Structural insight into SARS-CoV-2 neutralizing antibodies and modulation of syncytia.

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is initiated by binding of the viral Spike protein to host receptor angiotensin-converting enzyme 2 (ACE2), followed by fusion of viral and host membranes. Although antibodies that block this interaction are in emergency use as early coronavirus disease 2019 (COVID-19) therapies, the precise determinants of neutralization potency remain unknown. We discovered a series of antibodies that potently block ACE2 binding but exhibit divergent neutralization efficacy against the live virus. Strikingly, these neutralizing antibodies can inhibit or enhance Spike-mediated membrane fusion and formation of syncytia, which are associated with chronic tissue damage in individuals with COVID-19. As revealed by cryoelectron microscopy, multiple structures of Spike-antibody complexes have distinct binding modes that not only block ACE2 binding but also alter the Spike protein conformational cycle triggered by ACE2 binding. We show that stabilization of different Spike conformations leads to modulation of Spike-mediated membrane fusion with profound implications for COVID-19 pathology and immunity.

Hydroxychloroquine use against SARS-CoV-2 infection in non-human primates.

Coronavirus disease 2019 (COVID-19) has rapidly become a global pandemic and no antiviral drug or vaccine is yet available for the treatment of this disease1-3. Several clinical studies are ongoing to evaluate the efficacy of repurposed drugs that have demonstrated antiviral efficacy in vitro. Among these candidates, hydroxychloroquine (HCQ) has been given to thousands of individuals infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-the virus that causes COVID-19-worldwide but there is no definitive evidence that HCQ is effective for treating COVID-194-7. Here we evaluated the antiviral activity of HCQ both in vitro and in SARS-CoV-2-infected macaques. HCQ showed antiviral activity in African green monkey kidney cells (Vero E6) but not in a model of reconstituted human airway epithelium. In macaques, we tested different treatment strategies in comparison to a placebo treatment, before and after peak viral load, alone or in combination with azithromycin (AZTH). Neither HCQ nor the combination of HCQ and AZTH showed a significant effect on viral load in any of the analysed tissues. When the drug was used as a pre-exposure prophylaxis treatment, HCQ did not confer protection against infection with SARS-CoV-2. Our findings do not support the use of HCQ, either alone or in combination with AZTH, as an antiviral drug for the treatment of COVID-19 in humans.

Influenza viruses and coronaviruses: Knowns, unknowns, and common research challenges.

The development of safe and effective vaccines in a record time after the emergence of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a remarkable achievement, partly based on the experience gained from multiple viral outbreaks in the past decades. However, the Coronavirus Disease 2019 (COVID-19) crisis also revealed weaknesses in the global pandemic response and large gaps that remain in our knowledge of the biology of coronaviruses (CoVs) and influenza viruses, the 2 major respiratory viruses with pandemic potential. Here, we review current knowns and unknowns of influenza viruses and CoVs, and we highlight common research challenges they pose in 3 areas: the mechanisms of viral emergence and adaptation to humans, the physiological and molecular determinants of disease severity, and the development of control strategies. We outline multidisciplinary approaches and technological innovations that need to be harnessed in order to improve preparedeness to the next pandemic.

Interactions Between Severe Acute Respiratory Syndrome Coronavirus 2 Replication and Major Respiratory Viruses in Human Nasal Epithelium.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), along with extensive nonpharmacological interventions, have profoundly altered the epidemiology of major respiratory viruses. Some studies have described virus-virus interactions, particularly manifested by viral interference mechanisms at different scales. However, our knowledge of the interactions between SARS-CoV-2 and other respiratory viruses remains incomplete. Here, we studied the interactions between SARS-CoV-2 and several respiratory viruses (influenza, respiratory syncytial virus, human metapneumovirus, and human rhinovirus) in a reconstituted human epithelial airway model, exploring different scenarios affecting the sequence and timing of coinfections. We show that the virus type and sequence of infections are key factors in virus-virus interactions, the primary infection having a determinant role in the immune response to the secondary infection.

SARS-CoV-2 viral dynamics in non-human primates.

