A protectin DX (PDX) analog with in vitro activity against influenza A(H1N1) viruses.

Antiviral therapy based on neuraminidase (oseltamivir) or polymerase (baloxavir marboxil) inhibitors plays an important role in the management of influenza infections. However, the emergence of drug resistance and the uncontrolled inflammatory response are major limitations in the treatment of severe influenza disease. Protectins D1 (PD1) and DX (PDX), part of a family of pro-resolving mediators, have previously demonstrated anti-influenza activity as well as anti-inflammatory properties in various clinical contexts. Herein, we synthetized a series of simplified PDX analogs and assessed their in vitro antiviral activity against influenza A(H1N1) viruses, including oseltamivir- and baloxavir-resistant variants. In ST6GalI-MDCK cells, the PDX analog AN-137B reduced viral replication in a dose-dependent manner with IC50 values of 23.8 for A/Puerto Rico/8/1934 (H1N1) and between 32.6 and 36.7 µM for susceptible and resistant A(H1N1)pdm09 viruses. In MTS-based cell viability experiments, AN-137B showed a 50% cellular cytotoxicity (CC50 ) of 638.7 µM with a resulting selectivity index of 26.8. Of greater importance, the combination of AN-137B with oseltamivir or baloxavir resulted in synergistic and additive in vitro effects, respectively. Treatment of lipopolysaccharide (LPS)-stimulated macrophages with AN-137B resulted in a decrease of iNOS activity as shown by the reduction of nitrite production, suggesting an anti-inflammatory effect. In conclusion, our results indicate that the protectin analog AN-137B constitutes an interesting therapeutic modality against influenza A virus, warranting further evaluation in animal models.

Fitness of influenza A and B viruses with reduced susceptibility to baloxavir: A mini-review.

Neuraminidase inhibitors (NAIs), that currently include oseltamivir (Tamiflu® ), zanamivir (Relenza® ), peramivir (Rapivab® ) and laninamivir (Inavir® ), constitute an important class of antivirals recommended against seasonal influenza A and B infections. NAIs target the surface NA protein whose sialidase activity is responsible for virion release from infected cells. Because of their pivotal role in the transcription/translation process, the polymerase acidic (PA) and polymerase basic 1 and 2 (PB1 and PB2, respectively) internal proteins also constitute targets of interest for the development of additional anti-influenza agents. Baloxavir marboxil (BXM), an inhibitor of the cap-dependent endonuclease activity of the influenza PA protein, was approved in the United States and Japan in 2018. Baloxavir acid (BXA), the active compound of BXM, demonstrated a potent in vitro activity against different types/subtypes of influenza viruses including seasonal influenza A/B strains as well as avian influenza A viruses with a pandemic potential. A single oral dose of BXM provided virological and clinical benefits that were respectively superior or equal to those displayed by the standard (5 days, twice daily) oseltamivir regimen. Nevertheless, BXM-resistant variants have emerged at relatively high rates in BXM-treated children and adults. Consequently, there is a need to study the fitness (virulence and transmissibility) characteristics of mutants with a high potential to emerge as such variants can compromise the clinical usefulness of BXM. The purpose of this manuscript is to review the fitness properties of influenza A and B isolates harbouring mutations of reduced susceptibility to BXA.

Replication and transmission of an influenza A(H3N2) virus harboring the polymerase acidic I38T substitution, in guinea pigs.

The polymerase acidic (PA) I38T substitution is a dominant marker of resistance to baloxavir. We evaluated the impact of I38T on the fitness of a contemporary influenza A(H3N2) virus. Influenza A/Switzerland/9715293/2013 (H3N2) wild-type (WT) virus and its I38T mutant were rescued by reverse genetics. Replication kinetics were compared using ST6GalI-MDCK and A549 cells and infectivity/contact transmissibility were evaluated in guinea pigs. Nasal wash (NW) viral titres were determined by TCID50 ml-1 in ST6GalI-MDCK cells. Competition experiments were performed and the evolution of viral population was assessed by droplet digital RT-PCR. I38T did not alter in vitro replication. I38T induced comparable titres vs the WT in guinea pigs NWs and the two viruses transmitted equally by direct contact. However, a 50 %:50 % mixture inoculum evolved to mean WT/I38T ratios of 71 %:29 % and 66.4 %:33.6 % on days 4 and 6 p.i., respectively. Contemporary influenza A(H3N2)-I38T PA variants may conserve a significant level of viral fitness.

