ABSTRACT
The seasonal nature of outbreaks of respiratory viral infections with increased transmission during low temperatures has been well established. Accordingly, temperature has been suggested to play a role on the viability and transmissibility of SARS-CoV-2, the virus responsible for the COVID-19 pandemic. The receptor-binding domain (RBD) of the Spike glycoprotein is known to bind to its host receptor angiotensin-converting enzyme 2 (ACE2) to initiate viral fusion. Using biochemical, biophysical, and functional assays to dissect the effect of temperature on the receptor-Spike interaction, we observed a significant and stepwise increase in RBD-ACE2 affinity at low temperatures, resulting in slower dissociation kinetics. This translated into enhanced interaction of the full Spike glycoprotein with the ACE2 receptor and higher viral attachment at low temperatures. Interestingly, the RBD N501Y mutation, present in emerging variants of concern (VOCs) that are fueling the pandemic worldwide (including the B.1.1.7 (α) lineage), bypassed this requirement. This data suggests that the acquisition of N501Y reflects an adaptation to warmer climates, a hypothesis that remains to be tested.
Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/chemistry , COVID-19/pathology , COVID-19/virology , Calorimetry , Humans , Interferometry , Polymorphism, Single Nucleotide , Protein Binding , Protein Structure, Quaternary , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/chemistry , Temperature , ThermodynamicsABSTRACT
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.
Subject(s)
Antiviral Agents/pharmacology , Drug Resistance, Viral , Influenza B virus/drug effects , Influenza B virus/enzymology , Influenza, Human/epidemiology , Neuraminidase/antagonists & inhibitors , Viral Proteins/antagonists & inhibitors , Antiviral Agents/therapeutic use , Canada/epidemiology , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/therapeutic use , Female , Hemagglutinin Glycoproteins, Influenza Virus/genetics , Humans , Influenza B virus/classification , Influenza B virus/genetics , Influenza, Human/drug therapy , Influenza, Human/virology , Microbial Sensitivity Tests , Middle Aged , Mutation , Neuraminidase/genetics , Sequence Analysis, DNA , Viral Proteins/geneticsABSTRACT
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.
Subject(s)
Antiviral Agents/administration & dosage , Azithromycin/administration & dosage , Influenza A Virus, H1N1 Subtype/drug effects , Orthomyxoviridae Infections/drug therapy , Oseltamivir/administration & dosage , Animals , Antiviral Agents/adverse effects , Antiviral Agents/therapeutic use , Azithromycin/adverse effects , Drug Therapy, Combination , Humans , Influenza, Human/drug therapy , Influenza, Human/virology , Injections, Intraperitoneal , Lung/drug effects , Lung/virology , Mice , Mice, Inbred BALB C , Orthomyxoviridae Infections/virology , Oseltamivir/adverse effects , Viral LoadABSTRACT
The types of interactions between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other respiratory viruses are not well-characterized due to the low number of co-infection cases described since the onset of the pandemic. We have evaluated the interactions between SARS-CoV-2 (D614G mutant) and influenza A(H1N1)pdm09 or respiratory syncytial virus (RSV) in the nasal human airway epithelium (HAE) infected simultaneously or sequentially (24 h apart) with virus combinations. The replication kinetics of each virus were determined by RT-qPCR at different post-infection times. Our results showed that during simultaneous infection, SARS-CoV-2 interferes with RSV-A2 but not with A(H1N1)pdm09 replication. The prior infection of nasal HAE with SARS-CoV-2 reduces the replication kinetics of both respiratory viruses. SARS-CoV-2 replication is decreased by a prior infection with A(H1N1)pdm09 but not with RSV-A2. The pretreatment of nasal HAE with BX795, a TANK-binding kinase 1 inhibitor, partially alleviates the reduced replication of SARS-CoV-2 or influenza A(H1N1)pdm09 during sequential infection with both virus combinations. Thus, a prior infection of nasal HAE with SARS-CoV-2 interferes with the replication kinetics of A(H1N1)pdm09 and RSV-A2, whereas only A(H1N1)pdm09 reduces the subsequent infection with SARS-CoV-2. The mechanism involved in the viral interference between SARS-CoV-2 and A(H1N1)pdm09 is mediated by the production of interferon.