Non-human primates infected with SARS-CoV-2 exhibit mild clinical signs. Here we used a mathematical model to characterize in detail the viral dynamics in 31 cynomolgus macaques for which nasopharyngeal and tracheal viral load were frequently assessed. We identified that infected cells had a large burst size (>104 virus) and a within-host reproductive basic number of approximately 6 and 4 in nasopharyngeal and tracheal compartment, respectively. After peak viral load, infected cells were rapidly lost with a half-life of 9 hours, with no significant association between cytokine elevation and clearance, leading to a median time to viral clearance of 10 days, consistent with observations in mild human infections. Given these parameter estimates, we predict that a prophylactic treatment blocking 90% of viral production or viral infection could prevent viral growth. In conclusion, our results provide estimates of SARS-CoV-2 viral kinetic parameters in an experimental model of mild infection and they provide means to assess the efficacy of future antiviral treatments.

Human Respiratory Syncytial Virus-Induced Immune Signature of Infection Revealed by Transcriptome Analysis of Clinical Pediatric Nasopharyngeal Swab Samples.

Human respiratory syncytial virus (HRSV) constitutes one the main causes of respiratory infection in neonates and infants worldwide. Transcriptome analysis of clinical samples using high-throughput technologies remains an important tool to better understand virus-host complex interactions in the real-life setting but also to identify new diagnosis/prognosis markers or therapeutics targets. A major challenge when exploiting clinical samples such as nasal swabs, washes, or bronchoalveolar lavages is the poor quantity and integrity of nucleic acids. In this study, we applied a tailored transcriptomics workflow to exploit nasal wash samples from children who tested positive for HRSV. Our analysis revealed a characteristic immune signature as a direct reflection of HRSV pathogenesis and highlighted putative biomarkers of interest such as IP-10, TMEM190, MCEMP1, and TIMM23.

Human metapneumovirus activates NOD-like receptor protein 3 inflammasome via its small hydrophobic protein which plays a detrimental role during infection in mice.

NOD-like receptor protein 3 (NLRP3) inflammasome activation triggers caspase-1 activation-induced maturation of interleukin (IL)-1ß and IL-18 and therefore is important for the development of the host defense against various RNA viral diseases. However, the implication of this protein complex in human metapneumovirus (HMPV) disease has not been fully studied. Herein, we report that NLRP3 inflammasome plays a detrimental role during HMPV infection because NLRP3 inflammasome inhibition protected mice from mortality and reduced weight loss and inflammation without impacting viral replication. We also demonstrate that NLRP3 inflammasome exerts its deleterious effect via IL-1ß production since we observed reduced mortality, weight loss and inflammation in IL-1ß-deficient (IL-1ß-/-) mice, as compared to wild-type animals during HMPV infection. Moreover, the effect on these evaluated parameters was not different in IL-1ß-/- and wild-type mice treated with an NLRP3 inflammasome inhibitor. The production of IL-1ß was also abrogated in bone marrow derived macrophages deficient for NLRP3. Finally, we show that small hydrophobic protein-deleted recombinant HMPV (HMPV ΔSH) failed to activate caspase-1, which is responsible for IL-1ß cleavage and maturation. Furthermore, HMPV ΔSH-infected mice had less weight loss, showed no mortality and reduced inflammation, as compared to wild-type HMPV-infected mice. Thus, NLRP3 inflammasome activation seems to be triggered by HMPV SH protein in HMPV disease. In summary, once activated by the HMPV SH protein, NLRP3 inflammasome promotes the maturation of IL-1ß, which exacerbates HMPV-induced inflammation. Therefore, the blockade of IL-1ß production by using NLRP3 inflammasome inhibitors might be a novel potential strategy for the therapy and prevention of HMPV infection.

Antiviral Properties of the NSAID Drug Naproxen Targeting the Nucleoprotein of SARS-CoV-2 Coronavirus.

There is an urgent need for specific antiviral treatments directed against SARS-CoV-2 to prevent the most severe forms of COVID-19. By drug repurposing, affordable therapeutics could be supplied worldwide in the present pandemic context. Targeting the nucleoprotein N of the SARS-CoV-2 coronavirus could be a strategy to impede viral replication and possibly other essential functions associated with viral N. The antiviral properties of naproxen, a non-steroidal anti-inflammatory drug (NSAID) that was previously demonstrated to be active against Influenza A virus, were evaluated against SARS-CoV-2. Intrinsic fluorescence spectroscopy, fluorescence anisotropy, and dynamic light scattering assays demonstrated naproxen binding to the nucleoprotein of SARS-Cov-2 as predicted by molecular modeling. Naproxen impeded recombinant N oligomerization and inhibited viral replication in infected cells. In VeroE6 cells and reconstituted human primary respiratory epithelium models of SARS-CoV-2 infection, naproxen specifically inhibited viral replication and protected the bronchial epithelia against SARS-CoV-2-induced damage. No inhibition of viral replication was observed with paracetamol or the COX-2 inhibitor celecoxib. Thus, among the NSAID tested, only naproxen combined antiviral and anti-inflammatory properties. Naproxen addition to the standard of care could be beneficial in a clinical setting, as tested in an ongoing clinical study.

