[Show abstract][Hide abstract] ABSTRACT: Importance:
The number of humans infected with avian influenza viruses is increasing, raising concerns of the emergence of avian influenza viruses resistant to neuraminidase (NA) inhibitors (NAIs). As most studies have focused on NAI-resistance in human influenza viruses, here we investigated the molecular changes in NA that could confer NAI-resistance in avian viruses grown in immortalized monolayer cells, especially those of the N3, N7 and N9 subtypes, which have caused human infections. We identified not only numerous NAI-resistant substitutions previously reported in other NA subtypes but also several novel changes conferring reduced susceptibility to NAIs, which are subtype-specific. The findings indicate that some resistance markers are common across NA subtypes but other markers needs to be determined empirically for each subtype. The study also implies that antiviral surveillance monitoring could play a critical role in the clinical management of influenza infection and an essential component of pandemic preparedness.
Journal of Virology 08/2015; 89(21). DOI:10.1128/JVI.01514-15 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Human infection with avian influenza A(H7N9) virus is associated mainly with the exposure to infected poultry. The factors that allow interspecies transmission but limit human-to-human transmission are unknown. Here we show that A/Anhui/1/2013(H7N9) influenza virus infection of chickens (natural hosts) is asymptomatic and that it generates a high genetic diversity. In contrast, diversity is tightly restricted in infected ferrets, limiting further adaptation to a fully transmissible form. Airborne transmission in ferrets is accompanied by the mutations in PB1, NP and NA genes that reduce viral polymerase and neuraminidase activity. Therefore, while A(H7N9) virus can infect mammals, further adaptation appears to incur a fitness cost. Our results reveal that a tight genetic bottleneck during avian-to-mammalian transmission is a limiting factor in A(H7N9) influenza virus adaptation to mammals. This previously unrecognized biological mechanism limiting species jumps provides a measure of adaptive potential and may serve as a risk assessment tool for pandemic preparedness.
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Influenza A and B viruses are human pathogens that are regarded to cause almost equally significant disease burdens. Neuraminidase (NA) inhibitors (NAIs) are the only class of drugs available to treat influenza A and B virus infections, so the development of NAI-resistant viruses with superior fitness is a public health concern. The fitness of NAI-resistant influenza B viruses has not been widely studied. Here we examined the replicative capacity and relative fitness in normal human bronchial epithelial (NHBE) cells of recombinant influenza B/Yamanashi/166/1998 viruses containing a single amino acid substitution in NA generated by reverse genetics (rg) that is associated with NAI resistance. The replication in NHBE cells of viruses with reduced inhibition by oseltamivir (recombinant virus with the E119A mutation generated by reverse genetics [rg-E119A], rg-D198E, rg-I222T, rg-H274Y, rg-N294S, and rg-R371K, N2 numbering) or zanamivir (rg-E119A and rg-R371K) failed to be inhibited by the presence of the respective NAI. In a fluorescence-based assay, detection of rg-E119A was easily masked by the presence of NAI-susceptible virus. We coinfected NHBE cells with NAI-susceptible and -resistant viruses and used next-generation deep sequencing to reveal the order of relative fitness compared to that of recombinant wild-type (WT) virus generated by reverse genetics (rg-WT): rg-H274Y > rg-WT > rg-I222T > rg-N294S > rg-D198E > rg-E119A ≫ rg-R371K. Based on the lack of attenuated replication of rg-E119A in NHBE cells in the presence of oseltamivir or zanamivir and the fitness advantage of rg-H274Y over rg-WT, we emphasize the importance of these substitutions in the NA glycoprotein. Human infections with influenza B viruses carrying the E119A or H274Y substitution could limit the therapeutic options for those infected; the emergence of such viruses should be closely monitored.
