared it with several older v-ATPase inhibitors concanamycin A, bafilomycin A1, (BafA) and archazolid B targeting the subunit c of the V₀ sector.

EXPERIMENTAL APPROACH

An in vitro assay was devised to quantify the anti-influenza effect of v-ATPase inhibitors by measuring green fluorescent protein fluorescence of a reporter IAV. These data were combined with cytotoxicity testing to calculate selectivity indices. Data were validated by testing v-ATPase inhibitors against wild-type IAV in vitro and in vivo in mice.

KEY RESULTS

In vitro SaliPhe blocked the proliferation of pandemic and multidrug resistant viruses at concentrations up to 51-fold below its cytotoxic concentrations. At essentially non-toxic concentrations, SaliPhe protected 62.5% of mice against a lethal challenge of a mouse-adapted influenza strain, while BafA at cytotoxic concentrations showed essentially no protection against infection with IAV (SaliPhe vs. BafA P < 0.001).

CONCLUSIONS AND IMPLICATIONS

Our results show that a distinct binding site of the proton translocation domain of cellular v-ATPase can be selectively targeted by a new generation v-ATPase inhibitor with reduced toxicity to treat influenza virus infections, including multi-resistant strains. Treatment strategies against influenza that target host cellular proteins are expected to be more resistant to virus mutations than drugs blocking viral proteins.     

Inhibition of miR-93 promotes interferon effector signaling to suppress influenza A infection by upregulating JAK1

Type I interferons play a critical role in host defense against influenza virus infection. Interferon cascade induces the expression of interferon-stimulated genes then subsequently promotes antiviral immune responses. The microRNAs are important regulators of innate immunity, but microRNAs-mediated regulation of interferon cascade during influenza infection remains to be fully identified. Here we found influenza A virus (IAV) infection significantly inhibited miR-93 expression in alveolar epithelial type II cells through RIG-I/JNK pathway. IAV-induced downregulation of miR-93 was found to upregulate JAK1, the target of miR-93, and then feedback promote antiviral innate response by facilitating IFN effector signaling. Importantly, in vivo administration of miR-93 antagomiR markedly suppressed IAV infection, protecting mice form IAVs -associated death. Hence, the inducible downregulation of miR-93 feedback suppress IAV infection by strengthening IFN-JAK-STAT pathway via JAK1 upregulation, and in vivo inhibition of miR-93 bears considerable therapeutic potential for suppressing IAV infection.     

Antiviral activity of the proteasome inhibitor VL-01 against influenza A viruses

The appearance of highly pathogenic avian influenza A viruses of the H5N1 subtype being able to infect humans and the 2009 H1N1 pandemic reveals the urgent need for new and efficient countermeasures against these viruses. The long-term efficacy of current antivirals is often limited, because of the emergence of drug-resistant virus mutants. A growing understanding of the virus-host interaction raises the possibility to explore alternative targets involved in the viral replication. In the present study we show that the proteasome inhibitor VL-01 leads to reduction of influenza virus replication in human lung adenocarcinoma epithelial cells (A549) as demonstrated with three different influenza virus strains, A/Puerto Rico/8/34 (H1N1) (EC₅₀ value of 1.7 μM), A/Regensburg/D6/09 (H1N1v) (EC₅₀ value of 2.4 μM) and A/Mallard/Bavaria/1/2006 (H5N1) (EC₅₀ value of 0.8 μM). In in vivo experiments we could demonstrate that VL-01-aerosol-treatment of BALB/c mice with 14.1 mg/kg results in no toxic side effects, reduced progeny virus titers in the lung (1.1 ± 0.3 log₁₀ pfu) and enhanced survival of mice after infection with a 5-fold MLD₅₀ of the human influenza A virus strain A/Puerto Rico/8/34 (H1N1) up to 50%. Furthermore, treatment of mice with VL-01 reduced the cytokine release of IL-α/β, IL-6, MIP-1β, RANTES and TNF-α induced by LPS or highly pathogen avian H5N1 influenza A virus. The present data demonstrates an antiviral effect of VL-01 in vitro and in vivo and the ability to reduce influenza virus induced cytokines and chemokines.     

Therapeutic targeting of adenosine receptors in inflammatory diseases

A novel H7N9 avian influenza A virus (IAV) emerged in China in early 2013 causing > 450 cases of respiratory illness and 175 deaths within a 20-month period. Though avian viruses infect humans infrequently, the lack of human immunity to these viruses raises the possibility of a pandemic if they were to acquire the ability to transmit efficiently. Despite the fact that IAV pathogenicity results from the cytopathic effects and tissue damage caused by both viral replication and an overly robust immune response, current IAV therapeutics only target the viral proteins. This has led to the emergence of drug resistance due to the high mutation rates of viruses. The growing obsolescence of our current influenza therapeutics underscores the need for alternative treatment strategies. One promising area of research is the use of drugs that target the host response to IAV infection. This article describes how gene expression profiling can be used to predict drugs that reverse the destructive effects of the host response to H7N9 and other pathogenic influenza viruses.     