Role of p53/NF-κB functional balance in respiratory syncytial virus-induced inflammation response.

The interplay between respiratory syncytial virus (RSV) and the p53 pathway has only been reported in a limited number of studies, yet the underlying abrogation mechanisms of p53 activity during the time course of infection, possibly involving viral proteins, remained unclear. Here, we demonstrate that RSV infection impairs global p53 transcriptional activity, notably via its proteasome-dependent degradation at late stages of infection. We also demonstrate that NS1 and NS2 contribute to the abrogation of p53 activity, and used different experimental strategies (e.g. siRNA, small molecules) to underline the antiviral contribution of p53 in the context of RSV infection. Notably, our study highlights a strong RSV-induced disequilibrium of the p53/NF-κB functional balance, which appears to contribute to the up-regulation of the expression of several proinflammatory cytokines and chemokines.

The combination of oseltamivir with azithromycin does not show additional benefits over oseltamivir monotherapy in mice infected with influenza A(H1N1)pdm2009 virus.

The combination of azithromycin, an immunomodulator, with oseltamivir was compared to oseltamivir monotherapy in a lethal BALB/c model of influenza A(H1N1)pdm09 infection. Groups of 14-16 mice received oral oseltamivir (10 mg/kg once daily for 5 days, starting at day 2 post-inoculation) alone or combined to azithromycin (a single 100 mg/kg dose, injected intraperitoneally at day 3 post-inoculation). Based on survival rates, lung viral titers, and pro-inflammatory cytokine levels, the combination therapy did not provide obvious additional clinical/virological benefits over oseltamivir monotherapy. Additional studies are still needed to better define the potential role of adjunctive immunomodulatory therapy for severe influenza infections.

Impact of potential permissive neuraminidase mutations on viral fitness of the H275Y oseltamivir-resistant influenza A(H1N1)pdm09 virus in vitro, in mice and in ferrets.

Neuraminidase (NA) mutations conferring resistance to NA inhibitors (NAIs) generally compromise the fitness of influenza viruses. The only NAI-resistant virus that widely spread in the population, the A/Brisbane/59/2007 (H1N1) strain, contained permissive mutations that restored the detrimental effect caused by the H275Y change. Computational analysis predicted other permissive NA mutations for A(H1N1)pdm09 viruses. Here, we investigated the effect of T289M and N369K mutations on the viral fitness of the A(H1N1)pdm09 H275Y variant. Recombinant wild-type (WT) A(H1N1)pdm09 and the H275Y, H275Y/T289M, H275Y/N369K, and H275Y/V241I/N369K (a natural variant) NA mutants were generated by reverse genetics. Replication kinetics were performed by using ST6GalI-MDCK cells. Virulence was assessed in C57BL/6 mice, and contact transmission was evaluated in ferrets. The H275Y mutation significantly reduced viral titers during the first 12 to 36 h postinfection (p.i.) in vitro. Nevertheless, the WT and H275Y viruses induced comparable mortality rates, weight loss, and lung titers in mice. The T289M mutation eliminated the detrimental effect caused by the H275Y change in vitro while causing greater weight loss and mortality in mice, with significantly higher lung viral titers on days 3 and 6 p.i. than with the H275Y mutant. In index ferrets, the WT, H275Y, H275Y/T289M, and H275Y/V241I/N369K recombinants induced comparable fever, weight loss, and nasal wash viral titers. All tested viruses were transmitted at comparable rates in contact ferrets, with the H275Y/V241I/N369K recombinant demonstrating higher nasal wash viral titers than the H275Y mutant. Permissive mutations may enhance the fitness of A(H1N1)pdm09 H275Y viruses in vitro and in vivo. The emergence of such variants should be carefully monitored.

