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International audience; Influenza A viruses cause major morbidity and represent a severe global health problem. Current influenza vaccines are mainly egg-based products requiring the split of whole viruses using classical detergents such as Triton X-100, which implies certain limitations. Here, we report the use of the novel calixarene-based surfactant CALX133ACE as an alternative to classical detergents for influenza inactivated split vaccine preparation. We confirmed that CALX133ACE-based split HA antigens are fully functional and quantifiable by the "gold standard" method SRID. Additionally, as in the case of the Triton X-100-based split, the CALX133ACE-based split antigens are stable for at least 6 months at 4 °C. Moreover, immunization of mice with CALX133ACE-based split NYMC X-179A (H1N1) antigens harboring 10 to 30-fold less antigen than the commercialized trivalent inactivated vaccines Vaxigrip® or Fluviral® induced comparable efficient protection and neutralizing antibody responses against A(H1N1)pdm09 infection. Taken together, our results demonstrate for the first time the use of a calixarene-based detergent as an efficient splitting agent for the production of optimized influenza split antigens, paving the way for significant improvement in the vaccine manufacturing process, notably with regard to the current regulation on the prohibition of endocrine disruptors, such as Triton X-100.

International audience; 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.

Human metapneumovirus (HMPV) is a major pediatric respiratory pathogen for which there is currently no specific treatment or licensed vaccine. Different strategies have been evaluated to prevent this infection, including the use of live-attenuated vaccines (LAVs). However, further development of LAV approaches is often hampered by the lack of highly efficient and scalable cell-based production systems that support worldwide vaccine production. In this context, avian cell lines cultivated in suspension are currently competing with traditional cell platforms used for viral vaccine manufacturing. We investigated whether the DuckCelt®-T17 avian cell line (Vaxxel) we previously described as an efficient production system for several influenza strains could also be used to produce a new HMPV LAV candidate (Metavac®), an engineered SH gene-deleted mutant of the A1/C-85473 strain of HMPV. To that end, we characterized the operational parameters of multiplicity of infection (MOI), cell density, and trypsin addition to achieve optimal production of the LAV Metavac® in the DuckCelt®-T17 cell line platform. We demonstrated that the DuckCelt®-T17 cell line is permissive and is well adapted to the production of the wild-type A1/C-85473 HMPV and the Metavac® vaccine candidate. Moreover, our results confirmed that the LAV candidate produced in DuckCelt®-T17 cells conserves its advantageous replication properties in LLC-MK2 and 3D-reconstituted human airway epithelium models, as well as its capacity to induce efficient neutralizing antibodies in a mouse model. Our results suggest that the DuckCelt®-T17 avian cell line is a very promising platform for scalable in-suspension serum-free production of the HMPV-based LAV candidate Metavac®.

Inactivated influenza vaccines (IIVs) lack broad efficacy. Cellular immunity to a conserved internal antigen, the nucleoprotein (NP), has been correlated to protection against pandemic and seasonal influenza and thus could have the potential to broaden vaccine efficacy. We developed OVX836, a recombinant protein vaccine based on an oligomerized NP, which shows increased uptake by dendritic cells and immunogenicity compared with NP. Intramuscular immunization in mice with OVX836 induced strong NP-specific CD4+ and CD8+ T-cell systemic responses and established CD8+ tissue memory T cells in the lung parenchyma. Strikingly, OVX836 protected mice against viral challenge with three different influenza A subtypes, isolated several decades apart and induced a reduction in viral load. When co-administered with IIV, OVX836 was even more effective in reducing lung viral load., Influenza vaccine: engineered nucleoprotein with improved protective efficacy against multiple strains Circulating influenza A virus (IAV) strains differ in their surface proteins each year, and vaccines eliciting an immune response to these proteins are often only partially protective. Internal viral proteins, such as the nucleoprotein (NP), are highly conserved, and cellular immunity to NP has been correlated with protection from diverse strains. However, current IAV vaccines induce a poor immune response to NP. In this study, led by Fergal Hill from Osivax, researchers develop an oligomeric version of NP with improved immunogenicity. Vaccination of mice with oligomeric NP results in an improved NP-specific T-cell response, including CD8+ tissue memory T cells in the lung, and protects mice against three different IAV subtypes. Co-administration with the currently used inactivated influenza vaccine further improves protection against virus infection in mice. These results encourage further pre-clinical and clinical development for this vaccine candidate.

