Mutant Influenza Virus Research Articles

Dynamics analysis of an influenza epidemic model with virus mutation incorporating log-normal Ornstein–Uhlenbeck process

A stochastic influenza epidemic model where influenza virus can mutate into a mutant influenza virus is established to study the influence of environmental disturbance. And the transmission rate of the model is assumed to satisfy log-normal Ornstein–Uhlenbeck process. We verify that there exists a unique global positive solution to the stochastic model. By constructing proper Lyapunov functions, sufficient conditions under which the stationary distribution exists are obtained. In addition, we discuss the extinction of the disease. Furthermore, we get the accurate expression of probability density function near the endemic equilibrium of the stochastic model. Finally, several numerical simulations are carried out to verify theoretical results and examine the influence of environmental noise.

Development of Digital Droplet PCR Targeting the Influenza H3N2 Oseltamivir-Resistant E119V Mutation and Its Performance through the Use of Reverse Genetics Mutants.

The monitoring of antiviral-resistant influenza virus strains is important for public health given the availability and use of neuraminidase inhibitors and other antivirals to treat infected patients. Naturally occurring oseltamivir-resistant seasonal H3N2 influenza virus strains often carry a glutamate-to-valine substitution at position 119 in the neuraminidase (E119V-NA). Early detection of resistant influenza viruses is important for patient management and for the rapid containment of antiviral resistance. The neuraminidase inhibition assay allows the phenotypical identification of resistant strains; however, this test often has limited sensitivity with high variability depending on the virus strain, drugs and assays. Once a mutation such as E119V-NA is known, highly sensitive PCR-based genotypic assays can be used to identify the prevalence of such mutant influenza viruses in clinical samples. In this study, based on an existing reverse transcriptase real-time PCR (RT-qPCR) assay, we developed a reverse transcriptase droplet digital PCR assay (RT-ddPCR) to detect and quantify the frequency of the E119V-NA mutation. Furthermore, reverse genetics viruses carrying this mutation were created to test the performance of the RT-ddPCR assay and compare it to the standard phenotypic NA assay. We also discuss the advantage of using an RT-ddPCR instead of qPCR method in the context of viral diagnostics and surveillance.

Design of new chemical entities targeting both native and H275Y mutant influenza a virus by deep reinforcement learning

Influenza virus remains a major public health challenge due to its high morbidity and mortality and seasonal surge. Although antiviral drugs against the influenza virus are widely used as a first-line defense, the virus undergoes rapid genetic changes, resulting in the emergence of drug-resistant strains. Thus, new antiviral drugs that can outwit resistant strains are of significant importance. Herein, we used deep reinforcement learning (RL) algorithm to design new chemical entities (NCEs) that are able to bind to the native and H275Y mutant (oseltamivir-resistant) neuraminidases (NAs) of influenza A virus with better binding energy than oseltamivir. We generated more than 66211 NCEs, which were prioritized based on the filtering rules, structural alerts, and synthetic accessibility. Then, 18 NCEs with better MM/PBSA scores than oseltamivir were further analyzed in molecular dynamics (MD) simulations conducted for 100 ns. The MD experiments showed that 8 NCEs formed very stable complexes with the binding pocket of both native and H275Y mutant NAs of H1N1. Furthermore, most NCEs demonstrated much better binding affinity to group 2 (N2, N3, and N9) and influenza B virus NAs than oseltamivir. Although all 8 NCEs have non-sialic acid-like structures, they showed a similar binding mode as oseltamivir, indicating that it is possible to find new scaffolds with better binding and antiviral properties than sialic acid-like inhibitors. In conclusion, we have designed potential compounds as antiviral candidates for further synthesis and testing against wild and mutant influenza virus. Communicated by Ramaswamy H. Sarma

Zanamivir-Cholesterol Conjugate: A Long-Acting Neuraminidase Inhibitor with Potent Efficacy against Drug-Resistant Influenza Viruses.