Subject(s)
Epithelial Cells/virology , Influenza A Virus, H1N1 Subtype/physiology , Nasopharynx/cytology , Respiratory Syncytial Virus, Human/physiology , SARS-CoV-2/physiology , Viral Interference , Virus Replication , Coinfection , Humans , Microbial Interactions , Nasopharynx/virologyABSTRACT
Antimicrobial resistance has spread quickly on a worldwide scale, reducing therapeutic options for bacterial infections. CRISPR-Cas is an adaptive immune system found in many prokaryotes that can be designed to target bacterial genomes, leading to cell death. Repurposing the CRISPR-Cas system as a therapeutic strategy offers an attractive way to overcome antimicrobial resistance. However, this strategy requires efficient vectors for the CRISPR-Cas system to reach the bacterial genomes. Engineered phages offer an attractive option as cargo delivery vectors. In this review, we discuss the production of phage-based vectors and the relevance of using repurposed CRISPR-Cas systems as antimicrobials. We also discuss recent progress in phage engineering that can potentially overcome the limitations and increase the efficiency of CRISPR-Cas delivery.
Subject(s)
Bacteriophages , CRISPR-Cas Systems , Bacteriophages/genetics , CRISPR-Cas Systems/genetics , Clustered Regularly Interspaced Short Palindromic RepeatsABSTRACT
The seasonal nature in the outbreaks of respiratory viral infections with increased transmission during low temperatures has been well established. The current COVID-19 pandemic makes no exception, and temperature has been suggested to play a role on the viability and transmissibility of SARS-CoV-2. The receptor binding domain (RBD) of the Spike glycoprotein binds to the angiotensin-converting enzyme 2 (ACE2) to initiate viral fusion. Studying the effect of temperature on the receptor-Spike interaction, we observed a significant and stepwise increase in RBD-ACE2 affinity at low temperatures, resulting in slower dissociation kinetics. This translated into enhanced interaction of the full Spike to ACE2 receptor and higher viral attachment at low temperatures. Interestingly, the RBD N501Y mutation, present in emerging variants of concern (VOCs) that are fueling the pandemic worldwide, bypassed this requirement. This data suggests that the acquisition of N501Y reflects an adaptation to warmer climates, a hypothesis that remains to be tested.
ABSTRACT
Baloxavir marboxil (BXM) is a potent inhibitor of the polymerase acidic (PA) protein of influenza viruses. However, clinical trials predominantly involving influenza A(H1N1) and A(H3N2) infections showed that BXM exhibited a low barrier of resistance. Contrasting with influenza A viruses, BXM-resistant influenza B variants remain poorly documented. We evaluated the impact of I38 T/M and E23K PA substitutions, previously reported in influenza A viruses, on in vitro properties and virulence of contemporary influenza B recombinant viruses. Influenza B/Phuket/3073/2013 recombinant wild-type (WT) virus and the I38T, I38M and E23K PA mutants were assessed for their susceptibility to baloxavir acid (BXA), the active metabolite of BXM, by plaque reduction assays in ST6GalI-MDCK cells. Luciferase-based minigenome tests were performed to determine polymerase activity. Replication kinetics and genetic stability were evaluated in ST6GalI-MDCK cells. Virulence was evaluated in BALB/c mice. The I38T, I38M and E23K substitutions increased BXA IC50s values by 12.6-, 5.5-, and 2.6-fold, respectively, compared to the WT. Minigenome assays revealed a 46% loss of polymerase activity for the E23K substitution vs the WT while the I38T and I38M PA variants retained ≈80% of activity. Peak viral titers were comparable for the WT, I38T and I38M recombinants (7.95 ± 0.5, 7.45 ± 0.25 and 8.11 ± 0.28 logTCID50/mL), respectively, whereas it was significantly lower for the E23K mutant (6.28 ± 0.28 logTCID50/mL;P < 0.05 vs the WT). In mice, the WT, I38T and I38M recombinants induced mortality rates of 60%, 40% and 100%, respectively and similar lung viral titers were obtained for the three groups at days 3 and 6 p.i. In conclusion, the fitness of BXA-resistant I38T and I38M PA mutants appears unaltered in contemporary influenza B viruses warranting surveillance for their emergence.