Evolution of oseltamivir resistance mutations in Influenza A(H1N1) and A(H3N2) viruses during selection in experimentally infected mice.

The evolution of oseltamivir resistance mutations during selection through serial passages in animals is still poorly described. Herein, we assessed the evolution of neuraminidase (NA) and hemagglutinin (HA) genes of influenza A/WSN/33 (H1N1) and A/Victoria/3/75 (H3N2) viruses recovered from the lungs of experimentally infected BALB/c mice receiving suboptimal doses (0.05 and 1 mg/kg of body weight/day) of oseltamivir over two generations. The traditional phenotypic and genotypic methods as well as deep-sequencing analysis were used to characterize the potential selection of mutations and population dynamics of oseltamivir-resistant variants. No oseltamivir-resistant NA or HA changes were detected in the recovered A/WSN/33 viruses. However, we observed a positive selection of the I222T NA substitution in the recovered A/Victoria/3/75 viruses, with a frequency increasing over time and with an oseltamivir concentration from 4% in the initial pretherapy inoculum up to 28% after two lung passages. Although the presence of mixed I222T viral populations in mouse lungs only led to a minimal increase in oseltamivir 50% enzyme-inhibitory concentrations (IC50s) (by a mean of 5.7-fold) compared to that of the baseline virus, the expressed recombinant A/Victoria/3/75 I222T NA protein displayed a 16-fold increase in the oseltamivir IC50 level compared to that of the recombinant wild type (WT). In conclusion, the combination of serial in vivo passages under neuraminidase inhibitor (NAI) pressure and temporal deep-sequencing analysis enabled, for the first time, the identification and selection of the oseltamivir-resistant I222T NA mutation in an influenza H3N2 virus. Additional in vivo selection experiments with other antivirals and drug combinations might provide important information on the evolution of antiviral resistance in influenza viruses.

Mucosal bivalent live attenuated vaccine protects against human metapneumovirus and respiratory syncytial virus in mice.

Live-Attenuated Vaccines (LAVs) stimulate robust mucosal and cellular responses and have the potential to protect against Respiratory Syncytial Virus (RSV) and Human Metapneumovirus (HMPV), the main etiologic agents of viral bronchiolitis and pneumonia in children. We inserted the RSV-F gene into an HMPV-based LAV (Metavac®) we previously validated for the protection of mice against HMPV challenge, and rescued a replicative recombinant virus (Metavac®-RSV), exposing both RSV- and HMPV-F proteins at the virion surface and expressing them in reconstructed human airway epithelium models. When administered to BALB/c mice by the intranasal route, bivalent Metavac®-RSV demonstrated its capacity to replicate with reduced lung inflammatory score and to protect against both RSV and lethal HMPV challenges in vaccinated mice while inducing strong IgG and broad RSV and HMPV neutralizing antibody responses. Altogether, our results showed the versatility of the Metavac® platform and suggested that Metavac®-RSV is a promising mucosal bivalent LAV candidate to prevent pneumovirus-induced diseases.

Evaluation of recombinant 2009 pandemic influenza A (H1N1) viruses harboring zanamivir resistance mutations in mice and ferrets.

Recombinant influenza A(H1N1)pdm09 wild-type (WT) and zanamivir-resistant E119G and Q136K neuraminidase mutants were generated to determine their enzymatic and replicative properties in vitro, as well as their infectivity and transmissibility in mice and ferrets. Viral titers of recombinant E119G and Q136K mutants were significantly lower than those of the WT in the first 36 h postinoculation (p.i.) in vitro. The E119G and Q136K mutations were both associated with a significant reduction of total neuraminidase (NA) activity at the cell surface of 293T cells, with relative total NA activities of 14% (P < 0.01) and 20% (P < 0.01), respectively, compared to the WT. The E119G mutation significantly reduced the affinity (8-fold increase in Km) but not the Vmax. The Q136K mutation increased the affinity (5-fold decrease in Km) with a reduction in Vmax (8% Vmax ratio versus the WT). In mice, infection with the E119G and Q136K mutants resulted in lung viral titers that were significantly lower than those of the WT on days 3 p.i. (3.4 × 10(6) ± 0.8 × 10(6) and 2.1 × 10(7) ± 0.4 × 10(7) PFU/ml, respectively, versus 8.8 × 10(7) ± 1.1 × 10(7); P < 0.05) and 6 p.i. (3.0 × 10(5) ± 0.5 × 10(5) and 8.6 × 10(5) ± 1.4 × 10(5) PFU/ml, respectively, versus 5.8 × 10(7) ± 0.3 × 10(7); P < 0.01). In experimentally infected ferrets, the E119G mutation rapidly reverted to the WT in donor and contact animals. The Q136K mutation was maintained in ferrets, although nasal wash viral titers from the Q136K contact group were significantly lower than those of the WT on days 3 to 5 p.i. Our results demonstrate that zanamivir-resistant E119G and Q136K mutations compromise viral fitness and transmissibility in A(H1N1)pdm09 viruses.