Impact of the Baloxavir-Resistant Polymerase Acid I38T Substitution on the Fitness of Contemporary Influenza A(H1N1)pdm09 and A(H3N2) Strains.

BACKGROUND: Baloxavir is a cap-dependent inhibitor of the polymerase acid (PA) protein of influenza viruses. While appearing virologically superior to oseltamivir, baloxavir exhibits a low barrier of resistance. We sought to assess the impact of the common baloxavir-resistant I38T PA substitution on in vitro properties and virulence. METHODS: Influenza A/Quebec/144147/2009 (H1N1)pdm09 and A/Switzerland/9715293/2013 (H3N2) recombinant viruses and their I38T PA mutants were compared in single and competitive infection experiments in ST6GalI-MDCK cells and C57/BL6 mice. Virus titers in cell culture supernatants and lung homogenates were determined by virus yield assays. Ratios of wild-type (WT) and I38T mutant were assessed by digital RT-PCR. RESULTS: I38T substitution did not alter the replication kinetics of A(H1N1)pdm09 and A(H3N2) viruses. In competition experiments, a 50%:50% mixture evolved to 70%:30% (WT/mutant) for A(H1N1) and 88%:12% for A(H3N2) viruses after a single cell passage. The I38T substitution remained stable after 4 passages in vitro. In mice, the WT and its I38T mutant induced similar weight loss with comparable lung titers in both viral subtypes. The mutant virus tended to predominate over the WT in mouse competition experiments. CONCLUSION: The fitness of baloxavir-resistant I38T PA mutants appears relatively unaltered in seasonal subtypes warranting surveillance for its dissemination.

Reduced Susceptibility to Neuraminidase Inhibitors in Influenza B Isolate, Canada.

We identified an influenza B isolate harboring a Gly407Ser neuraminidase substitution in an immunocompromised patient in Canada before antiviral therapy. This mutation mediated reduced susceptibility to oseltamivir, zanamivir, and peramivir, most likely by preventing interaction with the catalytic Arg374 residue. The potential emergence of such variants emphasizes the need for new antivirals.

In vitro and in vivo evidence of a potential A(H1N1)pdm09 antigenic drift mediated by escape mutations in the haemagglutinin Sa antigenic site.

Influenza A(H1N1)pdm09 virus continues to circulate worldwide without evidence of significant antigenic drift between 2009 and 2016. By using escape mutants, we previously identified six haemagglutinin (HA) changes (T80R, G143E, G158E, N159D, K166E and A198E) that were located within antigenic sites. Combinations of these mutations were introduced into the A(H1N1)pdm09 HA plasmid by mutagenesis. Reassortant 6 : 2 viruses containing both the HA and NA genes of the A(H1N1)pdm09 and the six internal gene segments of A/PR/8/34 were rescued by reverse genetics. In vitro, HA inhibition and microneutralization assays showed that the HA hexa-mutant reassortant virus (RG1) escaped A(H1N1)pdm09 hyper-immune ferret antiserum recognition. C57Black/6 mice that received the vaccine formulated with A/California/07/09 were challenged with 2×104 p.f.u. of either the 6 : 2 wild-type (WT) or RG1 viruses. Reductions in body weight loss, mortality rate and lung viral titre were observed in immunized animals challenged with the 6 : 2 WT virus compared to non-immunized mice. However, immunization did not protect mice challenged with RG1 virus. To further characterize the mutations causing this antigenic change, 11 additional RG viruses whose HA gene contained single or combinations of mutations were evaluated in vitro. Although the RG1 virus was still the least reactive against hyper-immune serum by HAI testing, mutations G158E and N159D within the Sa antigenic site appeared to play the major role in the altered antigenicity of the A(H1N1)pdm09 virus. These results show that the Sa antigenic site contains the most prominent epitopes susceptible to cause an antigenic drift, escaping actual vaccine protection.