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., Author summary Non-human primates infected with SARS-CoV-2 develop a mild infection resembling asymptomatic human infection. Here we used viral dynamic modelling to characterize the nasopharyngeal and tracheal viral loads. We found that viral load rapidly declined after peak viral load despite the absence of association between model parameters and immune markers. The within-host reproductive basic reproduction number was approximately equal to 6 and 4 in nasopharynx and trachea suggesting that a prophylactic therapy blocking viral entry or production with 90% efficacy could be sufficient to prevent viral growth and peak viral load.

Superinfections of bacterial/fungal origin are known to affect the course and severity of respiratory viral infections. An increasing number of evidence indicate a relatively high prevalence of superinfections associated with COVID-19, including invasive aspergillosis, but the underlying mechanisms remain to be characterized. In the present study, to better understand the biological impact of superinfection we sought to determine and compare the host transcriptional response to SARS-CoV-2 versusAspergillussuperinfection, using a model of reconstituted humain airway epithelium. Our analyses reveal that both simple infection and superinfection induce a strong deregulation of core components of innate immune and inflammatory responses, with a stronger response to superinfection in the bronchial epithelial model compared to its nasal counterpart. Our results also highlight unique transcriptional footprints of SARS-CoV-2Aspergillussuperinfection, such as an imbalanced type I/type III IFN, and an induction of several monocyte- and neutrophil associated chemokines, that could be useful for the understanding ofAspergillus-associated COVID-19 and but also management of severe forms of aspergillosis in this specific context.

International audience; Two antiviral classes, the neuraminidase inhibitors (NAIs) and polymerase inhibitors (baloxavir marboxil and favipiravir) can be used to prevent and treat influenza infections during seasonal epidemics and pandemics. However, prolonged treatment may lead to the emergence of drug resistance. Therapeutic combinations constitute an alternative to prevent resistance and reduce antiviral doses. Therefore, we evaluated in vitro combinations of baloxavir acid (BXA) and other approved drugs against influenza A(H1N1)pdm09 and A(H3N2) subtypes. The determination of an effective concentration inhibiting virus cytopathic effects by 50% (EC50) for each drug and combination indexes (CIs) were based on cell viability. CompuSyn software was used to determine synergism, additivity or antagonism between drugs. Combinations of BXA and NAIs or favipiravir had synergistic effects on cell viability against the two influenza A subtypes. Those effects were confirmed using a physiological and predictive ex vivo reconstructed human airway epithelium model. On the other hand, the combination of BXA and ribavirin showed mixed results. Overall, BXA stands as a good candidate for combination with several existing drugs, notably oseltamivir and favipiravir, to improve in vitro antiviral activity. These results should be considered for further animal and clinical evaluations.

In vitro antibody selection against pathogens from naive combinatorial libraries can yield various classes of antigen-specific binders that are distinct from those evolved from natural infection 1–4 . Also, rapid neutralizing antibody discovery can be made possible by a strategy that selects for those interfering with pathogen and host interaction 5 . Here we report the discovery of antibodies that neutralize SARS-CoV-2, the virus responsible for the COVID-19 pandemic, from a highly diverse naive human Fab library. Lead antibody 5A6 blocks the receptor binding domain (RBD) of the viral spike from binding to the host receptor angiotensin converting enzyme 2 (ACE2), neutralizes SARS-CoV-2 infection of Vero E6 cells, and reduces viral replication in reconstituted human nasal and bronchial epithelium models. 5A6 has a high occupancy on the viral surface and exerts its neutralization activity via a bivalent binding mode to the tip of two neighbouring RBDs at the ACE2 interaction interface, one in the “up” and the other in the “down” position, explaining its superior neutralization capacity. Furthermore, 5A6 is insensitive to several spike mutations identified in clinical isolates, including the D614G mutant that has become dominant worldwide. Our results suggest that 5A6 could be an effective prophylactic and therapeutic treatment of COVID-19.