Role of permissive neuraminidase mutations in influenza A/Brisbane/59/2007-like (H1N1) viruses.

Neuraminidase (NA) mutations conferring resistance to NA inhibitors were believed to compromise influenza virus fitness. Unexpectedly, an oseltamivir-resistant A/Brisbane/59/2007 (Bris07)-like H1N1 H275Y NA variant emerged in 2007 and completely replaced the wild-type (WT) strain in 2008-2009. The NA of such variant contained additional NA changes (R222Q, V234M and D344N) that potentially counteracted the detrimental effect of the H275Y mutation on viral fitness. Here, we rescued a recombinant Bris07-like WT virus and 4 NA mutants/revertants (H275Y, H275Y/Q222R, H275Y/M234V and H275Y/N344D) and characterized them in vitro and in ferrets. A fluorometric-based NA assay was used to determine Vmax and Km values. Replicative capacities were evaluated by yield assays in ST6Gal1-MDCK cells. Recombinant NA proteins were expressed in 293T cells and surface NA activity was determined. Infectivity and contact transmission experiments were evaluated for the WT, H275Y and H275Y/Q222R recombinants in ferrets. The H275Y mutation did not significantly alter Km and Vmax values compared to WT. The H275Y/N344D mutant had a reduced affinity (Km of 50 vs 12 µM) whereas the H275Y/M234V mutant had a reduced activity (22 vs 28 U/sec). In contrast, the H275Y/Q222R mutant showed a significant decrease of both affinity (40 µM) and activity (7 U/sec). The WT, H275Y, H275Y/M234V and H275Y/N344D recombinants had comparable replicative capacities contrasting with H275Y/Q222R mutant whose viral titers were significantly reduced. All studied mutations reduced the cell surface NA activity compared to WT with the maximum reduction being obtained for the H275Y/Q222R mutant. Comparable infectivity and transmissibility were seen between the WT and the H275Y mutant in ferrets whereas the H275Y/Q222R mutant was associated with significantly lower lung viral titers. In conclusion, the Q222R reversion mutation compromised Bris07-like H1N1 virus in vitro and in vivo. Thus, the R222Q NA mutation present in the WT virus may have facilitated the emergence of NAI-resistant Bris07 variants.

Preclinical evaluation of PHH-1V vaccine candidate against SARS-CoV-2 in non-human primates.

SARS-CoV-2 emerged in December 2019 and quickly spread worldwide, continuously striking with an unpredictable evolution. Despite the success in vaccine production and mass vaccination programs, the situation is not still completely controlled, and therefore accessible second-generation vaccines are required to mitigate the pandemic. We previously developed an adjuvanted vaccine candidate coded PHH-1V, based on a heterodimer fusion protein comprising the RBD domain of two SARS-CoV-2 variants. Here, we report data on the efficacy, safety, and immunogenicity of PHH-1V in cynomolgus macaques. PHH-1V prime-boost vaccination induces high levels of RBD-specific IgG binding and neutralizing antibodies against several SARS-CoV-2 variants, as well as a balanced Th1/Th2 cellular immune response. Remarkably, PHH-1V vaccination prevents SARS-CoV-2 replication in the lower respiratory tract and significantly reduces viral load in the upper respiratory tract after an experimental infection. These results highlight the potential use of the PHH-1V vaccine in humans, currently undergoing Phase III clinical trials.

Prior SARS-CoV-2 infection enhances and reshapes spike protein-specific memory induced by vaccination.

