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.     

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.     

Susceptibility of highly pathogenic A(H5N1) avian influenza viruses to the neuraminidase inhibitors and adamantanes

Since 2003, highly pathogenic A(H5N1) influenza viruses have been the cause of large-scale death in poultry and the subsequent infection and death of over 140 humans. A group of 55 influenza A(H5N1) viruses isolated from various regions of South East Asia between 2004 and 2006 were tested for their susceptibility to the anti-influenza drugs the neuraminidase inhibitors and adamantanes. The majority of strains were found to be fully sensitive to the neuraminidase inhibitors oseltamivir carboxylate, zanamivir and peramivir; however two strains demonstrated increased IC50 values. Sequence analysis of these strains revealed mutations in the normally highly conserved residues 116 and 117 of the N1 neuraminidase. Sequence analysis of the M2 gene showed that all of the A(H5N1) viruses from Vietnam, Malaysia and Cambodia contained mutations (L26I and S31N) associated with resistance to the adamantane drugs (rimantadine and amantadine), while strains from Indonesia were found to be a mix of both adamantane resistant (S31N) and sensitive viruses. None of the A(H5N1) viruses from Myanmar contained mutations known to confer adamantane resistance. These results support the use of neuraminidase inhibitors as the most appropriate class of antiviral drug to prevent or treat human A(H5N1) virus infections.     

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.     

Susceptibility of recent Canadian influenza A and B virus isolates to different neuraminidase inhibitors

Forty-two influenza A and 23 influenza B isolates collected from untreated subjects during the 1999-2000 influenza season in Canada were tested for their susceptibility to three neuraminidase inhibitors (zanamivir, oseltamivir carboxylate and RWJ-270201 or BCX-1812) using a chemiluminescent neuraminidase assay. Influenza B isolates were less susceptible than A viruses to all tested drugs. RWJ-270201 was the most potent drug against both influenza A(H3N2) (mean IC(50): 0.60 nM) and B (mean IC(50): 0.87 nM) viruses. Oseltamivir carboxylate was more active than zanamivir for influenza A(H3N2) isolates (mean IC(50): 0.73 vs. 2.09 nM) whereas it was less potent against B viruses (mean IC(50): 11.53 vs. 4.15 nM).     

Sensitivity of influenza viruses to zanamivir and oseltamivir: a study performed on viruses circulating in France prior to the introduction of neuraminidase inhibitors in clinical practice

Influenza virus neuraminidase inhibitors (NAIs) were introduced in clinical practice in various parts of the world since 1999 but were only scarcely distributed in France. Prior to the generalization of zanamivir and oseltamivir utilization in our country, we decided to test a large panel of influenza strains to establish the baseline sensitivity of these viruses to anti-neuraminidase drugs, based upon a fluorometric neuraminidase enzymatic test. Our study was performed on clinical samples collected by practitioners of the GROG network (Groupe Régional d'Observation de la Grippe) in the south of France during the 2002-2003 influenza season. Out of 355 isolates tested in the fluorometric neuraminidase activity assay, 267 isolates could be included in inhibition assay against anti-neuraminidase drugs. Differences in IC50 range were found according to the subtype and the anti-neuraminidase drug. Influenza B and A/H1N1 viruses appeared to be more sensitive to zanamivir than to oseltamivir (mean B IC50 values: 4.19 nM versus 13 nM; mean H1N1 IC50 values: 0.92 nM versus 1.34 nM), while A/H1N2 and A/H3N2 viruses were more sensitive to oseltamivir than to zanamivir (mean H3N2 IC50 values: 0.67 nM versus 2.28 nM; mean H1N2 IC50 values: 0.9 nM versus 3.09 nM). Out of 128 N2 carrying isolates, 10 isolates had zanamivir or oseltamivir IC50 values in upper limits compared to their respective data range. Sequencing of the neuraminidase of these outliers N2 highlighted several mutations, but none of them were associated with resistance to neuraminidase inhibitors.     

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.     