COVID-19 has become a pandemic that has caused over 200,000 deaths worldwide, with no antiviral drug or vaccine yet available. 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 worldwide but definitive evidence for HCQ efficacy in treatment of COVID-19 is still missing.We evaluated the antiviral activity of HCQ both in vitro and in SARS-CoV-2-infected macaques. HCQ showed antiviral activity in monkey African green monkey kidney (VeroE6) cells but not in a model of reconstituted human airway epithelium. In macaques, we tested different treatment strategies in comparison to placebo, before and after peak viral load, alone or in combination with azithromycin (AZTH). Neither HCQ nor HCQ+AZTH showed a significant effect on the viral load levels in any of the tested compartments. When the drug was used as a pre-exposure prophylaxis (PrEP), HCQ did not confer protection against acquisition of infection.Our findings do not support the use of HCQ, either alone or in combination with AZTH, as an antiviral treatment for COVID-19 in humans.

Human metapneumovirus (HMPV) is a major pediatric respiratory pathogen with currently no specific treatment or licensed vaccine. Different strategies to prevent this infection have been evaluated, including live-attenuated vaccines (LAV) based on SH and/or G protein deletions. This approach showed promising outcomes but has not been evaluated further using different viral strains. In that regard, we previously showed that different HMPV strains harbor distinct in vitro fusogenic and in vivo pathogenic phenotypes, possibly influencing the selection of vaccine strains. In this study, we investigated the putative contribution of the low conserved SH or G accessory proteins in such strain-dependent phenotypes and generated recombinant wild type (WT) and SH- or G-deleted viruses derived from two different patient-derived HMPV strains, A1/C-85473 and B2/CAN98-75. The &Delta, SH and &Delta, G deletions led to different strain-specific phenotypes in both LLC-MK2 cell and reconstituted human airway epithelium models. More interestingly, the &Delta, G-85473 and especially &Delta, SH-C-85473 recombinant viruses conferred significant protection against HMPV challenge and induced immunogenicity against a heterologous strain. In conclusion, our results show that the viral genetic backbone should be considered in the design of live-attenuated HMPV vaccines, and that a SH-deleted virus based on the A1/C-85473 HMPV strain could be a promising LAV candidate as it is both attenuated and protective in mice while being efficiently produced in a cell-based system.

International audience; NOD-like receptor protein 3 (NLRP3) inflammasome activation triggers caspase-1 activation-induced maturation of interleukin (IL)-1β 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-1β production since we observed reduced mortality, weight loss and inflammation in IL-1β-deficient (IL-1β-/-) mice, as compared to wild-type animals during HMPV infection. Moreover, the effect on these evaluated parameters was not different in IL-1β-/- and wild-type mice treated with an NLRP3 inflammasome inhibitor. The production of IL-1β 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-1β 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-1β, which exacerbates HMPV-induced inflammation. Therefore, the blockade of IL-1β production by using NLRP3 inflammasome inhibitors might be a novel potential strategy for the therapy and prevention of HMPV infection.

International audience; Influenza infections remain a burden on health care systems despite vaccination programs and marketed antiviral drugs. Immunomodulation through activation of innate sensors could represent innovative approaches to fight the flu. This study evaluated the ability of flagellin, agonist of Toll-like receptor 5 (TLR5), to control the replication of influenza A virus (IAV) in mice. First, we showed that systemic or intranasal administration of flagellin activated transcription of anti-viral genes in lung tissue. Prophylactic and therapeutic flagellin administration resulted in decreased levels of viral RNA and infectious virus in the lungs of H3N2 IAV-infected mice. The effect of the flagellin on viral replication was also observed in Ifnar-/- and Il22-/- IAV-infected mice, suggesting a mechanism independent of type I interferon and interleukin 22 signaling. In addition, a combination therapy associating the neuraminidase inhibitor oseltamivir and flagellin was more effective than standalone treatments in reducing pulmonary viral replication. Thus, this study highlights the therapeutic potential of the flagellin to control the replication of the influenza virus.

International audience; Influenza viruses still constitute a real public health problem today. To cope with the emergence of new circulating strains, but also the emergence of resistant strains to classic antivirals, it is necessary to develop new antiviral approaches. This review summarizes the state-of-the-art of current antiviral options against influenza infection, with a particular focus on the recent advances of anti-influenza drug repurposing strategies and their potential therapeutic, regulatory and economic benefits. The review will illustrate the multiple ways to reposition molecules for the treatment of influenza, from adventitious discovery to in silico-based screening. These novel antiviral molecules, many of which targeting the host cell, in combination with conventional antiviral agents targeting the virus, will ideally enter the clinics and reinforce the therapeutic arsenal to combat influenza virus infections.