The diversity of vaccination modalities and infection history are both variables that have an impact on the immune memory of individuals vaccinated against SARS-CoV-2. To gain more accurate knowledge of how these parameters imprint on immune memory, we conducted a long-term follow-up of SARS-CoV-2 spike protein-specific immune memory in unvaccinated and vaccinated COVID-19 convalescent individuals as well as in infection-naïve vaccinated individuals. Here, we report that individuals from the convalescent vaccinated (hybrid immunity) group have the highest concentrations of spike protein-specific antibodies at 6 months after vaccination. As compared with infection-naïve vaccinated individuals, they also display increased frequencies of an atypical mucosa-targeted memory B cell subset. These individuals also exhibited enhanced TH1 polarization of their SARS-CoV-2 spike protein-specific follicular T helper cell pool. Together, our data suggest that prior SARS-CoV-2 infection increases the titers of SARS-CoV-2 spike protein-specific antibody responses elicited by subsequent vaccination and induces modifications in the composition of the spike protein-specific memory B cell pool that are compatible with enhanced functional protection at mucosal sites.

Therapeutic activity of intramuscular peramivir in mice infected with a recombinant influenza A/WSN/33 (H1N1) virus containing the H275Y neuraminidase mutation.

The therapeutic activity of intramuscular (IM) peramivir was evaluated in mice infected with a recombinant influenza A/WSN/33 virus containing the H275Y neuraminidase (NA) mutation known to confer oseltamivir resistance. Regimens consisted of single (90 mg/kg of body weight) or multiple (45 mg/kg daily for 5 days) IM peramivir doses that were initiated 24 h or 48 h postinfection (p.i.). An oral oseltamivir regimen (1 or 10 mg/kg daily for 5 days) was used for comparison. Untreated animals had a mortality rate of 75% and showed a mean weight loss of 16.9% on day 5 p.i. When started at 24 h p.i., both peramivir regimens prevented mortality and significantly reduced weight loss (P < 0.001) and lung viral titers (LVT) (P < 0.001). A high dose (10 mg/kg) of oseltamivir initiated at 24 h p.i. also prevented mortality and significantly decreased weight loss (P < 0.05) and LVT (P < 0.001) compared to the untreated group results. In contrast, a low dose (1 mg/kg) of oseltamivir did not show any benefits. When started at 48 h p.i., both peramivir regimens prevented mortality and significantly reduced weight loss (P < 0.01) and LVT (P < 0.001) whereas low-dose or high-dose oseltamivir regimens had no effect on mortality rates, body weight loss, and LVT. Our results show that single-dose and multiple-dose IM peramivir regimens retain clinical and virological activities against the A/H1N1 H275Y variant despite some reduction in susceptibility when assessed in vitro using enzymatic assays. IM peramivir could constitute an alternative for treatment of oseltamivir-resistant A/H1N1 infections, although additional studies are warranted to support such a recommendation.

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.

Generation and characterization of recombinant pandemic influenza A(H1N1) viruses resistant to neuraminidase inhibitors.

BACKGROUND: Neuraminidase inhibitors (NAIs) play a key role in the management of influenza epidemics and pandemics. Given the novel pandemic influenza A(H1N1) (pH1N1) virus and the restricted number of approved anti-influenza drugs, evaluation of potential drug-resistant variants is of high priority. METHODS: Recombinant pH1N1 viruses were generated by reverse genetics, expressing either the wild-type or any of 9 mutant neuraminidase (NA) proteins (N2 numbering: E119G, E119V, D198G, I222V, H274Y, N294S, S334N, I222V-H274Y, and H274Y-S334N). We evaluated these recombinant viruses for their resistance phenotype to 4 NAIs (oseltamivir, zanamivir, peramivir, and A-315675), NA enzymatic activity, and replicative capacity. RESULTS: The E119G and E119V mutations conferred a multidrug resistance phenotype to many NAIs but severely compromised viral fitness. The oseltamivir- and peramivir-resistance phenotype was confirmed for the H274Y and N294S mutants, although both viruses remained susceptible to zanamivir. Remarkably, the I222V mutation had a synergistic effect on the oseltamivir- and peramivir-resistance phenotype of H274Y and compensated for reduced viral fitness, raising concerns about the potential emergence and dissemination of this double-mutant virus. CONCLUSIONS: This study highlights the importance of continuous monitoring of antiviral drug resistance in clinical samples as well as the need to develop new agents and combination strategies.