Susceptibility of human influenza viruses from Australasia and South East Asia to the neuraminidase inhibitors zanamivir and oseltamivir

Human influenza viruses isolated from Australasia (Australia and New Zealand) and South East Asia were analysed to determine their sensitivity to the NA inhibitor drugs, zanamivir and oseltamivir. A total of 532 strains isolated between 1998 and 2002 were tested using a fluorescence-based assay to measure the relative inhibition of NA activity over a range of drug concentrations. Based on median IC50 values, influenza A viruses (with neuraminidase subtypes N1 and N2) were more sensitive to both the NA inhibitors than were influenza B strains. Influenza A viruses with a N1 subtype and influenza B strains both demonstrated a greater sensitivity to zanamivir than to oseltamivir carboxylate, whereas influenza A strains with a N2 subtype were more susceptible to oseltamivir carboxylate. For each of the neuraminidase types, IC50 values for viruses from Australasia and South East Asia were found to be comparable. Based on the data prior to and following the licensing of the drugs into the respective regions, the use of the NA inhibitors did not appear to have a significant impact on the susceptibility of the viruses tested to zanamivir or oseltamivir carboxylate.     

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.     

Current and future antiviral therapy of severe seasonal and avian influenza

The currently circulating H3N2 and H1N1 subtypes of influenza A virus cause a transient, febrile upper respiratory illness in most adults and children ("seasonal influenza"), but infants, the elderly, immunodeficient and chronically ill persons may develop life-threatening primary viral pneumonia or complications such as bacterial pneumonia. By contrast, avian influenza viruses such as the H5N1 virus that recently emerged in Southeast Asia can cause severe disease when transferred from domestic poultry to previously healthy people ("avian influenza"). Most H5N1 patients present with fever, cough and shortness of breath that progress rapidly to adult respiratory distress syndrome. In seasonal influenza, viral replication remains confined to the respiratory tract, but limited studies indicate that H5N1 infections are characterized by systemic viral dissemination, high cytokine levels and multiorgan failure. Gastrointestinal infection and encephalitis also occur. The licensed anti-influenza drugs (the M2 ion channel blockers, amantadine and rimantadine, and the neuraminidase inhibitors, oseltamivir and zanamivir) are beneficial for uncomplicated seasonal influenza, but appropriate dosing regimens for severe seasonal or H5N1 viral infections have not been defined. Treatment options may be limited by the rapid emergence of drug-resistant viruses. Ribavirin has also been used to a limited extent to treat influenza. This article reviews licensed drugs and treatments under development, including high-dose oseltamivir; parenterally administered neuraminidase inhibitors, peramivir and zanamivir; dimeric forms of zanamivir; the RNA polymerase inhibitor T-705; a ribavirin prodrug, viramidine; polyvalent and monoclonal antibodies; and combination therapies.     

Adamantane resistance in influenza A(H1) viruses increased in 2007 in South East Asia but decreased in Australia and some other countries

The adamantanes (amantadine and rimantadine) were the initial antivirals licensed for use against influenza A viruses and have been used in some countries to control seasonal influenza and have also been stockpiled for potential pandemic use. While high rates of resistance have been observed in recent years with A(H3) viruses, the rates of resistance with A(H1) viruses has varied widely. In this study we analysed 281 human influenza A viruses isolated in 2007 that were referred to the WHO Collaborating Centre for Reference and Research in Melbourne, mainly from Australia and the surrounding regions, for evidence of resistance to adamantanes and a subset of these was examined for resistance to the neuraminidase inhibitors (NIs). We found that the rates of adamantane resistance in A(H3) viruses continued to increase in most countries in 2007 but a distinct variation was seen with A(H1) resistance levels. A(H1) viruses from Australia, New Zealand and Europe had low rates of resistance (2-9%) whereas viruses from a number of South East (SE) Asian countries had high rates of resistance (33-100%). This difference can be attributed to the spread of A/Brisbane/59/2007-like viruses to many parts of the world with the exception of SE Asia where A/Hong Kong/2652/2006-like viruses continue to predominate. When these two A(H1) subgroups were compared for their in vitro sensitivity to the other class of influenza antiviral drugs, the neuraminidase inhibitors, no difference was seen between the groups with both showing normal levels of sensitivity to these drugs, The finding of reducing A(H1) resistance rates in Australia and rising levels in SE Asia in 2007, reverses the trend seen in 2006 when A(H1) resistance levels were rising in Australia and elsewhere but remained low in most of SE Asia.     