Antiviral therapy of influenza virus infections depends heavily on two viral neuraminidase (NA) inhibitors, oseltamivir (OSV) and zanamivir (ZNV). The efficacy of OSV is challenged by the development of viral resistance, while the clinical use of ZNV is limited by its poor pharmacokinetic profile and requirement for twice-daily intranasal administration. We have developed a novel NA inhibitor by conjugating ZNV to cholesterol. The ZNV-cholesterol conjugate showed markedly improved antiviral efficacy and plasma half-life compared with ZNV. Single-dose administration of the conjugate protected the mice from lethal challenges with wild-type or mutant H1N1 influenza viruses bearing an OSV-resistant H275Y-substitution. Mechanistic studies showed that the conjugate targeted the cell membrane and entered the host cells, thereby inhibiting the NA function and the assembly of progeny virions. The ZNV-cholesterol conjugate represents a potential new treatment for influenza infections with sustained effect. Cholesterol conjugation may be an effective strategy for improving the pharmacokinetics and efficacy of other small-molecule therapeutics.

Site-Specific Glycan-Masking/Unmasking Hemagglutinin Antigen Design to Elicit Broadly Neutralizing and Stem-Binding Antibodies Against Highly Pathogenic Avian Influenza H5N1 Virus Infections.

The highly pathogenic avian influenza (HPAI) H5N1 viruses with the capability of transmission from birds to humans have a serious impact on public health. To date, HPAI H5N1 viruses have evolved into ten antigenically distinct clades that could cause a mismatch of vaccine strains and reduce vaccine efficacy. In this study, the glycan masking and unmasking strategies on hemagglutinin antigen were used for designing two antigens: H5-dm/st2 and H5-tm/st2, and investigated for their elicited immunity using two-dose recombinant H5 (rH5) immunization and a first-dose adenovirus vector prime, followed by a second-dose rH5 protein booster immunization. The H5-dm/st2 antigen was found to elicit broadly neutralizing antibodies against different H5N1 clade/subclade viruses, as well as more stem-binding antibodies to inhibit HA-facilitated membrane fusion activity. Mice immunized with the H5-dm/st2 antigen had a higher survival rate when challenged with homologous and heterologous clades of H5N1 viruses. Mutant influenza virus replaced with the H5-dm/st2 gene generated by reverse genetics (RG) technology amplified well in MDCK cells and embryonated chicken eggs. Again, the inactivated H5N1-dm/st2 RG virus elicited more potent cross-clade neutralizing and anti-fusion antibodies in sera. Therefore, the H5N1-dm/st2 RG virus with the site-specific glycan-masking on the globular head and the glycan-unmasking on the stem region of H5 antigen can be used for further development of cross-protective H5N1 vaccines.

Design, synthesis, and bioassay of 4-thiazolinone derivatives as influenza neuraminidase inhibitors.

A series of 4-thiazolinone derivatives (D1-D58) were designed and synthesized. All of the derivatives were evaluated invitro for neuraminidase (NA) inhibitory activities against influenza virus A (H1N1), and the inhibitory activities of the five most potent compounds were further evaluated on NA from two different influenza viral subtypes (H3N2 and B), and then their invitro anti-viral activities were evaluated using the cytopathic effect (CPE) reduction assay. The results showed that the majority of the target compounds exhibited moderate to good NA inhibitory activity. Compound D18 presented the most potent inhibitory activity with IC50 values of 13.06μM against influenza H1N1 subtype. Among the selected compounds, D18 and D41 turned out to be the most potent inhibitors against influenza virus H3N2 subtype (IC50=15.00μM and IC50=14.97μM, respectively). D25 was the most potent compound against influenza B subtype (IC50=16.09μM). In addition, D41 showed low toxicity and greater potency than reference compounds Oseltamivir and Amantadine against N1-H275Y variant in cellular assays. The structure-activity relationship (SAR) analysis showed that introducing 4-CO2H, 4-OH, 3-OCH3-4-OH substituted benzyl methylene can greatly improve the activity of 4-thiazolinones. Further SAR analysis indicated that 4-thiazolinone and ferulic acid fragments are necessary fragments of target compounds for inhibiting NA. Molecular docking was performed to study the interaction between compound D41 and the active site of NA. This study may providing important information for new drug development for anti-influenza virus including mutant influenza virus.