Influenza B viruses are important human respiratory pathogens contributing to a significant portion of seasonal influenza virus infections worldwide. The development of resistance to a single class of available antivirals, the neuraminidase (NA) inhibitors (NAIs), is a public health concern. Amino acid substitutions in the NA glycoprotein of influenza B virus not only can confer antiviral resistance but also can alter viral fitness. Here we used normal human bronchial epithelial (NHBE) cells, a model of the human upper respiratory tract, to examine the replicative capacities and fitness of NAI-resistant influenza B viruses. We show that virus with an E119A NA substitution can replicate efficiently in NHBE cells in the presence of oseltamivir or zanamivir and that virus with the H274Y NA substitution has a relative fitness greater than that of the wild-type NAI-susceptible virus. This study is the first to use NHBE cells to determine the fitness of NAI-resistant influenza B viruses.
Journal of Virology 02/2015; 89(8). DOI:10.1128/JVI.02473-14 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Oseltamivir-resistant H1N1 influenza viruses carrying the H275Y neuraminidase mutation predominated worldwide during the 2007-2009 seasons. Although several neuraminidase substitutions were found to be necessary to counteract the adverse effects of H275Y, the order and impact of evolutionary events involved remain elusive. Here we reconstruct H1N1 neuraminidase phylogeny during 1999-2009, estimate the timing and order of crucial amino acid changes and evaluate their impact on the biological outcome of the H275Y mutation. Of the 12 neuraminidase substitutions that occurred during 1999-2009, 5 (chronologically, V234M, R222Q, K329E, D344N, H275Y and D354G) are necessary for maintaining full neuraminidase function in the presence of the H275Y mutation by altering protein accumulation or enzyme affinity/activity. The sequential emergence and cumulative effects of these mutations clearly illustrate a role for epistasis in shaping the emergence and subsequent evolution of a drug-resistant virus population, which can be useful in understanding emergence of novel viral phenotypes of influenza.
[Show abstract][Hide abstract] ABSTRACT: Influenza is an acute respiratory disease in mammals and domestic poultry that emerges from zoonotic reservoirs in aquatic birds and bats. Although influenza viruses are among the most intensively studied pathogens, existing control options require further improvement. Influenza vaccines must be regularly updated because of continuous antigenic drift and sporadic antigenic shifts in the viral surface glycoproteins. Currently, influenza therapeutics are limited to neuraminidase inhibitors; novel drugs and vaccine approaches are therefore urgently needed. Advances in vaccinology and structural analysis have revealed common antigenic epitopes on hemagglutinins across all influenza viruses and suggest that a universal influenza vaccine is possible. In addition, various immunomodulatory agents and signaling pathway inhibitors are undergoing preclinical development. Continuing challenges in influenza include the emergence of pandemic H1N1 influenza in 2009, human infections with avian H7N9 influenza in 2013, and sporadic human cases of highly pathogenic avian H5N1 influenza. Here, we review the challenges facing influenza scientists and veterinary and human public health officials; we also discuss the exciting possibility of achieving the ultimate goal of controlling influenza's ability to change its antigenicity.
Annals of the New York Academy of Sciences 05/2014; 1323(1). DOI:10.1111/nyas.12462 · 4.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: There is a need for new approaches for the control of influenza given the burden caused by annual seasonal outbreaks, the emergence of viruses with pandemic potential, and the development of resistance to current antiviral drugs. We show that multivalent biologics, engineered using carbohydrate-binding modules specific for sialic acid, mask the cell-surface receptor recognized by the influenza virus and protect mice from a lethal challenge with 2009 pandemic H1N1 influenza virus. The most promising biologic protects mice when given as a single 1-μg intranasal dose 7 d in advance of viral challenge. There also is sufficient virus replication to establish an immune response, potentially protecting the animal from future exposure to the virus. Furthermore, the biologics appear to stimulate inflammatory mediators, and this stimulation may contribute to their protective ability. Our results suggest that this host-targeted approach could provide a front-line prophylactic that has the potential to protect against any current and future influenza virus and possibly against other respiratory pathogens that use sialic acid as a receptor.
