New insights into the neuraminidase-mediated hemagglutination activity of influenza A(H3N2) viruses.

Influenza virus neuraminidase (NA) can act as a receptor-binding protein, a role commonly attributed to hemagglutinin (HA). In influenza A(H3N2) viruses, three NA amino acid residues have previously been associated with NA-mediated hemagglutination: T148, D151, and more recently, H150. These residues are part of the 150-loop of the NA monomer. Substitutions at 148 and 151 arise from virus propagation in laboratory cell cultures, whereas changes at 150 occurred during virus evolution in the human host. In this study, we examined the effect of natural amino acid polymorphism at position 150 on NA-mediated hemagglutination. Using the A/Puerto Rico/8/34 backbone, we generated a comprehensive panel of recombinant A(H3N2) viruses that have different NAs but shared an HA that displays poor binding to red blood cells (RBCs). None of the tested substitutions at 150 (C, H, L, R, and S) promoted NA-binding. However, we identified two new determinants of NA-binding, Q136K and T439R, that emerged during virus culturing. Similar to T148I, both Q136K and T439R reduced NA enzyme activity by 48-86% and inhibition (14- to 173-fold) by the NA inhibitor zanamivir. NA-binding was observed when a virus preparation contained approximately 10% of NA variants with either T148I or T439R, highlighting the benefit of using deep sequencing in virus characterization. Taken together, our findings provide new insights into the molecular mechanisms underlying the ability of NA to function as a binding protein. Information gained may aid in the design of new and improved NA-targeting antivirals.

Resistance profiles for the investigational neuraminidase inhibitor AV5080 in influenza A and B viruses.

Neuraminidase inhibitors (NAIs) are recommended for influenza treatment and prevention worldwide. The most widely prescribed NAI is oral oseltamivir, while inhaled zanamivir is less commonly used. Using phenotypic neuraminidase (NA) enzymatic assays and molecular modeling approaches, we examined the ability of the investigational orally-dosed NAI AV5080 to inhibit viruses of the influenza A(H1N1)pdm09, A(H3N2), A(H5N1), and A(H7N9) subtypes and the influenza B/Victoria- and B/Yamagata-lineages containing NA substitutions conferring oseltamivir or zanamivir resistance including: NA-R292K, NA-E119G/V, NA-H274Y, NA-I122L/N, and NA-R150K. Broadly, AV5080 showed enhanced in vitro efficacy when compared with oseltamivir and/or zanamivir. Reduced AV5080 inhibition was determined for influenza A viruses with NA-E119G and NA-R292K, and for B/Victoria-lineage viruses with NA-I122N/L and B/Yamagata-lineage virus with NA-R150K. Molecular modeling suggested loss of the short hydrogen bond to the carboxyl group of AV5080 affected inhibition of NA-R292K viruses, whereas loss of the salt bridge with the guanidine group of AV5080 affected inhibition of NA-E119G. The resistance profiles and predicted binding modes of AV5080 and zanamivir are most similar, but dissimilar to those of oseltamivir, in part because of a guanidine moiety compensatory binding effect. Overall, our data suggests that AV5080 is a promising orally-dosed NAI that exhibited similar or superior in vitro efficacy against viruses with reduced or highly reduced inhibition phenotypes with respect to currently approved NAIs.

Antiviral susceptibility of clade 2.3.4.4b highly pathogenic avian influenza A(H5N1) viruses isolated from birds and mammals in the United States, 2022.