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.     

Single-stranded DNA aptamer that specifically binds to the influenza virus NS1 protein suppresses interferon antagonism

Non-structural protein 1 (NS1) of the influenza A virus (IAV) inhibits the host’s innate immune response by suppressing the induction of interferons (IFNs). Therefore, blocking NS1 activity can be a potential strategy in the development of antiviral agents against IAV infection. In the present study, we selected a single-stranded DNA aptamer specific to the IAV NS1 protein after 15 cycles of systematic evolution of ligands by exponential enrichment (SELEX) procedure and examined the ability of the selected aptamer to inhibit the function of NS1. The selected aptamer binds to NS1 with a K_d of 18.91 ± 3.95 nM and RNA binding domain of NS1 is determined to be critical for the aptamer binding. The aptamer has a G-rich sequence in the random sequence region and forms a G-quadruplex structure. The localization of the aptamer bound to NS1 in cells was determined by confocal images, and flow cytometry analysis further demonstrated that the selected aptamer binds specifically to NS1. In addition, luciferase reporter gene assay, quantitative RT-PCR, and enzyme-linked immunosorbent assay (ELISA) experiments demonstrated that the selected aptamer had the ability to induce IFN-β by suppressing the function of NS1. Importantly, we also found that the selected aptamer was able to inhibit the viral replication without affecting cell viability. These results indicate that the selected ssDNA aptamer has strong potential to be further developed as a therapeutic agent against IAV.     

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.     

Heme oxygenase activity and hemoglobin neurotoxicity are attenuated by inhibitors of the MEK/ERK pathway

Hemoglobin breakdown produces an iron-dependent neuronal injury after experimental CNS hemorrhage that may be attenuated by heme oxygenase (HO) inhibitors. The HO enzymes are phosphoproteins that are activated by phosphorylation in vitro. While testing the effect of kinase inhibitors in cortical cell cultures, we observed that HO activity was consistently decreased by the MEK inhibitor U0126. The present study tested the hypothesis that MEK/ERK pathway inhibitors reduce HO activity and neuronal vulnerability to hemoglobin. The MEK inhibitors U0126 and SL327 and the ERK inhibitor FR180204 reduced baseline culture HO activity by 35-50%, without altering the activity of recombinant HO-1 or HO-2; negative control compounds U0124 and FR180289 had no effect. Hemoglobin exposure for 16 h produced widespread neuronal injury, manifested by release of 59.2 ± 7.8% of neuronal lactate dehydrogenase and a twelve-fold increase in malondialdehyde; kinase inhibitors were highly protective. HO-1 induction after hemoglobin treatment was also decreased by U0126, SL327, and FR180204. These results suggest that reduction in HO activity may contribute to the protective effect of MEK and ERK inhibitors against heme-mediated neuronal injury.     

Type II transmembrane serine proteases as potential target for anti-influenza drug discovery

Introduction: The outbreak of an influenza pandemic as well as the continued circulation of seasonal influenza highlights the need for effective antiviral therapies. The emergence of drug-resistant strains further necessitates the development of novel antivirals that target the host factors crucial for viral replication.

Area covered: This review summarizes the current understanding of the structural and functional properties of type II transmembrane serine proteases (TTSPs) as a proteolytic activator of influenza virus infection and discusses their potential as antiviral targets. It also explores the experimental evidence accumulated for inhibitors of TTSPs as novel, broad-spectrum antivirals against various influenza virus subtypes. The review also provides an overview of the properties of small molecules, proteins, and peptides that efficiently inhibit the proteolytic activation of the influenza virus.

Expert opinion: TTSPs activate a wide range of influenza virus subtypes including avian influenza viruses, both in vitro and in vivo, via proteolytic cleavage of influenza hemagglutinin (HA) into infection-competent fusogenic conformation. Other viruses such as SARS-, MERS-coronaviruses and human metapneumoviruses may use the same host cell proteases for activation, implying that TTSP inhibition might be a novel strategy for developing broad-spectrum antiviral agents for respiratory viral infections.     

Antiviral effects of Psidium guajava Linn. (guava) tea on the growth of clinical isolated H1N1 viruses: its role in viral hemagglutination and neuraminidase inhibition