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.

Droplet digital PCR to investigate quasi-species at codons 119 and 275 of the A(H1N1)pdm09 neuraminidase during zanamivir and oseltamivir therapies.

The H275Y and E119D neuraminidase (NA) mutations constitute important molecular markers of resistance to NA inhibitors in A(H1N1) pdm09 viruses. We used reverse transcriptase-droplet digital PCR amplification (RT-ddPCR) to analyze quasi-species at codons 275 and 119 of the NA in A(H1N1) pdm09 viruses recovered from an immuncompromised patient who received oseltamivir and zanamivir therapies. RT-ddPCR assays detected and quantified H275Y and E119D mutations with an efficiency that was comparable to that of high throughput sequencing (HiSeq 2500 Illumina, San Diego, CA) technology. With its sensitivity and reproducibility, RT-ddPCR could be a reliable method for accurate detection and quantification of major NAI-resistance mutations in clinical settings. J. Med. Virol. 89:737-741, 2017. © 2016 Wiley Periodicals, Inc.

E119D Neuraminidase Mutation Conferring Pan-Resistance to Neuraminidase Inhibitors in an A(H1N1)pdm09 Isolate From a Stem-Cell Transplant Recipient.

BACKGROUND: An influenza A(H1N1)pdm09 infection was diagnosed in a hematopoietic stem cell transplant recipient during conditioning regimen. He was treated with oral oseltamivir, later combined with intravenous zanamivir. The H275Y neuraminidase (NA) mutation was first detected, and an E119D NA mutation was identified during zanamivir therapy. METHODS: Recombinant wild-type (WT) E119D and E119D/H275Y A(H1N1)pdm09 NA variants were generated by reverse genetics. Susceptibility to NA inhibitors (NAIs) was evaluated with a fluorometric assay using the 2'-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (MUNANA) substrate. Susceptibility to favipiravir (T-705) was assessed using plaque reduction assays. The NA affinity and velocity values were determined with NA enzymatic studies. RESULTS: We identified an influenza A(H1N1)pdm09 E119D mutant that exhibited a marked increase in the 50% inhibitory concentrations against all tested NAIs (827-, 25-, 286-, and 702-fold for zanamivir, oseltamivir, peramivir, and laninamivir, respectively). The double E119D/H275Y mutation further increased oseltamivir and peramivir 50% inhibitory concentrations by 790- and >5000-fold, respectively, compared with the WT. The mutant viruses remained susceptible to favipiravir. The NA affinity and velocity values of the E119D variant decreased by 8.1-fold and 4.5-fold, respectively, compared with the WT. CONCLUSIONS: The actual emergence of a single NA mutation conferring pan-NAI resistance in the clinical setting reinforces the pressing need to develop new anti-influenza strategies.

Heterosubtypic protection conferred by the human monoclonal antibody PN-SIA28 against influenza A virus lethal infections in mice.

PN-SIA28 is a human monoclonal antibody (Hu-MAb) targeting highly conserved epitopes within the stem portion of the influenza virus hemagglutinin (HA) (N. Clementi, et al, PLoS One 6:e28001, 2011, http://dx.doi.org/10.1371/journal.pone.0028001). Previous in vitro studies demonstrated PN-SIA28 neutralizing activities against phylogenetically divergent influenza A subtypes. In this study, the protective activity of PN-SIA28 was evaluated in mice inoculated with lethal influenza A/WSN/33 (H1N1), A/Quebec/144147/09 (H1N1)pdm09, and A/Victoria/3/75 (H3N2) viruses. At 24 h postinoculation (p.i.), animals received PN-SIA28 intraperitoneally (1 or 10 mg/kg of body weight) or 10 mg/kg of unrelated Hu-MAb (mock). Body weight loss and mortality rate (MR) were recorded for 14 days postinfection (p.i.). Lung viral titers (LVT) were determined at day 5 p.i. In A/WSN/33 (H1N1)-infected groups, all untreated and mock-receiving mice died, whereas MRs of 87.5% and 25% were observed in mice that received PN-SIA28 1 and 10 mg/kg, respectively. In influenza A(H1N1) pdm09-infected groups, an MR of 75% was recorded for untreated and mock-treated groups, whereas the PN-SIA28 1-mg/kg and 10-mg/kg groups had rates of 62.5% and 0%, respectively. In A/Victoria/3/75 (H3N2)-infected animals, untreated and mock-treated animals had MRs of 37.5% and 25%, respectively, and no mortalities were recorded after PN-SIA28 treatments. Accordingly, PN-SIA28 treatments significantly reduced weight losses and resulted in a ≥ 1-log reduction in LVT compared to the control in all infection groups. This study confirms that antibodies targeting highly conserved epitopes in the influenza HA stem region, like PN-SIA28, not only neutralize influenza A viruses of clinically relevant subtypes in vitro but also, more importantly, protect from a lethal influenza virus challenge in vivo.