Clade 2.3.4.4b highly pathogenic avian influenza (HPAI) A(H5N1) viruses that are responsible for devastating outbreaks in birds and mammals pose a potential threat to public health. Here, we evaluated their susceptibility to influenza antivirals. Of 1,015 sequences of HPAI A(H5N1) viruses collected in the United States during 2022, eight viruses (∼0.8%) had a molecular marker of drug resistance to an FDA-approved antiviral: three adamantane-resistant (M2-V27A), four oseltamivir-resistant (NA-H275Y), and one baloxavir-resistant (PA-I38T). Additionally, 31 viruses contained mutations that may reduce susceptibility to inhibitors of neuraminidase (NA) (n = 20) or cap-dependent endonuclease (CEN) (n = 11). A panel of 22 representative viruses was tested phenotypically. Overall, clade 2.3.4.4b A(H5N1) viruses lacking recognized resistance mutations were susceptible to FDA-approved antivirals. Oseltamivir was least potent at inhibiting NA activity, while the investigational NA inhibitor AV5080 was most potent, including against NA mutants. A novel NA substitution T438N conferred 12-fold reduced inhibition by zanamivir, and in combination with the known marker N295S, synergistically affected susceptibility to all five NA inhibitors. In cell culture-based assays HINT and IRINA, the PA-I38T virus displayed 75- to 108-fold and 37- to 78-fold reduced susceptibility to CEN inhibitors, baloxavir and the investigational AV5116, respectively. Viruses with PA-I38M or PA-A37T showed 5- to 10-fold reduced susceptibilities. As HPAI A(H5N1) viruses continue to circulate and evolve, close monitoring of drug susceptibility is needed for risk assessment and to inform decisions regarding antiviral stockpiling.

Effect of E23G/K, F36V, N37T, E119D, and E199G polymerase acidic protein substitutions on the replication and baloxavir susceptibility of influenza B viruses.

Baloxavir marboxil (baloxavir) is a highly effective, single-dose influenza therapeutic. Identification of molecular markers in the target polymerase acidic (PA) protein that can diminish baloxavir efficacy is an ongoing goal of the scientific community. In this study, we generated recombinant Victoria-lineage and Yamagata-lineage influenza B viruses (IBVs) containing 6 substitutions (E23G/K, F36V, N37T, E119D, and E199G) spanning the endonuclease domain of the PA. Although 5 of these PA substitutions negatively impacted in vitro polymerase activity and replication kinetics, particularly in the Victoria-lineage IBV background, viruses with E119D exhibited activity levels comparable to those of wild-type viruses. Moreover, only E119D moderately decreased the susceptibility of IBVs to baloxavir (resulting in ∼2.0-fold to 2.6-fold elevated EC50s); viruses with the other substitutions exhibited normal drug inhibition. These results show that E119D may reduce the baloxavir susceptibility of IBVs without compromising their replicative fitness. Overall, this study expands the molecular landscape of PA substitutions potentially affecting baloxavir efficacy against IBV.

Influenza A virus polymerase acidic protein E23G/K substitutions weaken key baloxavir drug-binding contacts with minimal impact on replication and transmission.

Baloxavir marboxil (BXM) is approved for treating uncomplicated influenza. The active metabolite baloxavir acid (BXA) inhibits cap-dependent endonuclease activity of the influenza virus polymerase acidic protein (PA), which is necessary for viral transcription. Treatment-emergent E23G or E23K (E23G/K) PA substitutions have been implicated in reduced BXA susceptibility, but their effect on virus fitness and transmissibility, their synergism with other BXA resistance markers, and the mechanisms of resistance have been insufficiently studied. Accordingly, we generated point mutants of circulating seasonal influenza A(H1N1)pdm09 and A(H3N2) viruses carrying E23G/K substitutions. Both substitutions caused 2- to 13-fold increases in the BXA EC50. EC50s were higher with E23K than with E23G and increased dramatically (138- to 446-fold) when these substitutions were combined with PA I38T, the dominant BXA resistance marker. E23G/K-substituted viruses exhibited slightly impaired replication in MDCK and Calu-3 cells, which was more pronounced with E23K. In ferret transmission experiments, all viruses transmitted to direct-contact and airborne-transmission animals, with only E23K+I38T viruses failing to infect 100% of animals by airborne transmission. E23G/K genotypes were predominantly stable during transmission events and through five passages in vitro. Thermostable PA-BXA interactions were weakened by E23G/K substitutions and further weakened when combined with I38T. In silico modeling indicated this was caused by E23G/K altering the placement of functionally important Tyr24 in the endonuclease domain, potentially decreasing BXA binding but at some cost to the virus. These data implicate E23G/K, alone or combined with I38T, as important markers of reduced BXM susceptibility, and such mutants could emerge and/or transmit among humans.

Ancestral sequence reconstruction pinpoints adaptations that enable avian influenza virus transmission in pigs.