Subject(s)
Antiviral Agents/pharmacology , Dibenzothiepins/pharmacology , Drug Resistance, Viral/genetics , Influenza B virus/drug effects , Influenza B virus/genetics , Morpholines/pharmacology , Pyridones/pharmacology , Triazines/pharmacology , Animals , Clinical Trials as Topic , Dogs , Female , Genome, Viral , Inhibitory Concentration 50 , Madin Darby Canine Kidney Cells , Mice , Mice, Inbred BALB C , Mutation , Orthomyxoviridae Infections/drug therapy , Orthomyxoviridae Infections/virology , Reassortant Viruses/drug effects , Recombination, Genetic , Virus ReplicationABSTRACT
As part of a 2015-2018 clinical trial of peramivir treatment for acute influenza infections in the elderly, an influenza B/Yamagata/16/1988-like isolate harbouring a Val430Ile neuraminidase (NA) substitution was recovered from a single patient. This substitution was detected in respiratory samples collected before and during peramivir treatment. In NA inhibition assays, oseltamivir, zanamivir and peramivir IC50s of the Val430Ile isolate were 4-, 15- and 16-fold higher compared to a wild-type (WT) strain. In reverse genetics experiments, the Ile430Val reversion restored the drug susceptible phenotype. The Val430Ile mutant and the WT strain had comparable replication kinetics in ST6GalI-MDCK cells and the NA mutation was stable after four passages in that cell line. Molecular dynamics simulations suggested that Val430Ile impacts the NA binding through a mechanism involving the catalytic Arg116 residue. The potential of some NA mutations not part of the active site to alter the susceptibility to NA inhibitors highlights the need to develop novel antiviral strategies against influenza B infections.
Subject(s)
Amino Acid Substitution , Antiviral Agents/pharmacology , Enzyme Inhibitors/pharmacology , Neuraminidase/genetics , Acids, Carbocyclic , Amino Acid Sequence , Animals , Clinical Trials, Phase III as Topic , Cyclopentanes/therapeutic use , Dogs , Guanidines/therapeutic use , Humans , Influenza B virus , Inhibitory Concentration 50 , Madin Darby Canine Kidney Cells , Molecular Dynamics Simulation , Multicenter Studies as Topic , Mutation , Neuraminidase/antagonists & inhibitors , Neuraminidase/chemistry , Reverse Genetics , Virus Replication/drug effectsABSTRACT
BACKGROUND: Neuraminidase (NA) inhibitors (NAIs), including oseltamivir and zanamivir, play an important therapeutic role against influenza infections in immunocompromised patients. In such settings, however, NAI therapy may lead to the emergence of resistance involving mutations within the influenza surface genes. The aim of this study was to investigate the evolution of NA and haemagglutinin (HA) genes of influenza A(H1N1)pdm09 virus in an immunocompromised patient receiving oseltamivir then zanamivir therapies. METHODS: Nasopharyngeal swab (NPS) samples were collected between 27 January 2018 and 11 April 2018 from a haematopoietic stem cell transplant recipient. These include 10 samples collected either pre-therapy, during oseltamivir and zanamivir treatment as well as after therapy. The A(H1N1)pdm09 HA/NA genes were sequenced. The H275Y NA substitution was quantified by droplet digital RT-PCR assay. A(H1N1)pdm09 recombinant viruses containing HA mutations were tested by HA elution experiments to investigate in vitro binding properties. RESULTS: Oseltamivir rapidly induced the H275Y NA mutation which constituted 98.33% of the viral population after 15 days of oseltamivir treatment. The related HA gene contained S135A and P183S substitutions within the receptor-binding site. After a switch to zanamivir, 275H/Y and 119E/G/D mixed populations were detected. In the last samples, the double H275Y-E119G NA variant dominated with S135A and P183S HA substitutions. CONCLUSIONS: This report confirms that oseltamivir can rapidly induce the emergence of the H275Y substitution in A(H1N1)pdm09 viruses and subsequent switch to zanamivir can lead to additional substitutions at codon E119 resulting in multi-drug resistance. Such data additionally suggest a potential compensatory role for HA substitutions near the receptor binding site.