Selection and antigenic characterization of immune-escape mutants of H7N2 low pathogenic avian influenza virus using homologous polyclonal sera

Understanding the dynamics of the selection of influenza A immune escape variants by serum antibody is critical for designing effective vaccination programs for animals, especially poultry where large populations have a short generation time and may be vaccinated with high frequency. In this report, immune-escape mutants of A/turkey/New York/4450/1994 H7N2 low pathogenic avian influenza virus, were selected by serially passaging the virus in the presence of continuously increasing concentrations of homologous chicken polyclonal sera. Amino acid mutations were identified by sequencing the parental hemagglutinin (HA) gene and every 10 passages by both Sanger and deep sequencing, and the antigenic distance of the mutants to the parent strain was determined. Progressively, a total of five amino acid mutations were observed over the course of 30 passages. Based on their absence from the parental virus with deep sequencing, the mutations appear to have developed de novo. The antigenic distance between the selected mutants and the parent strain increased as the number of amino acid mutations accumulated and the concentration of antibodies had to be periodically increased to maintain the same reduction in virus titer during selection. This selection system demonstrates how H7 avian influenza viruses behave under selection with homologous sera, and provides a glimpse of their evolutionary dynamics, which can be applied to developing vaccination programs that maximize the effectiveness of a vaccine over time.

Design, Synthesis, and Biological Evaluation of Novel Acylhydrazone Derivatives as Potent Neuraminidase Inhibitors.

Neuraminidase (NA) is an important target for current research on anti-influenza drugs. The acylhydrazone derivatives containing the -CONHN=CH- framework have been shown to have good NA inhibitory activity. In this paper, a series of novel acylhydrazone NA inhibitors (9a-9n) were designed and synthesized, and the inhibitory activities against NA were evaluated in vitro. The NA inhibition results showed that compound 9j has the most potent inhibitory activity (IC50 = 0.6 μM) against NA, which is significantly lower than that of the positive control oseltamivir carboxylic acid (OSC) (IC50 = 17.00 μM). Molecular docking analysis indicates that the acylhydrazone group plays an important role in compound 9j, which can bind well to the residues Arg371 and Arg292 in the S1 subsite of NA. The good potency of 9j may be also ascribed to the extending of morpholinyl ring into the 430-cavity. The results of this work may contribute to the development of more potent NA inhibitors to against mutant influenza viruses.

A Replication-Defective Influenza Virus Vaccine Confers Complete Protection against H7N9 Viral Infection in Mice.

Avian influenza H7N9 viruses continue to pose a great threat to public health, which is evident by their high case-fatality rates. Although H7N9 was first isolated in humans in China in 2013, to date, there is no commercial vaccine available against this particular strain. Our previous studies developed a replication-defective influenza virus through mutation of the hemagglutinin (HA) cleavage site from a trypsin-sensitive to an elastase-sensitive motif. In this study, we report the development of a reassortant mutant influenza virus derived from the human isolate A/British Columbia/01/2015 (H7N9) [BC15 (H7N9)], which is the QVT virus. The HA gene of this virus possesses three mutations at the cleavage site, Lys-Gly-Arg were mutated to Gln-Thr-Val at amino acid (aa) positions 337, 338, and 339, respectively. We report this virus to rely on elastase in vitro, possess unaltered replication abilities when elastase was provided compared to the wild type virus in vitro, and to be non-virulent and replication-defective in mice. In addition, we report this virus to induce significant levels of antibodies and IFN-γ and IL-5 secreting cells, and to protect mice against a lethal challenge of the BC15 (H7N9) virus. This protection is demonstrated through the lack of body weight loss, 100% survival rate, and the prevention of BC15 (H7N9) viral replication as well as the reduction of proinflammatory cytokines induced in the mouse lung associated with the influenza disease. Therefore, these results provide strong evidence for the use of this reassortant mutant H7N9 virus as a replication-defective virus vaccine candidate against H7N9 viruses.