Understanding the evolutionary adaptations that enable avian influenza viruses to transmit in mammalian hosts could allow better detection of zoonotic viruses with pandemic potential. We applied ancestral sequence reconstruction to gain viruses representing different adaptive stages of the European avian-like (EA) H1N1 swine influenza virus as it transitioned from avian to swine hosts since 1979. Ancestral viruses representing the avian-like precursor virus and EA swine influenza viruses from 1979-1983, 1984-1987 and 1988-1992 were reconstructed and characterized. Glycan-binding analyses showed stepwise changes in the haemagglutinin receptor-binding specificity of the EA swine influenza viruses-that is, from recognition of both α2,3- and α2,6-linked sialosides to recognition of α2,6-linked sialosides only; however, efficient transmission in piglets was enabled by adaptive changes in the viral polymerase protein and nucleoprotein, which have been fixed since 1983. PB1-Q621R and NP-R351K increased viral replication and transmission in piglets when introduced into the 1979-1983 ancestral virus that lacked efficient transmissibility. The stepwise adaptation of an avian influenza virus to a mammalian host suggests that there may be opportunities to intervene and prevent interspecies jumps through strategic coordination of surveillance and risk assessment activities.

Baloxavir Treatment Delays Influenza B Virus Transmission in Ferrets and Results in Limited Generation of Drug-Resistant Variants.

Clinical efficacy of the influenza antiviral baloxavir marboxil (baloxavir) is compromised by treatment-emergent variants harboring a polymerase acidic protein I38T (isoleucine-38-threonine) substitution. However, the fitness of I38T-containing influenza B viruses (IBVs) remains inadequately defined. After the pharmacokinetics of the compound were confirmed in ferrets, animals were injected subcutaneously with 8 mg/kg of baloxavir acid (BXA) at 24 h postinoculation with recombinant BXA-sensitive (BXA-Sen, I38) or BXA-resistant (BXA-Res, I38T) B/Brisbane/60/2008 (Victoria lineage) virus. BXA treatment of donor ferrets reduced virus replication and delayed transmission of the BXA-Sen but not the BXA-Res IBV. The I38 genotype remained dominant in the BXA-Sen-infected animals, even with BXA treatment. In competitive-mixture experiments, no transmission to aerosol contacts was seen from BXA-treated donors coinfected with the BXA-Sen and BXA-Res B/Brisbane/60/2008 viruses. However, in parallel mixed infections with the B/Phuket/3073/2013 (Yamagata lineage) virus background, BXA treatment failed to block airborne transmission of the BXA-Res virus, and the I38T genotype generally predominated. Therefore, the relative fitness of BXA-Res IBVs is complex and dependent on the virus backbone and within-host virus competition. BXA treatment of single-virus-infected ferrets hampers aerosol transmission of the BXA-Sen virus and does not readily generate BXA-Res variants, whereas mixed infections may result in propagation of BXA-Res IBVs of the Yamagata lineage. Our findings confirm the antiviral potency of baloxavir against IBVs, while supporting optimization of the dosing regimen to maximize clinical benefit.

Pathogenic assessment of avian influenza viruses in migratory birds.

ABSTRACTSeveral subtypes of avian influenza (AI) viruses have caused human infections in recent years; however, there is a severe knowledge gap regarding the capacity of wild bird viruses to infect mammals. To assess the risk of mammalian infection by AI viruses from their natural reservoirs, a panel of isolates from 34 wild birds was examined in animal models. All selected AI virus subtypes were found to predominantly possess Eurasian lineage, although reassortment with North American lineage AI viruses was also noted in some isolates. When used to infect chickens, 20 AI isolates could be recovered from oropharyngeal swabs at 5 days post-infection (dpi) without causing significant morbidity. Similarly, mild to no observable disease was observed in mice infected with these viruses although the majority replicated efficiently in murine lungs. As expected, wild bird AI isolates were found to recognize avian-like receptors, while a few strains also exhibited detectable human-like receptor binding. Selected strains were further tested in ferrets, and 15 out of 20 were found to shed the virus in the upper respiratory tract until 5 dpi. Overall, we demonstrate that a diversity of low-pathogenic AI viruses carried by wild migratory birds have the capacity to infect land-based poultry and mammalian hosts while causing minimal signs of clinical disease. This study reiterates that there is a significant capacity for interspecies transmission of AI viruses harboured by wild aquatic birds. Thus, these viruses pose a significant threat to human health underscoring the need for continued surveillance.