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.

Characterization of drug-resistant influenza virus A(H1N1) and A(H3N2) variants selected in vitro with laninamivir.

Neuraminidase inhibitors (NAIs) play a major role for managing influenza virus infections. The widespread oseltamivir resistance among 2007-2008 seasonal A(H1N1) viruses and community outbreaks of oseltamivir-resistant A(H1N1)pdm09 strains highlights the need for additional anti-influenza virus agents. Laninamivir is a novel long-lasting NAI that has demonstrated in vitro activity against influenza A and B viruses, and its prodrug (laninamivir octanoate) is in phase II clinical trials in the United States and other countries. Currently, little information is available on the mechanisms of resistance to laninamivir. In this study, we first performed neuraminidase (NA) inhibition assays to determine the activity of laninamivir against a set of influenza A viruses containing NA mutations conferring resistance to one or many other NAIs. We also generated drug-resistant A(H1N1) and A(H3N2) viruses under in vitro laninamivir pressure. Laninamivir demonstrated a profile of susceptibility that was similar to that of zanamivir. More specifically, it retained activity against oseltamivir-resistant H275Y and N295S A(H1N1) variants and the E119V A(H3N2) variant. In vitro, laninamivir pressure selected the E119A NA substitution in the A/Solomon Islands/3/2006 A(H1N1) background, whereas E119K and G147E NA changes along with a K133E hemagglutinin (HA) substitution were selected in the A/Quebec/144147/2009 A(H1N1)pdm09 strain. In the A/Brisbane/10/2007 A(H3N2) background, a large NA deletion accompanied by S138A/P194L HA substitutions was selected. This H3N2 variant had altered receptor-binding properties and was highly resistant to laninamivir in plaque reduction assays. Overall, we confirmed the similarity between zanamivir and laninamivir susceptibility profiles and demonstrated that both NA and HA changes can contribute to laninamivir resistance in vitro.

Epitope mapping of the 2009 pandemic and the A/Brisbane/59/2007 seasonal (H1N1) influenza virus haemagglutinins using mAbs and escape mutants.

mAbs constitute an important biological tool for influenza virus haemagglutinin (HA) epitope mapping through the generation of escape mutants, which could provide insights into immune evasion mechanisms and may benefit the future development of vaccines. Several influenza A (H1N1) pandemic 2009 (pdm09) HA escape mutants have been recently described. However, the HA antigenic sites of the previous seasonal A/Brisbane/59/2007 (H1N1) (Bris07) virus remain poorly documented. Here, we produced mAbs against pdm09 and Bris07 HA proteins expressed in human HEK293 cells. Escape mutants were generated using mAbs that exhibited HA inhibition and neutralizing activities. The resulting epitope mapping of the pdm09 HA protein revealed 11 escape mutations including three that were previously described (G172E, N173D and K256E) and eight novel ones (T89R, F128L, G157E, K180E, A212E, R269K, N311T and G478E). Among the six HA mutations that were part of predicted antigenic sites (Ca1, Ca2, Cb, Sa or Sb), three (G172E, N173D and K180E) were within the Sa site. Eight escape mutations (H54N, N55D, N55K, L60H, N203D, A231T, V314I and K464E) were obtained for Bris07 HA, and all but one (N203D, Sb site) were outside the predicted antigenic sites. Our results suggest that the Sa antigenic site is immunodominant in pdm09 HA, whereas the N203D mutation (Sb site), present in three different Bris07 escape mutants, appears as the immunodominant epitope in that strain. The fact that some mutations were not part of predicted antigenic sites reinforces the necessity of further characterizing the HA of additional H1N1 strains.