Examining immune arms in mice immunized with site-specific influenza virus mutants

Site-specific mutants as candidates for live influenza vaccines were resulted from directly introducing into the genome of the pathogenic influenza virus A/WSN/33 (H1N1) strain ts mutations derived from the genes encoding the polymerase complex proteins from some cold-adapted strains serving as attenuation donor. Here we present the data of a comparative study examining immune system arms in mice immunized intranasally with influenza virus mutants and classical cold-adapted reassortant obtained by crossing cold-adapted strain Donor A/Krasnodar/101/35/59 (H2N2) with strain A/WSN/33 (H1N1) bearing surface antigens (hemagglutinin and neuraminidase) similar to mutants. Immunophenotyping mononuclear leukocytes from immunized mice indicated at moderate suppressive effect after using site-specific mutant and the HA reassortant viruses on some immune cell subsets. All viruses in immunized mice resulted in activation of certain lymphocyte subsets including MHC II-positive cells, CD45+/CD19+ B lymphocytes and natural killer cells (CD16/32+/CD3–). Timescale and magnitude of activation markedly differed for each cell subsets. Mice immunized with mutants M26 and U2 peaked with count of CD16/32+/CD3– expressing cells on day 2 after the second immunization compared with control (p 0.05) that may suggest about an important role for NK cells in activating immune response. In contrast, no significant changes were observed during the study in percentage of CD4+/CD25+/Fox P3 regulatory T cells, CD4+ T helpers and CD8+ cytotoxic cells, except for a sharply decreased count of activated CD4+/CD25+ cells (4-fold) on day 7 after immunization with mutant virus M26. Moreover, mutants U2 and M26 more moderately increased percentage of TLR2- and TLR4-positive cells. The viruses studied ambiguously affected count of TLR9-expressing cells in immunized animals. All viruses increased phagocytic activity in monocytes, but not neutrophils. Despite the moderate activation of innate and adaptive immunity arms, site-specific mutants more profoundly affected humoral reactions inducing increased antibody titers, so that immunogenicity of mutant viruses was higher than that of the cold-adapted reassortant. Thus, the findings hold a promise of using site-specific mutants as live influenza vaccines.

Influenza Reverse Genetics-Historical Perspective.

The generation of wild-type, mutant, and reassortant influenza viruses from viral cDNAs (reverse genetics) is now a basic molecular virology technique in many influenza virus laboratories. Here, I describe the original RNA polymerase I reverse genetics system and the modifications that have been developed in past years. Together, these technologies have made possible many advances in basic and applied influenza virology that would not have been otherwise attainable, including the revival and study of extinct influenza viruses, the rapid characterization of emerging influenza viruses, the generation of conventional influenza vaccines, and the development of novel influenza vaccines.

A Comparative Analysis of Risk Perception and Coping Behaviors among Chinese Poultry Farmers Regarding Human and Poultry Infection with Avian Influenza.

Poultry farmers face a dual risk when mutant avian influenza (AI) virus exhibits zoonotic characteristics. A/H5N1 and A/H7N9 are two principal strains of the AI virus that have captured public attention in recent years, as they have both been reported and can infect poultry and humans, respectively. Previous studies have focused either on poultry farmers’ risk perception and biosecurity preventive behaviors (BPBs) against A/H5N1 infection with poultry, or on their risk perception and personal protective behaviors (PPBs) against human infection with A/H7N9, even though these two strains often appear simultaneously. To bridge this research gap, a survey (N = 426) was conducted in the Chinese provinces of Jiangsu and Anhui to assess risk perception and coping behaviors adopted by poultry farmers facing the dual risk of these two viral strains. Paired sample t-tests revealed that farmers’ perceived risk of poultry infection with A/H5N1 was significantly higher than their perceived risk of human infection with A/H7N9, and that their reported frequency of BPBs against A/H5N1 was significantly higher than the frequency of PPBs against A/H7N9. Moreover, farmers were less familiar with AI infection in human beings compared to that in poultry, but they felt a higher sense of control regarding human AI infection. Multivariate regression analyses showed that farm size and perceived risks of both human and poultry infection with AI were positively associated with BPBs and PPBs. The findings of this research suggest that a campaign to spread knowledge about human AI infection among poultry farmers is needed, and that a policy incentive to encourage large-scale poultry farming could be effective in improving implementation of BPBs and PPBs.