Monoclonal Antibody Therapy Protects Pharmacologically Immunosuppressed Mice from Lethal Infection with Influenza B Virus.

Human influenza A and B viruses are highly contagious and cause similar illnesses and seasonal epidemics. Currently available antiviral drugs have limited efficacy in humans with compromised immune systems; therefore, alternative strategies for protection are needed. Here, we investigated whether monoclonal antibodies (MAbs) targeting hemagglutinin (HA) and/or neuraminidase (NA) proteins would protect immunosuppressed mice from severe infections with influenza B virus. Pharmacologically immunosuppressed BALB/c mice were inoculated with B/Brisbane/60/2008 (BR/08) influenza virus and were treated with a single dose of 1, 5, or 25 mg/kg of body weight per day of either an anti-HA MAb (1D2) or an anti-NA MAb (1F2) starting at 24 hours postinoculation (hpi). Monotherapy with 1D2 or 1F2 MAbs provided dose-dependent protection of mice, with decreased BR/08 virus replication and spread in the mouse lungs, compared with those of controls. Combination treatment with 1D2 and 1F2 provided greater protection than did monotherapy, even when started at 48 hpi. Virus spread was also efficiently restrained within the lungs, being limited to 6%, 10%, and 10% of that seen in active infection when treatment was initiated at 24, 48, and 72 hpi, respectively. In most cases, the expression of cytokines and chemokines was altered according to when treatment was initiated. Higher expression of proinflammatory IP-10 and MCP-1 in combination-treatment groups, but not in monotherapy groups, to some extent, promoted better control of virus spread within the lungs. This study demonstrates the potential value of MAb immunotherapy in treating influenza in immunocompromised hosts who are at increased risk of severe disease.

Influenza A and B viruses with reduced baloxavir susceptibility display attenuated in vitro fitness but retain ferret transmissibility.

Baloxavir marboxil (BXM) was approved in 2018 for treating influenza A and B virus infections. It is a first-in-class inhibitor targeting the endonuclease activity of the virus polymerase acidic (PA) protein. Clinical trial data revealed that PA amino acid substitutions at residue 38 (I38T/F/M) reduced BXM potency and caused virus rebound in treated patients, although the fitness characteristics of the mutant viruses were not fully defined. To determine the fitness impact of the I38T/F/M substitutions, we generated recombinant A/California/04/2009 (H1N1)pdm09, A/Texas/71/2017 (H3N2), and B/Brisbane/60/2008 viruses with I38T/F/M and examined drug susceptibility in vitro, enzymatic properties, replication efficiency, and transmissibility in ferrets. Influenza viruses with I38T/F/M substitutions exhibited reduced baloxavir susceptibility, with 38T causing the greatest reduction. The I38T/F/M substitutions impaired PA endonuclease activity as compared to that of wild-type (I38-WT) PA. However, only 38T/F A(H3N2) substitutions had a negative effect on polymerase complex activity. The 38T/F substitutions decreased replication in cells among all viruses, whereas 38M had minimal impact. Despite variable fitness consequences in vitro, all 38T/M viruses disseminated to naive ferrets by contact and airborne transmission, while 38F-containing A(H3N2) and B viruses failed to transmit via the airborne route. Reversion of 38T/F/M to I38-WT was rare among influenza A viruses in this study, suggesting stable retention of 38T/F/M genotypes during these transmission events. BXM reduced susceptibility-associated mutations had variable effects on in vitro fitness of influenza A and B viruses, but the ability of these viruses to transmit in vivo indicates a risk of their spreading from BXM-treated individuals.

Influenza B viruses from different genetic backgrounds are variably impaired by neuraminidase inhibitor resistance-associated substitutions.