Proceedings of the National Academy of Sciences 04/2014; 111(17). DOI:10.1073/pnas.1404205111 · 9.67 Impact Factor
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Viruses modulate cellular signaling pathways at almost every step of the infection cycle. Cellular signaling pathways activated at later times of influenza infection have previously been investigated; however, early influenza virus-host cell interactions remain understudied. Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that regulates phosphatidylinositol 3-kinase (PI3K) activation and actin reorganization, two critical processes during influenza A virus (IAV) infection in most cell types. Using 6 influenza A virus strains (A/Puerto Rico/8/1934, A/Aichi/2/1968 × A/Puerto Rico/8/1934 reassortant [X-31], A/California/04/2009, mouse-adapted A/California/04/2009, A/WSN/1933, and A/New Caledonia/20/1999), we examined the role of FAK during IAV entry. We found that influenza virus attachment induced PI3K-dependent FAK-Y397 phosphorylation. Pharmacological FAK inhibition or expression of a kinase-dead mutant of FAK led to disruption of the actin meshwork that resulted in sequestration of IAV at the cell periphery and reduced virion localization to early endosomes. Additionally, FAK inhibition impeded viral RNA replication at later times of infection and ultimately resulted in significantly reduced viral titers in both A549 and differentiated normal human bronchial epithelial (NHBE) cells. Although not all tested strains activated FAK, all of them exhibited a reduction in viral replication in response to inhibition of FAK signaling. These findings highlight novel biphasic roles of FAK activation during IAV infection and indicate that FAK serves as a central link between receptor-mediated PI3K activation and actin reorganization during IAV infection.
We found that FAK links early activation of PI3K and actin reorganization, thereby regulating influenza virus entry. Surprisingly, we also found that FAK can regulate viral RNA replication independently of its role in entry. Our study addresses a knowledge gap in the understanding of signaling events triggered by influenza virus that mediate its internalization and initiation of the infection cycle. Understanding of these fundamental molecular events will be necessary to identify novel host targets, such as FAK, and development of future anti-influenza virus therapeutics.
Journal of Virology 04/2014; 88(12). DOI:10.1128/JVI.00530-14 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Influenza B viruses cause annual outbreaks of respiratory illness in humans and are increasingly recognized as a major cause of influenza-associated pediatric mortality. Neuraminidase (NA) inhibitors (NAIs) are the only available therapy for patients infected with influenza B viruses and the potential emergence of NAI-resistant viruses is a public health concern. The NA substitutions located within enzyme active site could not only reduce NAI susceptibility of influenza B virus but also affect virus fitness. In this study we investigated the effect of single NA substitutions on fitness of influenza B/Yamanashi/166/1998 viruses (Yamagata lineage). We generated recombinant viruses containing either WT NA or NA with a substitution in the catalytic (R371K) or framework residues (E119A, D198E, D198Y, I222T, H274Y, N294S). We assessed NAI susceptibility, NA biochemical properties, NA protein expression, and virus replication in vitro and in differentiated normal human bronchial epithelial (NHBE) cells. Our results showed that four NA substitutions (D198E, I222T, H274Y, and N294S) conferred reduced inhibition by oseltamivir and 3 (E119A, D198Y, and R371K) conferred highly reduced inhibition by oseltamivir, zanamivir, and peramivir. All NA substitutions, except for D198Y and R371K, were genetically stable after 7 passages in MDCK cells. Cell surface NA protein expression was significantly increased by H274Y and N294S substitutions. Viruses with E119A, I222T, H274Y, or N294S substitutions were not attenuated in replication efficiency in vitro or in NHBE cells. Overall, viruses with E119A or H274Y NA substitutions possess fitness comparable to NAI-susceptible virus and their acquisition by influenza B viruses should be closely monitored.