The Role of EGFR in Influenza Pathogenicity: Multiple Network-Based Approaches to Identify a Key Regulator of Non-lethal Infections.

Despite high sequence similarity between pandemic and seasonal influenza viruses, there is extreme variation in host pathogenicity from one viral strain to the next. Identifying the underlying mechanisms of variability in pathogenicity is a critical task for understanding influenza virus infection and effective management of highly pathogenic influenza virus disease. We applied a network-based modeling approach to identify critical functions related to influenza virus pathogenicity using large transcriptomic and proteomic datasets from mice infected with six influenza virus strains or mutants. Our analysis revealed two pathogenicity-related gene expression clusters; these results were corroborated by matching proteomics data. We also identified parallel downstream processes that were altered during influenza pathogenesis. We found that network bottlenecks (nodes that bridge different network regions) were highly enriched in pathogenicity-related genes, while network hubs (highly connected network nodes) were significantly depleted in these genes. We confirmed that this trend persisted in a distinct virus: Severe Acute Respiratory Syndrome Coronavirus (SARS). The role of epidermal growth factor receptor (EGFR) in influenza pathogenesis, one of the bottleneck regulators with corroborating signals across transcript and protein expression data, was tested and validated in additional mouse infection experiments. We demonstrate that EGFR is important during influenza infection, but the role it plays changes for lethal versus non-lethal infections. Our results show that by using association networks, bottleneck genes that lack hub characteristics can be used to predict a gene’s involvement in influenza virus pathogenicity. We also demonstrate the utility of employing multiple network approaches for analyzing host response data from viral infections.

Diagnosis of Tamiflu-Resistant Influenza Virus in Human Nasal Fluid and Saliva Using Surface-Enhanced Raman Scattering.

Influenza viruses cause respiratory infection, spread through respiratory secretions, and are shed into the nasal secretion and saliva specimens. Therefore, nasal fluid and saliva are effective clinical samples for the diagnosis of influenza virus-infected patients. Although several methods have been developed to detect various types of influenza viruses, approaches for detecting mutant influenza viruses in clinical samples are rarely reported. Herein, we report for the first time a surface-enhanced Raman scattering (SERS)-based sensing platform for oseltamivir-resistant pandemic H1N1 (pH1N1) virus detection in human nasal fluid and saliva. By combining SERS-active urchin Au nanoparticles and oseltamivir hexylthiol, an excellent receptor for the pH1N1/H275Y mutant virus, we detected the pH1N1/H275Y virus specifically and sensitively in human saliva and nasal fluid samples. Considering that the current influenza virus infection testing methods do not provide information on the antiviral drug resistance of the virus, the proposed SERS-based diagnostic test for the oseltamivir-resistant virus will inform clinical decisions about the treatment of influenza virus infections, avoiding the unnecessary prescription of ineffective drugs and greatly improving therapy.

Study of the biological properties of attenuated variants of the virulent A/WSN/33 strain of influenza virus, obtained by the site-specific mutagenesis of PB2-gene

Aim. Study of biological properties of attenuated variants of the virulent A/WSN/33 strain of influenza virus, obtained by the site-specific mutagenesis of PB2-gene. Materials and methods. Site-specific mutants of A/WSN/33 of influenza virus, having in PB2-gene ts-mutations from genome of cold-adapted (CA) master-strains: A/Ann Arbor/6/60 (H2N2); A/Leningrad/134/17/57 (H2N2); A/Krasnodar/101/35/59 (H2N2) were obtained with help of reverse genetics methods. The ts-phenotype, att-phenotype, immunogenicity and protective efficacy in homologous and heterologous control infections were studied in the obtained site-specific mutants. Results. It was shown that the inclusion in the PB2-gene of the virulent A/WSN/33 strain as single mutations and a combination of mutations from the genomes of CA donor-strains leads to a change in the ts-phenotype and att-phenotype of the mutants obtained. These mutants had high protective efficacy in homologous and heterologous control infection. Conclusion. The results obtained allow us to consider the site-specific mutants of influenza virus as possible candidates for live influenza vaccines.