Identifying evolutionary routes to antiviral resistance among influenza viruses informs molecular-based resistance surveillance and clinical decisions. To improve antiviral management and understand whether clinically identified neuraminidase (NA) inhibitor (NAI) resistance-associated markers affect influenza B viruses of the Victoria- or Yamagata-lineages differentially, we generated a panel of NAI-resistant viruses (carrying E105K, G145E, R150K, D197N, I221 L/N/T/V, H273Y, N294S, or G407S substitutions; B numbering) in B/Brisbane/60/2008 (BR/08) and B/Phuket/3073/2013 (PH/13). In both backgrounds, I221 L/N/T/V resulted in reduced or highly reduced inhibition (HRI) by one to three currently available NAIs. D197N reduced inhibition by all NAIs in BR/08 but only by oseltamivir and peramivir in PH/13; R150K caused HRI by all NAIs in PH/13. Although PH/13 generally retained or enhanced NA activity in the presence of the substitutions, enzymatic activity in BR/08 was detrimentally affected. Similarly, substrate affinity and catalysis were relatively stable in PH/13, but not in the BR/08 variants. E105K, R150K, and D197N attenuated replication efficiency of BR/08 in vitro and in mice; only E105K had this effect in PH/13. Notably, the I221 L/N/T/V substitutions did not severely impair replication, particularly in PH/13. Overall, our data show differential effects of NA substitutions in representative Victoria- and Yamagata-lineage viruses, suggesting distinct evolution of these viruses caused variable fitness and NAI susceptibility profiles when similar key NA substitutions arise. Because the viruses harboring the I221 NA substitutions displayed undiminished fitness and are commonly reported, this position is likely to be the most clinically relevant marker for NAI resistance among contemporary influenza B viruses.

A Novel Neuraminidase-Dependent Hemagglutinin Cleavage Mechanism Enables the Systemic Spread of an H7N6 Avian Influenza Virus.

In this study, we demonstrate a novel mechanism for hemagglutinin (HA) activation in a naturally occurring H7N6 avian influenza A virus strain, A/mallard duck/Korea/6L/2007 (A/Mdk/6L/07). This novel mechanism allows for systemic infection of chickens, ducks, and mice, and A/Mdk/6L/07 can replicate in vitro without exogenous trypsin and exhibits broad tissue tropism in animals despite the presence of a monobasic HA cleavage motif (PEIPKGR/G). The trypsin-independent growth phenotype requires the N6 neuraminidase and the specific recognition of glycine at the P2 position of the HA cleavage motif by a thrombin-like protease. Correspondingly, viral growth is significantly attenuated by the addition of a thrombin-like protease inhibitor (argatroban). These data provide evidence for a previously unrecognized virus replication mechanism and support the hypothesis that thrombin-mediated HA cleavage is an important virulence marker and potential therapeutic target for H7 influenza viruses.IMPORTANCE The identification of virulence markers in influenza viruses underpins risk assessment programs and the development of novel therapeutics. The cleavage of the influenza virus HA is a required step in the viral life cycle, and phenotypic differences in viruses can be caused by changes in this process. Here, we describe a novel mechanism for HA cleavage in an H7N6 influenza virus isolated from a mallard duck. The mechanism requires the N6 protein and full activity of thrombin-like proteases and allows the virus to cause systemic infection in chickens, ducks, and mice. The thrombin-mediated cleavage of HA is thus a novel virulence determinant of avian influenza viruses.

Optimizing T-705 (favipiravir) treatment of severe influenza B virus infection in the immunocompromised mouse model.

BACKGROUND: Influenza B virus infections remain insufficiently studied and antiviral management in immunocompromised patients is not well defined. The treatment regimens for these high-risk patients, which have elevated risk of severe disease-associated complications, require optimization and can be partly addressed via animal models. METHODS: We examined the efficacy of monotherapy with the RNA-dependent RNA polymerase inhibitor T-705 (favipiravir) in protecting genetically modified, permanently immunocompromised BALB scid mice against lethal infection with B/Brisbane/60/2008 (BR/08) virus. Beginning at 24 h post-infection, BALB scid mice received oral T-705 twice daily (10, 50 or 250 mg/kg/day) for 5 or 10 days. RESULTS: T-705 had a dose-dependent effect on survival after BR/08 challenge, resulting in 100% protection at the highest dosages. With the 5 day regimens, dosages of 50 or 250 mg/kg/day reduced the peak lung viral titres within the treatment window, but could not efficiently clear the virus after completion of treatment. With the 10 day regimens, dosages of 50 or 250 mg/kg/day significantly suppressed virus replication in the lungs, particularly at 45 days post-infection, limiting viral spread and pulmonary pathology. No T-705 regimen decreased virus growth in the nasal turbinates of mice, which potentially contributed to the viral dynamics in the lungs. The susceptibility of influenza B viruses isolated from T-705-treated mice remained comparable to that of viruses from untreated control animals. CONCLUSIONS: T-705 treatment is efficacious against lethal challenge with BR/08 virus in immunocompromised mice. The antiviral benefit was greatest when longer T-705 treatment was combined with higher dosages.