We recently isolated an influenza A(H1N1)pdm09 E119D/H275Y neuraminidase (NA) variant from an immunocompromised patient who received oseltamivir and zanamivir therapies. This variant demonstrated cross resistance to zanamivir, oseltamivir, peramivir and laninamivir. In this study, the viral fitness of the recombinant wild-type (WT), E119D and E119D/H275Y A(H1N1)pdm09 viruses was evaluated in vitro and in experimentally-infected C57BL/6 mice and guinea pigs. In replication kinetics experiments, viral titers obtained with the E119D and E119D/H275Y recombinants were up to 2- and 4-log lower compared to the WT virus in MDCK and ST6GalI-MDCK cells, respectively. Enzymatic studies revealed that the E119D mutation significantly decreased the surface NA activity. In experimentally-infected mice, a 50% mortality rate was recorded in the group infected with the WT recombinant virus whereas no mortality was observed in the E119D and E119D/H275Y groups. Mean lung viral titers on day 5 post-inoculation for the WT (1.2 ± 0.57 × 10(8) PFU/ml) were significantly higher than those of the E119D (9.75 ± 0.41 × 10(5) PFU/ml, P

International audience; 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-\kappaB functional balance, which appears to contribute to the up-regulation of the expression of several proinflammatory cytokines and chemokines.

Permissive neuraminidase (NA) substitutions such as R222Q, V234M and D344N have facilitated the emergence and worldwide spread of oseltamivir-resistant influenza A/Brisbane/59/2007 (H1N1)-H275Y viruses. However, the potential contribution of genetic changes in other viral segments on viral fitness remains poorly investigated. A series of recombinant A(H1N1)pdm09 and A/WSN/33 7:1 reassortants containing the wild-type (WT) A/Brisbane/59/2007 NA gene or its single (H275Y) and double (H275Y/Q222R, H275Y/M234V and H275Y/N344D) variants were generated and their replicative properties were assessed in vitro. The Q222R reversion substitution significantly reduced viral titers when evaluated in both A(H1N1)pdm09 and A/WSN/33 backgrounds. The permissive role of the R222Q was further confirmed using A/WSN/33 7:1 reassortants containing the NA gene of the oseltamivir-susceptible or oseltamivir-resistant influenza A/Mississippi/03/2001 strains. Therefore, NA permissive substitutions play a dominant role for improving viral replication of oseltamivir-resistant A (H1N1)-H275Y viruses in vitro.

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 (IC 50 s) (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 IC 50 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.

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. This article is protected by copyright. All rights reserved

The efficacy of oseltamivir-zanamivir combination therapy compared to that of monotherapy was evaluated in mice infected with influenza A(H3N2) or A(H1N1)pdm09 viruses. For A(H3N2) virus, zanamivir monotherapy and oseltamivir-zanamivir combination showed significant reduction of mean weight loss compared to oseltamivir. Zanamivir monotherapy also conferred decreased mortality, weight loss and lung viral titers (LVT) compared to oseltamivir for A(H1N1)pdm09 wild-type virus. Intermediate benefits were observed for the oseltamivir-zanamivir combination. For the oseltamivir-resistant A(H1N1)pdm09 H275Y virus, the efficacy of oseltamivir-zanamivir was comparable to that of zanamivir and significantly higher than that of oseltamivir in terms of survival, weight loss and LVT.

In addition to immunization programs, antiviral agents can play a major role for the control of seasonal influenza epidemics and may also provide prophylactic and therapeutic benefits during an eventual pandemic. The purpose of this article is to review the mechanism of action, pharmacokinetics and clinical indications of neuraminidase inhibitors (NAIs) with an emphasis on the emergence of antiviral drug resistance. There are two approved NAIs compounds in US: inhaled zanamivir and oral oseltamivir, which have been commercially available since 1999-2000. In addition, two other NAIs, peramivir (an intravenous cyclopentane derivative) and laninamivir (a long-acting NAI administered by a single nasal inhalation) have been approved in certain countries and are under clinical evaluations in others. As for other antivirals, the development and dissemination of drug resistance is a significant threat to the clinical utility of NAIs. The emergence and worldwide spread of oseltamivir-resistant seasonal A(H1N1) viruses during the 2007-2009 seasons emphasize the need for continuous monitoring of antiviral drug susceptibilities. Further research priorities should include a better understanding of the mechanisms of resistance to existing antivirals, the development of novel compounds which target viral or host proteins and the evaluation of combination therapies for improved treatment of severe influenza infections, particularly in immunocompromised individuals. This article forms part of a symposium in Antiviral Research on "Treatment of influenza: targeting the virus or the host."