Structural basis for influenza virus NS1 protein block of mRNA nuclear export.

Influenza viruses antagonize key immune defense mechanisms via the virulence factor NS1 protein. A major NS1 strategy is to block nuclear export of host mRNAs including those encoding immune factors1–3; however, the direct cellular target of NS1 and the mechanism of host mRNA export inhibition are not known. Here, we identify the target of NS1 as the mRNA export receptor NXF1•NXT1, which is the principal receptor to mediate docking and translocation of mRNAs through the nuclear pore complex by interacting with nucleoporins4,5. We determined the crystal structure of NS1 in complex with NXF1•NXT1 at 3.8 Å resolution. The structure reveals that NS1 prevents binding of NXF1•NXT1 to nucleoporins, thereby inhibiting mRNA export through the nuclear pore complex into the cytoplasm for translation. We demonstrate that a mutant influenza virus deficient in binding NXF1•NXT1 does not block host mRNA export and is attenuated. This attenuation is marked by the release of mRNAs encoding immune factors from the nucleus. Together, our study uncovers the molecular basis of a major nuclear function of influenza NS1 protein that causes potent blockage of host gene expression and contributes to inhibition of host immunity.

Chinese poultry farmers' decision-making for avian influenza prevention: a qualitative analysis.

Poultry farmers faced dual risk when mutant avian influenza (AI) virus showed the zoonotic characteristics. A/H5N1 and A/H7N9 were two dominant AI virus strains that have captured the attention of the public over the years for they have been reported to bring about greater loss to poultry and human, respectively. Previous studies mainly used quantitative methods investigating either the means that poultry farmers adopted for protecting their poultry against A/H5N1 infection or the poultry farmers' self-protective behaviours against A/H7N9 infection. We sought insights into the underlying factors influencing Chinese poultry farmers' protective behaviours in response to the dual risk of AI by a qualitative way. Semi-structured in-depth interviews were conducted with 25 Chinese chicken farmers recruited by purposive sampling between November 2016 and May 2017, the peak season of AI. All interviews were audio-taped, transcribed and analysed using a grounded theory approach. From participants' experiences, we revealed five main themes: Measures adopted for protecting poultry and farmers, Emotional response to the AI epidemic, Perceived risk of AI, Perceived effectiveness of the preventive measures adopted and Perceived self-efficacy to take preventive measures. The information of AI outbreak directly triggered Chinese chicken farmers' emotional response and thereafter preventive actions. Compared to the perceived risk of poultry infection with A/H5N1 which mainly connected to economic loss, participants perceived much lower risk of human infection with A/H7N9. AI epidemic information played a key role triggering poultry farmers' response behaviours. Chinese poultry farmers weighted more attention on the risk of poultry infection which was highly associated with economic losses. The government should build and improve an early AI warning and information transmission network to poultry farmers. Further reinforcement of related self-protective and preventive knowledge training towards poultry farmers is necessary.

DYNAMICAL BEHAVIORS OF AN INFLUENZA EPIDEMIC MODEL WITH VIRUS MUTATION

Influenza virus mutates frequently. This poses immense challenges to prevent the spread of influenza. This paper aims to investigate an influenza epidemic model in which influenza virus can mutate into a mutant influenza virus. We find a threshold condition that determines the stability of the disease-free equilibrium. Two equilibria may also exist: mutant-dominant equilibrium and endemic equilibrium. We show that the mutant-dominant equilibrium is globally asymptotically stable under some biological feasible conditions. Furthermore, the influenza is endemic in the sense of permanence if and only if the endemic equilibrium exists. Numerical simulations are also performed to illustrate theoretical results and demonstrate the effects of disease-induced death on the dynamics of the model.

Screening for Neuraminidase Inhibitor Resistance Markers among Avian Influenza Viruses of the N4, N5, N6, and N8 Neuraminidase Subtypes.