A pharmacologically immunosuppressed mouse model for assessing influenza B virus pathogenicity and oseltamivir treatment.

Immunocompromised patients are highly susceptible to influenza virus infections. Although neuraminidase inhibitor (NAI) therapy has proved effective in these patients, the treatment regimens require optimization, which can be partly addressed via animal models. Here, we describe a pharmacologically immunosuppressed mouse model for studying the pathogenesis of influenza B viruses and evaluating the efficacy of antiviral treatment. We modeled clinical regimens for dexamethasone and cyclophosphamide to immunosuppress BALB/c mice that were then inoculated with B/Phuket/3073/2013 (Yamagata lineage) or B/Brisbane/60/2008 (BR/08, Victoria lineage) virus. Although both viruses caused morbidity and mortality in immunosuppressed mice, BR/08 was more virulent, consistently inducing greater morbidity and 100% lethality in mice inoculated with at least 103 TCID50/mouse. The replication of both viruses was prolonged in the lungs of immunosuppressed mice, but the extent of pulmonary inflammation in these mice was markedly less than that in immunocompetent animals. Most of the examined cytokines, including IFN-γ, IL-1ß, and RANTES, were significantly decreased in the lungs of immunosuppressed mice, as compared to immunocompetent animals, until at least 10 days post-infection. Treatment with the NAI oseltamivir for 8 or 16 days increased the mean survival time and reduced virus spread in the lungs of immunosuppressed mice challenged with a lethal dose of BR/08 but did not completely provide protection or decrease the virus titers. Our data suggests that the synergy of the viral load and aberrant immune responses is a key contributor to the severity of infection, as well as the limited efficacy of oseltamivir, which in immunosuppressed mice curtails virus release without clearing infected cells.

Pathogenicity and peramivir efficacy in immunocompromised murine models of influenza B virus infection.

Influenza B viruses are important human pathogens that remain inadequately studied, largely because available animal models are poorly defined. Here, we developed an immunocompromised murine models for influenza B virus infection, which we subsequently used to study pathogenicity and to examine antiviral efficacy of the neuraminidase inhibitor peramivir. We studied three influenza B viruses that represent both the Yamagata (B/Massachusetts/2/2012 and B/Phuket/3073/2013) and Victoria (B/Brisbane/60/2008, BR/08) lineages. BR/08 was the most pathogenic in genetically modified immunocompromised mice [BALB scid and non-obese diabetic (NOD) scid strains] causing lethal infection without prior adaptation. The immunocompromised mice demonstrated prolonged virus shedding with modest induction of immune responses compared to BALB/c. Rather than severe virus burden, BR/08 virus-associated disease severity correlated with extensive virus spread and severe pulmonary pathology, stronger and persistent natural killer cell responses, and the extended induction of pro-inflammatory cytokines and chemokines. In contrast to a single-dose treatment (75 mg/kg/day), repeated doses of peramivir rescued BALB scid mice from lethal challenge with BR/08, but did not result in complete virus clearance. In summary, we have established immunocompromised murine models for influenza B virus infection that will facilitate evaluations of the efficacy of currently available and investigational anti-influenza drugs.

Avian Influenza A Viruses: Evolution and Zoonotic Infection.

Although efficient human-to-human transmission of avian influenza virus has yet to be seen, in the past two decades avian-to-human transmission of influenza A viruses has been reported. Influenza A/H5N1, in particular, has repeatedly caused human infections associated with high mortality, and since 1998 the virus has evolved into many clades of variants with significant antigenic diversity. In 2013, three (A/H7N9, A/H6N1, and A/H10N8) novel avian influenza viruses (AIVs) breached the animal-human host species barrier in Asia. In humans, roughly 35% of A/H7N9-infected patients succumbed to the zoonotic infection, and two of three A/H10N8 human infections were also lethal; however, neither of these viruses cause influenza-like symptoms in poultry. While most of these cases were associated with direct contact with infected poultry, some involved sustained human-to-human transmission. Thus, these events elicited concern regarding potential AIV pandemics. This article reviews the human incursions associated with AIV variants and the potential role of pigs as an intermediate host that may hasten AIV evolution. In addition, we discuss the known influenza A virus virulence and transmission factors and their evaluation in animal models. With the growing number of human AIV infections, constant vigilance for the emergence of novel viruses is of utmost importance. In addition, careful characterization and pathobiological assessment of these novel variants will help to identify strains of particular concern for future pandemics.