[Show abstract][Hide abstract] ABSTRACT: H2N2 influenza A viruses were the cause of the 1957-1958 pandemic. Historical evidence demonstrates they arose from avian virus ancestors, and while the H2N2 subtype has disappeared from humans, it persists in wild and domestic birds. Re-emergence of H2N2 in humans is a significant threat due to the absence of humoral immunity in individuals under the age of 50. Thus, examination of these viruses, particularly those from the avian reservoir, must be addressed through surveillance, characterization, and antiviral testing. The data presented here are a risk assessment of 22 avian H2N2 viruses isolated from wild and domestic birds over 6 decades. Our data showed that they have a low rate of genetic and antigenic evolution and remained similar to isolates circulating near the time of the pandemic. Most isolates replicated in mice and human bronchial epithelial cells, but replication in swine tissues was low or absent. Multiple isolates replicated in ferrets, and 3 viruses were transmitted to direct-contact cagemates. Markers of mammalian adaptation in HA and PB2 proteins were absent from all isolates, and they retained a preference for avian-like α2-3 linked sialic acid receptors. Most isolates remained antigenically similar to pandemic A/Singapore/1/57 (H2N2) virus, suggesting they could be controlled by the pandemic vaccine candidate. All viruses were susceptible to neuraminidase inhibitors and adamantanes. Nonetheless, the sustained pathogenicity of avian H2N2 viruses in multiple mammalian models elevates their risk potential for human infections and stresses the need for continual surveillance as a component of pre-pandemic planning.
Journal of Virology 11/2013; 88(2). DOI:10.1128/JVI.02526-13 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: nfluenza A (H9N2) viruses are a genetically diverse population that infects wild and domestic avian species and mammals and contributed the internal gene segments to the A/H5N1 and A/H7N9 viruses associated with lethal human infections. Here we comprehensively assess the potential risk to mammals of a diverse panel of A/H9N2 viruses, representing the major H9N2 clades, using a combination of in vitro assays (e.g., antiviral susceptibility and virus growth in primary differentiated human airway cells) and in vivo assays (e.g., replication, transmission and/or pathogenicity of viruses in ducks, pigs, mice and ferrets). We observed that viruses isolated from humans, A/Hong Kong/1073/1999 and A/Hong Kong/33982/2009, had the highest risk potential. However, the A/swine/ Hong Kong/9A-1/1998 and A/chicken/Hong Kong/G9/1997 viruses also displayed several features suggesting a fitness profile adapted to human infection and transmission. The North American avian H9N2 clade virus had the lowest risk profile, and the other viruses tested displayed various levels of fitness across individual assays. In many cases, the known genotypic polymorphisms alone were not sufficient to accurately predict the virus’ phenotype. Therefore, we conclude that comprehensive risk analyses based on surveillance of circulating influenza virus strains are necessary to assess the potential for human infection by emerging influenza A viruses.
Emerging Microbes and Infections 11/2013; DOI:10.1038/emi.2013.75 · 2.26 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background:
High mortality and uncertainty about the effectiveness of neuraminidase inhibitors (NAIs) in humans infected with influenza A(H7N9) viruses are public health concerns.
Susceptibility of N9 viruses to NAIs was determined in a fluorescence-based assay. The NAI oseltamivir (5, 20, or 80 mg/kg/day) was administered to BALB/c mice twice daily starting 24, 48, or 72 hours after A/Anhui/1/2013 (H7N9) virus challenge.
All 12 avian N9 and 3 human H7N9 influenza viruses tested were susceptible to NAIs. Without prior adaptation, A/Anhui/1/2013 (H7N9) caused lethal infection in mice that was restricted to the respiratory tract and resulted in pulmonary edema and acute lung injury with hyaline membrane formation, leading to decreased oxygenation, all characteristics of human acute respiratory distress syndrome. Oseltamivir at 20 and 80 mg/kg protected 80% and 88% of mice when initiated after 24 hours, and the efficacy decreased to 70% and 60%, respectively, when treatment was delayed by 48 hours. Emergence of oseltamivir-resistant variants was not detected.
H7N9 viruses are comparable to currently circulating influenza A viruses in susceptibility to NAIs. Based on these animal studies, early treatment is associated with improved outcomes.