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.

Background The D222G (H1 numbering) hemagglutinin (HA) mutation within the receptor-binding site was detected with higher frequencies in severe cases of 2009 pandemic H1N1 (pH1N1) infections. We investigated the impact of this mutation in vitro and in animal models using recombinant pH1N1 viruses. Methods The recombinant D222G HA mutant was generated from a wild-type (WT) clinical strain by using reverse genetics and site-directed mutagenesis. Replicative capacities were determined in MDCK and MDCK-α2,6 cells. Antigenicity was characterized by HA inhibition and microneutralization assays. HA titers were determined using human, chicken, and resialylated turkey red blood cells (RBCs). Virulence and contact-transmissibility were analyzed in mice and ferrets. Results The recombinant D222G virus grew to significantly higher titers and generated larger viral plaques compared with the WT in MDCK but not in MDCK-α2,6 cells. The mutant also showed a significant reduction in HA titers using α2,6-expressing RBCs. The 2 recombinants were antigenically similar. The D222G mutant virus induced higher lung viral titers and alveolar inflammation in mice whereas the 2 recombinants had similar impacts in ferrets. Conclusions The D222G HA mutation alters receptor binding specificity, resulting in higher lung titers in mice. This could contribute to the higher case fatality rates reported in humans.

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.

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.

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 K m ) but not the V max . The Q136K mutation increased the affinity (5-fold decrease in K m ) with a reduction in V max (8% V max 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.

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., Author Summary The H275Y neuraminidase (NA) mutation conferring resistance to oseltamivir was shown to impair old influenza H1N1 strains both in vitro and in vivo. By contrast, 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. This discrepancy could be attributed to permissive NA mutations (R222Q, V234M and D344N) that were identified in most Bris07-like oseltamivir-resistant variants. To verify this hypothesis, we developed a reverse genetics system for a sensitive Bris07-like isolate (275H) whose NA protein contains the 3 permissive mutations (222Q, 234M, 344N). Using mutagenesis, we first introduced the H275Y then reverted codons at positions 222, 234 and 344. The resulting 5 recombinants (WT, H275Y, H275Y/Q222R, H275Y/M234V and H275Y/N344D) were compared with regard to NA enzyme properties, replicative capacities in vitro as well as infectivity and contact-transmissibility in ferrets. Among the studied permissive mutations, Q222R was associated with a significant reduction of both affinity and activity of the NA enzyme resulting in a virus with a reduced replicative capacity in vitro and decreased replication in lungs of ferrets. Thus, the R222Q mutation may have been the major permissive NA change that facilitated the emergence and spread of NAI-resistant Bris07 variants.

Influenza viruses are major human pathogens with a global distribution, accounting for more than 500,000 annual deaths worldwide and with considerable impact on the quality of life and productivity of the society. Due to the limited efficacy of vaccination, antiviral drugs constitute a complementary approach in the control and prevention of influenza infections and thus play an important role in the management of influenza outbreaks and pandemics. Currently, adamantanes and neuraminidase inhibitors (NAIs) are the only two classes of anti-influenza agents approved for clinical use. However, the worldwide emergence and high prevalence of adamantane-resistant virus variants has discouraged the use of the former drugs. NAIs have proved to be very effective against influenza A and B viruses. Nevertheless, oseltamivir-resistant strains have also been reported quite frequently, as in the case of seasonal H1N1 viruses that circulated between 2007 and 2009. Indeed, the emergence of drug-resistant virus variants is always a matter of concern because it could significantly compromise the usefulness of such intervention. This highlights the need for continuous monitoring of resistance markers, as well as the development of new anti-influenza drugs and combination therapies.