Subject(s)
Antiviral Agents/therapeutic use , Drug Resistance, Multiple, Viral , Immunocompromised Host , Influenza A Virus, H1N1 Subtype/drug effects , Influenza, Human/drug therapy , Aged , Antiviral Agents/administration & dosage , Antiviral Agents/pharmacology , Enzyme Inhibitors/administration & dosage , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/therapeutic use , Hematopoietic Stem Cell Transplantation , Humans , Influenza A Virus, H1N1 Subtype/genetics , Influenza, Human/virology , Male , Neuraminidase/antagonists & inhibitors , Oseltamivir/administration & dosage , Oseltamivir/pharmacology , Oseltamivir/therapeutic use , Transplant Recipients , Zanamivir/administration & dosage , Zanamivir/pharmacology , Zanamivir/therapeutic useABSTRACT
Three neuraminidase inhibitors (NAIs: Oseltamivir, zanamivir and peramivir) are currently approved in many countries for the treatment of influenza A and B infections. The emergence of influenza B viruses (IBVs) containing mutations of cross-resistance to these NAIs constitutes a serious clinical threat. Herein, we used a reverse genetics system for the current B/Phuket/3073/2013 vaccine strain to investigate the impact on in vitro properties and virulence of H136N, R152K, D198E/N, I222T and N294S NA substitutions (N2 numbering), reported by the World Health Organization (WHO) as clinical markers of reduced or highly-reduced inhibition (RI/HRI) to multiple NAIs. Recombinant viruses were tested by NA inhibition assays. Their replicative capacity and virulence were evaluated in ST6GalI-MDCK cells and BALB/c mice, respectively. All NA mutants (excepted D198E/N) showed RI/HRI phenotypes against ≥ 2 NAIs. These mutants grew to comparable titers of the recombinant wild-type (WT) IBV in vitro, and some of them (H136N, I222T and N294S mutants) induced more weight loss and mortality in BALB/c mice in comparison to the recombinant WT IBV. These results demonstrate that, in contemporary IBVs, some NA mutations may confer RI/HRI phenotypes to existing NAIs without altering the viral fitness. This reinforces the need for development of novel antiviral strategies with different mechanisms of action.
Subject(s)
Drug Resistance, Viral/genetics , Enzyme Inhibitors/pharmacology , Influenza B virus/genetics , Influenza B virus/pathogenicity , Orthomyxoviridae Infections/drug therapy , Amino Acid Substitution , Animals , Antiviral Agents/pharmacology , Dogs , Female , HEK293 Cells , Humans , Influenza B virus/drug effects , Madin Darby Canine Kidney Cells , Mice, Inbred BALB C , Mutation , Neuraminidase/genetics , Reverse Genetics , Virulence , Virus ReplicationABSTRACT
Neuraminidase (NA) mutations conferring resistance to NA inhibitors (NAIs) are expected to occur at framework or catalytic residues of the NA enzyme. Numerous clinical and in vitro reports already described NAI-resistant A(H1N1)pdm09 variants harboring various framework NA substitutions. By contrast, variants with NA catalytic changes remain poorly documented. Herein, we investigated the effect of R152K and R368K NA catalytic mutations on the NA enzyme properties, in vitro replicative capacity and virulence of A(H1N1)pdm09 recombinant viruses. In NA inhibition assays, the R152K and R368K substitutions resulted in reduced inhibition [10- to 100-fold increases in IC50 vs the wild-type (WT)] or highly reduced inhibition (>100-fold increases in IC50) to at least 3 approved NAIs (oseltamivir, zanamivir, peramivir and laninamivir). Such resistance phenotype correlated with a significant reduction of affinity observed for the mutants in enzyme kinetics experiments [increased Km from 20⯱â¯1.77 for the WT to 200.8⯱â¯10.54 and 565.2⯱â¯135⯵Mâ¯(Pâ¯<â¯0.01) for the R152K and R368K mutants, respectively]. The R152K and R368K variants grew at comparable or even higher titers than the WT in both MDCK and ST6GalI-MDCK cells. In experimentally-infected C57BL/6 mice, the recombinant WT and the R152K and R368K variants induced important signs of infection (weight loss) and resulted in mortality rates of 87.5%, 37.5% and 100%, respectively. The lung viral titers were comparable between the three infected groups. While the NA mutations were stable, an N154I substitution was detected in the HA2 protein of the R152K and R368K variants after in vitro passages as well as in lungs of infected mice. Due to the multi-drug resistance phenotypes and conserved fitness, the emergence of NA catalytic mutations accompanied with potential compensatory HA changes should be carefully monitored in A(H1N1)pdm09 viruses.