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.

Emergence of an oseltamivir-resistant influenza A/H3N2 virus in an elderly patient receiving a suboptimal dose of antiviral prophylaxis.

We report the emergence of an influenza virus A/H3N2-E119V neuraminidase variant from an elderly patient with renal dysfunction who received a suboptimal dose of oseltamivir prophylaxis. In neuraminidase inhibition assays, the E119V variant showed a 413-fold increase in the 50% inhibitory oseltamivir concentration and grew at titers comparable to those of the wild type in vitro.

The H275Y neuraminidase mutation of the pandemic A/H1N1 influenza virus lengthens the eclipse phase and reduces viral output of infected cells, potentially compromising fitness in ferrets.

The H275Y amino acid substitution of the neuraminidase gene is the most common mutation conferring oseltamivir resistance in the N1 subtype of the influenza virus. Using a mathematical model to analyze a set of in vitro experiments that allow for the full characterization of the viral replication cycle, we show that the primary effects of the H275Y substitution on the pandemic H1N1 (H1N1pdm09) strain are to lengthen the mean eclipse phase of infected cells (from 6.6 to 9.1 h) and decrease (by 7-fold) the viral burst size, i.e., the total number of virions produced per cell. We also find, however, that the infectious-unit-to-particle ratio of the H275Y mutant strain is 12-fold higher than that of the oseltamivir-susceptible strain (0.19 versus 0.016 per RNA copy). A parallel analysis of the H275Y mutation in the prior seasonal A/Brisbane/59/2007 background shows similar changes in the infection kinetic parameters, but in this background, the H275Y mutation also allows the mutant to infect cells five times more rapidly. Competitive mixed-strain infections in vitro, where the susceptible and resistant H1N1pdm09 strains must compete for cells, are characterized by higher viral production by the susceptible strain but suggest equivalent fractions of infected cells in the culture. In ferrets, however, the mutant strain appears to suffer a delay in its infection of the respiratory tract that allows the susceptible strain to dominate mixed-strain infections.

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.

Viral Fitness of Baloxavir-Resistant Recombinant Influenza B/Victoria- and B/Yamagata-like Viruses Harboring the I38T PA Change, In Vitro, Ex Vivo and in Guinea Pigs.

Seasonal influenza A and B viruses may cause severe infections requiring therapeutic interventions. Baloxavir, the latest antiviral drug approved against those infections, targets the endonuclease activity encoded by the polymerase acidic (PA) protein. While appearing effective at cessation of viral shedding, baloxavir demonstrated a low barrier of resistance. Herein, we aimed to assess the impact of PA-I38T substitution, a major marker of baloxavir-resistance, on the fitness of contemporary influenza B viruses. Recombinant wild-type (WT) influenza B/Phuket/2073/13 (B/Yamagata/16/88-like) and B/Washington/02/19 (B/Victoria/2/87-like) viruses and their respective PA-I38T mutants were used to evaluate replication kinetics in vitro, using A549 and Calu3 cells, and ex vivo, using nasal human airway epithelium (HAE) cells. Infectivity was also assessed in guinea pigs. In the B/Washington/02/19 background, there were no major differences between the recombinant WT virus and its I38T mutant when viral replication kinetics were evaluated in human lung cell lines and HAE as well as in nasal washes of experimentally infected guinea pigs. By contrast, the I38T mutation moderately impacted the B/Phuket/2073/13 viral fitness. In conclusion, contemporary influenza B viruses that may acquire baloxavir-resistance through the PA-I38T substitution could retain a significant level of fitness, highlighting the importance of monitoring the emergence of such variant.

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.