Several subtypes of avian influenza viruses (AIVs) are emerging as novel human pathogens, and the frequency of related infections has increased in recent years. Although neuraminidase (NA) inhibitors (NAIs) are the only class of antiviral drugs available for therapeutic intervention for AIV-infected patients, studies on NAI resistance among AIVs have been limited, and markers of resistance are poorly understood. Previously, we identified unique NAI resistance substitutions in AIVs of the N3, N7, and N9 NA subtypes. Here, we report profiles of NA substitutions that confer NAI resistance in AIVs of the N4, N5, N6, and N8 NA subtypes using gene-fragmented random mutagenesis. We generated libraries of mutant influenza viruses using reverse genetics (RG) and selected resistant variants in the presence of the NAIs oseltamivir carboxylate and zanamivir in MDCK cells. In addition, two substitutions, H274Y and R292K (N2 numbering), were introduced into each NA gene for comparison. We identified 37 amino acid substitutions within the NA gene, 16 of which (4 in N4, 4 in N5, 4 in N6, and 4 in N8) conferred resistance to NAIs (oseltamivir carboxylate, zanamivir, or peramivir) as determined using a fluorescence-based NA inhibition assay. Substitutions conferring NAI resistance were mainly categorized as either novel NA subtype specific (G/N147V/I, A246V, and I427L) or previously reported in other subtypes (E119A/D/V, Q136K, E276D, R292K, and R371K). Our results demonstrate that each NA subtype possesses unique NAI resistance markers, and knowledge of these substitutions in AIVs is important in facilitating antiviral susceptibility monitoring of NAI resistance in AIVs.IMPORTANCE The frequency of human infections with avian influenza viruses (AIVs) has increased in recent years. Despite the availability of vaccines, neuraminidase inhibitors (NAIs), as the only available class of drugs for AIVs in humans, have been constantly used for treatment, leading to the inevitable emergence of drug-resistant variants. To screen for substitutions conferring NAI resistance in AIVs of N4, N5, N6, and N8 NA subtypes, random mutations within the target gene were generated, and resistant viruses were selected from mutant libraries in the presence of individual drugs. We identified 16 NA substitutions conferring NAI resistance in the tested AIV subtypes; some are novel and subtype specific, and others have been previously reported in other subtypes. Our findings will contribute to an increased and more comprehensive understanding of the mechanisms of NAI-induced inhibition of influenza virus and help lead to the development of drugs that bind to alternative interaction motifs.

Quantifying the relative impact of influenza virus mutations on viral fitness and escape from cellular immunity

Abstract The relative conservation of T cell epitopes in human influenza viruses compared to antibody epitopes may be explained by two mechanisms: (1) mutations of the T cell epitopes are constrained by their potential to decrease viral fitness by altering protein function, or (2) the selection benefits gained by ‘escaping’ mutations are small due to the breadth of MHC polymorphisms among human population. The level of influenza-specific cellular immunity may further complicate the relative contribution of these two mechanisms. We developed a mathematical model incorporating the mutation cost, selection benefit, and allele frequency of ‘escapable’ MHC that indicated certain combinations of these three parameters determined the fate of influenza viruses harboring mutations in T cell epitopes. To measure the mutation cost and selection benefit, we developed a novel digital droplet PCR method for the simultaneous detection of wild-type and mutant influenza viruses harboring mutations in the immunodominant NP366–374 epitope. Primary infections with WT, NP-mutated, and 1:1 mixture of WT and mutated x31 influenza viruses showed no significant difference between the two viruses, while the secondary infection of PR8-primed B6 mice with the 1:1 mixture resulted in a 10-fold decrease in the peak of viral load of WT compared to NP-mutated viruses. Together, these data show that the mutation of a single immunodominant T cell epitope provides significant selection benefit with minimal fitness cost to the virus. Future studies in mice with diverse MHC alleles are underway to quantify the impact of varying levels of cellular immune pressure on the in vivo ability of mutant influenza viruses to escape T cell immunity.