Rapid evolution of influenza RNA virus has resulted in limitation of vaccine effectiveness, increased emergence of drug-resistant viruses and occurrence of pandemics. A new effective antiviral is therefore needed for control of the highly mutative influenza virus. Teas prepared by the infusion method were tested for their anti-influenza activity against clinical influenza A (H1N1) isolates by a 19-h influenza growth inhibition assay with ST6Gal I-expressing MDCK cells (AX4 cells) using fluorogenic quantification and chromogenic visualization. Guava tea markedly inhibited the growth of A/Narita/1/2009 (amantadine-resistant pandemic 2009 strain) at an IC(50) of 0.05% and the growth of A/Yamaguchi/20/06 (sensitive strain) and A/Kitakyushu/10/06 (oseltamivir-resistant strain) at similar IC(50) values ranging from 0.24% to 0.42% in AX4 cells, being 3.4- to 5.4-fold more potent than green tea (IC(50) values: 0.27% for the 2009 pandemic strain and 0.91% to 1.44% for the seasonal strains). In contrast to both teas, oseltamivir carboxylate (OC) demonstrated high potency against the growth of A/Narita/1/09 (IC(50) of 3.83nM) and A/Yamaguchi/20/06 (IC(50) of 11.57nM) but not against that of A/Kitakyushu/10/06 bearing a His274-to-Tyr substitution (IC(50) of 15.97μM). Immunofluorescence analysis under a confocal microscope indicated that both teas inhibited the most susceptible A/Narita/1/2009 virus at the initial stage of virus infection. This is consistent with results of direct inhibition assays showing that both teas inhibited viral hemagglutination at concentrations comparable to their growth inhibition concentrations but inhibited sialidase activity at about 8-times higher concentrations. Guava tea shows promise to be efficacious for control of epidemic and pandemic influenza viruses including oseltamivir-resistant strains, and its broad target blockage makes it less likely to lead to emergence of viral resistance.     

Characterization of a fully human monoclonal antibody against extracellular domain of matrix protein 2 of influenza A virus

The extra-cellular domain of the influenza virus matrix protein 2 (M2e) is highly conserved between influenza A virus strains compared to hemagglutinin and neuraminidase, and has long been viewed as a potential and universal vaccine target. M2e induces no or only weak and transient immune responses following infection, making it difficult to detect M2e-specific antibodies producing B-cells in human peripheral blood lymphocytes. Recently, using a single-cell manipulation method, immunospot array assay on a chip (ISAAC), we obtained an M2e-specific human antibody (Ab1-10) from the peripheral blood of a healthy volunteer. In this report, we have demonstrate that Ab1-10 reacted not only to seasonal influenza A viruses, but also to pandemic (H1N1) 2009 virus (2009 H1N1) and highly pathogenic avian influenza A virus, and that the antibody-bound M2e of 2009 H1N1 inactivated the virus with high affinity (∼10⁻¹⁰ M). More importantly, it inhibited 2009 H1N1 viral propagation in vitro. These results suggest that Ab1-10 might be a potential candidate for antibody therapeutics for a wide range of influenza A viruses.     

Vitisin B inhibits influenza A virus replication by multi-targeting neuraminidase and virus-induced oxidative stress

The development of drug-resistant influenza and new pathogenic virus strains underscores the need for antiviral therapeutics. Currently, neuraminidase(NA)inhibitors are commonly used antiviral drugs approved by the US Food and Drug Administration(FDA)for the prevention and treatment of influenza. Here, we show that vitisin B(VB)inhibits NA activity and suppresses H1N1 viral replication in MDCK and A549 cells. Reactive oxygen species(ROS), which frequently occur during viral infection,increase vi...     

Progress in identifying virulence determinants of the 1918 H1N1 and the Southeast Asian H5N1 influenza A viruses

The 1918 pandemic H1N1 influenza virus and the recently emerged Southeast Asian H5N1 avian influenza virus are unique among influenza A virus isolates in their high virulence for humans and their lethality for a variety of animal species without prior adaptation. Reverse genetic studies have implicated several viral genes as virulence determinants. For both the 1918 and H5N1 viruses, the hemagglutinin and the polymerase complex contribute to high virulence. Non-structural proteins NS1 and PB1-F2, which block host antiviral responses, also influence pathogenesis. Additionally, recent studies correlate high levels of viral replication and induction of strong proinflammatory responses with the high virulence of these viruses. Defining how individual viral proteins promote enhanced replication, inflammation and severe disease will provide insight into the pathogenesis of severe influenza virus infections and suggest novel therapeutic approaches.     

Chinese herbal medicine compound Yi-Zhi-Hao pellet inhibits replication of influenza virus infection through activation of heme oxygenase-1

As a leading cause of respiratory disease, influenza A virus (IAV) presents a pandemic threat in annual seasonal outbreaks. Given the limitation of existing anti-influenza therapies, there remains to be a requirement for new drugs. Compound Yi-Zhi-Hao pellet (CYZH) is a famous traditional Chinese medicine (TCM) used in the clinic, whose formula has been recorded in Complication of National Standard for Traditional Chinese Medicine to treat common cold. In this study, we found that CYZH exhibited a broad-spectrum anti-influenza activity and inhibited the expression of viral RNA and proteins in vitro. Mechanistically, CYZH had no inhibitory activities against viral protein hemagglutinin and IAV RNA-dependent RNA polymerase. Instead, it induced activation of erythroid 2-related factor 2 (Nrf2) and nuclear factor kappa B (NF-κB), which subsequently upregulated heme oxygenase-1 (HO-1) expression. Also, CYZH protected cells from oxidative damage induced by reactive oxygen series. In conclusions, CYZH inhibits IAV replication in vitro, at least partly by activating expression of the Nrf2/HO-1 pathway.     

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.     

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.     

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。