In vitro generation and characterisation of an influenza B variant with reduced sensitivity to neuraminidase inhibitors

A contemporary influenza type B virus was passaged in vitro in the presence of increasing concentrations of the neuraminidase inhibitors, zanamivir and oseltamivir carboxylate (0.1-1000 microM over nine passages). After the fifth passage in the presence of zanamivir (10 microM), the virus acquired a Glu 119 Asp neuraminidase mutation (influenza A N2 subtype numbering) in the enzyme active site. After a further three passages, in which growth occurred in 100 microM of zanamivir, a Gln 218 Lys mutation (A (H3) numbering) in the HA1 domain of the haemagglutinin was found. In a fluorescence-based neuraminidase inhibition assay, viruses with the Glu 119 Asp NA mutation had a 32,000-fold reduction in sensitivity to the NA inhibitor zanamivir compared to the wild-type virus, while the mutation resulted in a 105-fold reduction in sensitivity to oseltamivir carboxylate. Viruses grown in the presence of 1000 microM oseltamivir carboxylate did not acquire any neuraminidase mutations but did have a His 103 Gln substitution (A (H3) numbering) in the HA1 region of the haemagglutinin which was demonstrated to significantly reduce receptor binding strength in vitro. Tissue culture assays demonstrated that the HA mutation caused a seven-fold reduction in sensitivity to oseltamivir carboxylate, and a 90-fold reduction in sensitivity to zanamivir.     

Characterization of drug-resistant recombinant influenza A/H1N1 viruses selected in vitro with peramivir and zanamivir

There is a limited information with regard to the neuraminidase (NA) mutations conferring resistance to peramivir and zanamivir in the influenza N1 background. In this study, an influenza A/WSN/33 (H1N1) recombinant virus was passaged under peramivir or zanamivir pressure. The peramivir-selected variant had a H274Y mutation in the neuraminidase (NA) gene conferring resistance to peramivir and oseltamivir but susceptibility to zanamivir. The zanamivir-selected variant had a massive deletion in the region encoding the NA active center and an A200T hemagglutinin mutation. This variant exhibited reduced susceptibility to zanamivir with a drug-dependent phenotype.     

Antiviral agents active against influenza A viruses

The recent outbreaks of avian influenza A (H5N1) virus, its expanding geographic distribution and its ability to transfer to humans and cause severe infection have raised serious concerns about the measures available to control an avian or human pandemic of influenza A. In anticipation of such a pandemic, several preventive and therapeutic strategies have been proposed, including the stockpiling of antiviral drugs, in particular the neuraminidase inhibitors oseltamivir (Tamiflu; Roche) and zanamivir (Relenza; GlaxoSmithKline). This article reviews agents that have been shown to have activity against influenza A viruses and discusses their therapeutic potential, and also describes emerging strategies for targeting these viruses.     

Oseltamivir for treatment and prophylaxis of influenza infection

Background: Influenza infection is a global problem affecting millions of people worldwide, despite efficacious vaccines. Treatment and prophylaxis against influenza have been successful using antiviral medications such as adamantanes and neuraminidase inhibitors. Objective: To review the antiviral agents and specifically the neuraminidase inhibitor, oseltamivir, for use in treatment and prophylaxis of influenza infection. Methods: This review focuses on published literature regarding the clinical use of oseltamivir, as well as discussing emerging threats such as avian influenza, antiviral resistance, and strategies such as combination antiviral treatment to mitigate these threats. Results: Oseltamivir is effective in reducing symptom burden in those with influenza A or B infection, and is preventative against developing infection after exposure. Emergence of naturally occurring or post-treatment oseltamivir-resistant influenza as well as an avian influenza pandemic may limit its future use as a monotherapeutic antiviral treatment agent.     