The Journal of Infectious Diseases 10/2013; 209(9). DOI:10.1093/infdis/jit554 · 6.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Many aspects of the biology and epidemiology of influenza B viruses are far less studied than for influenza A viruses, and one of these aspects is effectiveness and resistance to the clinically available antiviral drugs, the neuraminidase (NA) inhibitors (NAIs). Acute respiratory infections are one of the leading causes of death in children and adults, and influenza is among the few respiratory infections that can be prevented and treated by vaccination and antiviral treatment. Recent data has suggested that influenza B virus infections are of specific concern to pediatric patients because of the increased risk of severe disease. Treatment of influenza B is a challenging task for the following reasons: This review presents current knowledge of the effectiveness of NAIs for influenza B virus and antiviral resistance in clinical, surveillance, and experimental studies.
Antiviral research 09/2013; 100(2). DOI:10.1016/j.antiviral.2013.08.023 · 3.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The influenza neuraminidase (NA) enzyme cleaves terminal sialic acid residues from cellular receptors, a process required for the release of newly synthesized virions. A balance of NA activity with sialic acid binding affinity of hemagglutinin (HA) is important for optimal virus replication. NA sequence evolution through genetic shift and drift contributes to the continuous modulation of influenza virus fitness and pathogenicity. A simple and reliable method for the determination of kinetic parameters of NA activity could add significant value to global influenza surveillance and provide parameters for the projection of fitness and pathogenicity of emerging virus variants. The use of fluorogenic substrate 2'-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (MUNANA) and cell- or egg-grown whole influenza virus preparations have been attractive components of NA enzyme activity investigations. We describe important criteria to be addressed when determining Km and Vmax kinetic parameters using this method: (1) determination of the dynamic range of MUNANA and 4-methylumbelliferone product (4-MU) fluorescence for the instrument used; (2) adjustment of reaction conditions to approximate initial rate conditions, i.e. ≤15% of substrate converted during the reaction, with signal-to-noise ratio ≥10; (3) correction for optical interference and inner filter effect caused by increasing concentrations of MUNANA substrate. The results indicate a significant interference of MUNANA with 4-MU fluorescence determination. The criteria proposed enable an improved rapid estimation of NA kinetic parameters and facilitate comparison of data between laboratories.
PLoS ONE 08/2013; 8(8):e71401. DOI:10.1371/journal.pone.0071401 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Since the end of March 2013, avian a influenza viruses of the H7N9 subtype have caused more than 130 human cases of infection in China, many of which were severe, resulting in 43 fatalities. Although this A(H7N9) virus outbreak is now under control, the virus (or one with similar properties) could reemerge as winter approaches. To better assess the pandemic threat posed by A(H7N9) viruses, NIAID/NIH Centers of Excellence in Influenza Research and Surveillance (CEIRS) investigators and other expert laboratories in China and elsewhere have characterized the wild-type avian A(H7N9) viruses in terms of host range, virulence, and transmission, and are evaluating the effectiveness of antiviral drugs and vaccine candidates. However, to fully assess the potential risk associated with these novel viruses, there is a need for additional research including experiments that may be classified as "gain-of-function" (GOF). Here, we outline the aspects of the current situation that most urgently require additional research, our proposed studies, and risk-mitigation strategies.
[Show abstract][Hide abstract] ABSTRACT: Highly pathogenic (HP) H5N1 influenza viruses are evolving pathogens with the potential to cause sustained human-to-human transmission and pandemic virus spread. Specific antiviral drugs can play an important role in the early stages of a pandemic, but the emergence of drug-resistant variants can limit control options. The available data on the susceptibility of HP H5N1 influenza viruses to neuraminidase (NA) inhibitors and adamantanes is scarce, and there is no extensive analysis. Here, we systematically examined the prevalence of NA inhibitor and adamantane resistance among HP H5N1 influenza viruses that circulated worldwide during 2002-2012. The phenotypic fluorescence-based assay showed that both human and avian HP H5N1 viruses are susceptible to NA inhibitors oseltamivir and zanamivir with little variability over time and ∼5.5-fold less susceptibility to oseltamivir of viruses of hemagglutinin (HA) clade 2 than of clade 1. Analysis of available sequence data revealed a low incidence of NA inhibitor-resistant variants. The established markers of NA inhibitor resistance (E119A, H274Y, and N294S, N2 numbering) were found in 2.4% of human and 0.8% of avian isolates, and the markers of reduced susceptibility (I117V, K150N, I222V/T/K, and S246N) were found in 0.8% of human and 2.9% of avian isolates. The frequency of amantadine-resistant variants was higher among human (62.2%) than avian (31.6%) viruses with disproportionate distribution among different HA clades. As in human isolates, avian H5N1 viruses carry double L26I and S31N M2 mutations more often than a single S31N mutation. Overall, both human and avian HP H5N1 influenza viruses are susceptible to NA inhibitors; some proportion is still susceptible to amantadine in contrast to ∼100% amantadine resistance among currently circulating seasonal human H1N1 and H3N2 viruses. Continued antiviral susceptibility monitoring of H5N1 viruses is needed to maintain therapeutic approaches for control of disease.