Subject(s)
Antiviral Agents/pharmacology , Drug Resistance, Viral/genetics , Enzyme Inhibitors/pharmacology , Influenza A Virus, H1N1 Subtype/enzymology , Neuraminidase/genetics , Amino Acid Substitution , Animals , Catalytic Domain , Dogs , Female , Genetic Fitness/drug effects , Influenza A Virus, H1N1 Subtype/drug effects , Lung/virology , Madin Darby Canine Kidney Cells , Mice, Inbred C57BL , Mutation , Neuraminidase/antagonists & inhibitors , Virulence/geneticsABSTRACT
After 6 years of circulation in humans, a novel antigenic variant of influenza A(H1N1)pdm09 (i.e., A/Michigan/45/2015) emerged in 2015-16 and has predominated thereafter worldwide. Herein, we compared in vitro and in vivo properties of 2016 wild-type (WT) A/Michigan/45/15-like isolate and its H275Y neuraminidase (NA) variant to the original A/California/07/09-like counterparts. The H275Y mutation induced comparable levels of resistance to oseltamivir and peramivir without altering zanamivir susceptibility in both 2009 and 2016 isolates. In vitro, the two WT isolates had comparable replicative properties. The 2016-H275Y isolate had lower titers at 36â¯h post-inoculation (PI) (Pâ¯<â¯0.05) while the 2009-H275Y titers were lower at both 24â¯h (Pâ¯<â¯0.01) and 36â¯h PI (Pâ¯<â¯0.001) vs the respective WTs. In mice, the 2016-WT isolate caused less weight losses (Pâ¯<â¯0.001) and lower lung viral titers (LVTs) (Pâ¯<â¯0.01) vs the 2009-WT. The LVTs of 2016-WT and 2016-H275Y groups were comparable whereas the 2009-H275Y LVTs were lower vs the respective WT (Pâ¯<â¯0.01). Ferrets infected with the 2016-WT isolate and their contacts had higher nasal viral titers (NVTs) at early time points vs the 2009-WT group (Pâ¯<â¯0.01). Also, NVTs of 2016-H275Y animals were lower vs the 2016-WT group at early time points in both infected (Pâ¯<â¯0.01) and contact animals (Pâ¯<â¯0.001). In conclusion, while the H275Y mutation similarly impacts the A/California/07/2009- and A/Michigan/45/2015-like A(H1N1)pdm09 NAs, the fitness of these isolates differs according to animal models with the 2016 virus being less virulent in mice but slightly more virulent in ferrets, potentially reflecting a period of cumulative changes in surface and internal genes.
Subject(s)
Influenza A Virus, H1N1 Subtype/enzymology , Influenza A Virus, H1N1 Subtype/genetics , Influenza, Human/virology , Mutation , Neuraminidase/genetics , Viral Proteins/genetics , Animals , Antiviral Agents/pharmacology , Disease Models, Animal , Drug Resistance, Multiple, Viral , Female , Ferrets , Humans , Mice , Mice, Inbred BALB C , Orthomyxoviridae Infections/virology , Oseltamivir/pharmacologyABSTRACT
BACKGROUND: Peramivir is a parenteral neuraminidase inhibitor (NAI) approved for treating influenza infections in a few countries. We determined peramivir susceptibilities of several uncharacterized influenza A and B neuraminidase (NA) and haemagglutinin (HA) mutants selected with different NAIs. METHODS: Recombinant wild-type (WT) and mutant NA proteins were expressed in 293T cells and susceptibility to peramivir, oseltamivir and zanamivir was determined by NA inhibition assay using the MUNANA substrate. Recombinant/reassortant influenza A(H1N1), A(H3N2) and B HA mutants were rescued by reverse genetics and assessed by plaque size or viral yield assays for drug susceptibility. RESULTS: Recombinant R152K, I222K/T, G248R+I266V, Q312R+I427T and R371K (A[H1N1]pdm09); E41G, 1222L/V, Q226H and S247P (A[H3N2]) and D198Y, A246D/S/T and G402S (B) mutant NA proteins (N2 numbering) were analysed. Peramivir exhibited the lowest IC50 values against both influenza A and B WT NAs. Peramivir and oseltamivir generally shared similar phenotypes. Of note, peramivir retained activity against I222K/T (A[H1N1]pdm09), I222L/V (A[H3N2]) and A246T (B) mutants, which had reduced inhibition (RI) or highly RI (HRI) against oseltamivir. Cross-RI/HRI against the three NAIs was observed for R152K, R371K and Q312R+I427T (A[H1N1]pdm09); S247P (A[H3N2]) and D198Y (B) mutants. All tested recombinant/reassortant R208K (A/Puerto Rico/8/34 [H1N1]); A28T, R124M and K189E (A/Victoria/3/75 [H3N2]) and T139N (B/Phuket/3073/13) HA mutants were susceptible to peramivir in cell culture experiments. CONCLUSIONS: Peramivir is highly active against seasonal influenza subtypes. Although peramivir and oseltamivir generally share similar phenotypes, peramivir still possesses activity against some variants with RI/HRI against oseltamivir. Finally, NAI-induced HA substitutions alone did not significantly impact NAI susceptibility.