The hydrophobic side chain of oseltamivir influences type A subtype selectivity of neuraminidase inhibitors

Neuraminidase, which plays a critical role in the influenza virus life cycle, is a target for new therapeutic agents. The study of structure–activity relationships revealed that the C‐5 position amino group of oseltamivir was pointed to 150‐cavity of the neuraminidase in group 1. This cavity is important for selectivity of inhibitors against N1 versus N2 NA. A serial of influenza neuraminidase inhibitors with the oseltamivir scaffold containing lipophilic side chains at the C‐5 position have been synthesized and evaluated for their influenza neuraminidase inhibitory activity and selectivity. The results indicated that compound 13o (H5N1 IC₅₀ = 0.1 ± 0.04 μm , H3N2 IC₅₀ = 0.26 ± 0.18 μm ) showed better inhibitory activity and selectivity against the group 1 neuraminidase. This study may provide a clue to design of better group 1 neuraminidase inhibitors.     

Influenza virus transmission: basic science and implications for the use of antiviral drugs during a pandemic

Recent and ongoing zoonotic infections of humans with avian influenza viruses have highlighted the importance of transmission in the development of an influenza pandemic. Despite the ability of H5N1 influenza viruses to grow to high titers and cause severe disease in human hosts, these viruses do not spread efficiently from human-to-human. The question of what viral, host and environmental factors are required to render an influenza virus transmissible has therefore become very topical. Recent work in the ferret model has suggested that receptor binding specificity is an important factor, but that the trait of human-like receptor recognition alone is not sufficient to confer a transmissible phenotype. In addition to the ferret, the guinea pig has been identified as a useful model host for transmission studies. Further research using these models is needed, toward understanding the molecular circumstances under which transmission can occur. A crucial role of antiviral drugs in mitigating an influenza pandemic will be to slow the spread of infection while an appropriate vaccine is in production. The efficacy of antivirals in preventing transmission is therefore of great importance. While the adamantanes, amantadine and rimantadine, have been found to fail in this respect due to the high transmissibility of drug resistant variants, the neuraminidase inhibitors, oseltamivir and zanamivir, show more promise. Anti-influenza drugs in development which show efficacy in terms of mitigating disease or viral growth should also be tested for their potential to block transmission.     

Neuraminidase inhibitors and risk of H5N1 influenza

Oseltamivir and zanamivir are highly potent inhibitors of influenza A and B neuraminidase and operate by inhibiting viral replication, and more specifically, the release and the movement of the virus through mucus. Neuraminidase inhibitors reduce the severity and duration of symptoms, and prevent clinical influenza as post-exposure and seasonal prophylaxis. Both have similar efficacy; oseltamivir has a more convenient route of administration, and zanamivir a more favourable resistance profile. Pending availability of effective vaccines, neuraminidase inhibitors are the only specific antiviral drugs which might be opposed to a possible pandemic that could emerge from the current highly pathogenic H5N1 virus. Although the effectiveness of oseltamivir and zanamivir for the therapy of clinical H5N1 influenza is questionable, simulation models suggest that a combination of targeted antiviral prophylaxis and quarantine might be able to contain an emerging influenza strain at the source. As a consequence, after an initial lack of commercial success probably related to the mild intensity of seasonal influenza during the last winters, neuraminidase inhibitors are now stockpiled by many countries to prepare for an outbreak.     

Zanamivir for the treatment of avian influenza infections in humans

Zanamivir is currently not recommended by the WHO for the first-line treatment of human influenza A (H5N1) infections. Zanamivir pharmacokinetics and antiviral efficacy were reviewed to assess the basis for this recommendation. Following inhalation of 10 mg zanamivir in healthy humans, the drug is widely distributed throughout the lungs. Mean lung sputum concentrations of zanamivir are 1441 and 235 ng/ml at 6 and 12 h postinhalation, respectively. Following repeated dosing to patients, mean steady-state C(max) and C(min) plasma zanamivir concentrations are approximately 60 and 4 ng/ml, respectively. Zanamivir has high in vitro activity against H5N1 strains isolated from patients (IC(50) 0.3-0.7 ng/ml) including oseltamivir-resistant strains. These data support a reassessment of zanamivir's role in the treatment of human H5N1 infections.