Antiviral research 02/2013; 98(2). DOI:10.1016/j.antiviral.2013.02.013 · 3.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Several novel anti-influenza compounds are in various phases of clinical development. One of these, T-705, has a mechanism of action that is not fully understood but is suggested to target influenza virus RNA-dependent RNA polymerase. We investigated the mechanism of T-705 activity against influenza A (H1N1) viruses by applying selective drug pressure over multiple sequential passages in MDCK cells. We found that T-705 treatment did not select specific mutations in potential target proteins, including PB1, PB2, PA, and NP. Phenotypic assays based on cell viability confirmed that no T-705-resistant variants were selected. In the presence of T-705, titers of infectious virus decreased significantly (P<0.0001) during serial passage in MDCK cells inoculated with seasonal influenza A (H1N1) viruses at a low multiplicity of infection (MOI=0.0001 PFU/cell) or with 2009 pandemic H1N1 viruses at a high MOI (10 PFU/cell). There was no corresponding decrease in the number of viral RNA copies; therefore, specific virus infectivity (the ratio of infectious virus yield to viral RNA copy number) was reduced. Sequence analysis showed enrichment of G-to-A and C-to-T transversion mutations, increased mutation frequency, and a shift of the nucleotide profiles of individual NP gene clones under drug selection pressure. Our results demonstrate that T-705 induces a rapid rate of mutation that generates a non-viable viral phenotype and that lethal mutagenesis is a key antiviral mechanism of T-705. Our findings also explain the broad spectrum of activity of T-705 against viruses of multiple families.
Journal of Virology 01/2013; 87(7). DOI:10.1128/JVI.02346-12 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The rarely identified influenza A viruses of H15 hemagglutinin subtype have been isolated exclusively in Australia. Here we report the isolation of an H15N4 influenza A virus (A/teal/Chany/7119/2008) in Western Siberia, Russia. Phylogenetic analysis demonstrated that the internal genes of the A/teal/Chany/7119/2008 belong to the Eurasian clade, and that the H15 and N4 genes were introduced into the gene pool of circulating endemic avian influenza viruses through reassortment events.
Journal of Virology 01/2013; 87(6). DOI:10.1128/JVI.02521-12 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Please cite this paper as: Govorkova EA. (2012) Consequences of resistance: in vitro fitness, in vivo infectivity, and transmissibility of oseltamivir-resistant influenza A viruses. Influenza and Other Respiratory Viruses 7(Suppl. 1), 50-57. The development of drug resistance is a major drawback to any antiviral therapy, and the specific anti-influenza drugs, the neuraminidase (NA) inhibitors (NAIs), are not excluded from this rule. The impact of drug resistance depends on the degree of reduction in fitness of the particular drug-resistant virus. If the resistance mutations lead to only a modest biological fitness cost and the virus remains highly transmissible, the effectiveness of antiviral use is likely to be reduced. This review focuses on the fitness of oseltamivir-resistant seasonal H1N1 and H3N2, 2009 pandemic H1N1 (H1N1pdm09), and highly pathogenic H5N1 influenza A viruses carrying clinically derived NAI resistance-associated NA mutations.
Influenza and Other Respiratory Viruses 01/2013; 7 Suppl 1(s1):50-7. DOI:10.1111/irv.12044 · 2.20 Impact Factor