Subject(s)
Antiviral Agents/pharmacology , Cyclopentanes/pharmacology , Guanidines/pharmacology , Influenza A virus/drug effects , Influenza A virus/genetics , Influenza B virus/drug effects , Influenza B virus/genetics , Neuraminidase/antagonists & inhibitors , Acids, Carbocyclic , Humans , Inhibitory Concentration 50 , Microbial Sensitivity Tests , Mutation , Neuraminidase/genetics , Neuraminidase/metabolism , Reassortant Viruses , Recombination, Genetic , Viral Proteins/geneticsABSTRACT
Emergence of pan neuraminidase inhibitor (NAI)-resistant variants constitutes a serious clinical concern. An influenza A(H1N1)pdm09 variant containing the I427T/Q313R neuraminidase (NA) substitutions was previously identified in a surveillance study. Although these changes are not part of the NA active site, the variant showed reduced susceptibility to many NAIs. In this study, we investigated the mechanism of resistance for the I427T/Q313R substitution and its impact on the NA enzyme and viral fitness. Recombinant wild-type (WT), I427T/Q313R and I427T A(H1N1)pdm09 viruses were generated by reverse genetics and tested for their drug susceptibilities, enzymatic properties and replication kinetics in vitro as well as their virulence in mice. Molecular dynamics (MD) simulations were performed for NA structural analysis. The I427T substitution, which was responsible for the resistance phenotype observed in the double (I427T/Q313R) mutant, induced 17-, 56-, 7-, and 14-fold increases in IC50 values against oseltamivir, zanamivir, peramivir and laninamivir, respectively. The I427T substitution alone or combined to Q313R significantly reduced NA affinity. The I427T/Q313R and to a lesser extent I427T recombinant viruses displayed reduced viral titers vs WT in vitro. In experimentally-infected mice, the mortality rates were 62.5%, 0% and 14.3% for the WT, I417T/Q313R and I427T viruses, respectively. There were about 2.5- and 2-Log reductions in mean lung viral titers on day 5 post-infection for the I427T/Q313R and I427T mutants, respectively, compared to WT. Results from simulations revealed that the I427T change indirectly altered the stability of the catalytic R368 residue of the NA enzyme causing its reduced binding to the substrate/inhibitor. This study demonstrates that the I427T/Q313R mutant, not only alters NAI susceptibility but also compromises NA properties and viral fitness, which could explain its infrequent detection in clinic.
Subject(s)
Antiviral Agents/pharmacology , Genetic Fitness , Influenza A Virus, H1N1 Subtype/genetics , Neuraminidase/chemistry , Neuraminidase/genetics , Acids, Carbocyclic , Amino Acid Substitution , Animals , Catalytic Domain , Computer Simulation , Cyclopentanes/pharmacology , Drug Resistance, Viral , Enzyme Inhibitors/pharmacology , Guanidines/pharmacology , Influenza A Virus, H1N1 Subtype/drug effects , Influenza A Virus, H1N1 Subtype/enzymology , Inhibitory Concentration 50 , Mice , Molecular Dynamics Simulation , Neuraminidase/metabolism , Orthomyxoviridae Infections/virology , Oseltamivir/pharmacology , Reverse GeneticsABSTRACT
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 < 0.01) and the E119D/H275Y (1.47 ± 0.61 × 10(6) PFU/ml, P < 0.01) groups. In guinea pigs, comparable seroconversion rates and viral titers in nasal washes (NW) were obtained for the WT and mutant index and contact groups. However, the D119E reversion was observed in most NW samples of the E119D and E119D/H275Y animals. In conclusion, the E119D NA mutation that could emerge in A(H1N1)pdm09 viruses during zanamivir therapy has a significant impact on viral fitness and such mutant is unlikely to be highly transmissible.