Competitive Fitness of Influenza B Viruses Possessing E119A and H274Y Neuraminidase Inhibitor Resistance-Associated Substitutions in Ferrets.

Neuraminidase (NA) inhibitors (NAIs) are the only antiviral drugs recommended for influenza treatment and prophylaxis. Although NAI-resistant influenza B viruses that could pose a threat to public health have been reported in the field, their fitness is poorly understood. We evaluated in ferrets the pathogenicity and relative fitness of reverse genetics (rg)-generated influenza B/Yamanashi/166/1998-like viruses containing E119A or H274Y NA substitutions (N2 numbering). Ferrets inoculated with NAI-susceptible rg-wild-type (rg-WT) or NAI-resistant (rg-E119A or rg-H274Y) viruses developed mild infections. Growth of rg-E119A virus in the nasal cavities was delayed, but the high titers at 3 days post-inoculation (dpi) were comparable to those of the rg-WT and rg-H274Y viruses (3.6-4.1 log10TCID50/mL). No virus persisted beyond 5 dpi and replication did not extend to the trachea or lungs. Positive virus antigen-staining of the nasal turbinate epithelium was intermittent with the rg-WT and rg-H274Y viruses; whereas antigen-staining for the rg-E119A virus was more diffuse. Virus populations in ferrets coinoculated with NAI-susceptible and -resistant viruses (1:1 mixture) remained heterogeneous at 5 dpi but were predominantly rg-WT (>70%). Although the E119A substitution was associated with delayed replication in ferrets, the H274Y substitution did not measurably affect viral growth properties. These data suggest that rg-H274Y has undiminished fitness in single virus inoculations, but neither rg-E119A nor rg-H274Y gained a fitness advantage over rg-WT in direct competition experiments without antiviral drug pressure. Taken together, our data suggest the following order of relative fitness in a ferret animal model: rg-WT > rg-H274Y > rg-E119A.

A Novel Endonuclease Inhibitor Exhibits Broad-Spectrum Anti-Influenza Virus Activity In Vitro.

Antiviral drugs are important in preventing and controlling influenza, particularly when vaccines are ineffective or unavailable. A single class of antiviral drugs, the neuraminidase inhibitors (NAIs), is recommended for treating influenza. The limited therapeutic options and the potential risk of antiviral resistance are driving the search for additional small-molecule inhibitors that act on influenza virus proteins. The acid polymerase (PA) of influenza viruses is a promising target for new antivirals because of its essential role in initiating virus transcription. Here, we characterized a novel compound, RO-7, identified as a putative PA endonuclease inhibitor. RO-7 was effective when added before the cessation of genome replication, reduced polymerase activity in cell-free systems, and decreased relative amounts of viral mRNA and genomic RNA during influenza virus infection. RO-7 specifically inhibited the ability of the PA endonuclease domain to cleave a nucleic acid substrate. RO-7 also inhibited influenza A viruses (seasonal and 2009 pandemic H1N1 and seasonal H3N2) and B viruses (Yamagata and Victoria lineages), zoonotic viruses (H5N1, H7N9, and H9N2), and NAI-resistant variants in plaque reduction, yield reduction, and cell viability assays in Madin-Darby canine kidney (MDCK) cells with nanomolar to submicromolar 50% effective concentrations (EC50s), low toxicity, and favorable selective indices. RO-7 also inhibited influenza virus replication in primary normal human bronchial epithelial cells. Overall, RO-7 exhibits broad-spectrum activity against influenza A and B viruses in multiple in vitro assays, supporting its further characterization and development as a potential antiviral agent for treating influenza.

Growth and Pathogenic Potential of Naturally Selected Reassortants after Coinfection with Pandemic H1N1 and Highly Pathogenic Avian Influenza H5N1 Viruses.

Coinfection of ferrets with H5N1 and pH1N1 viruses resulted in two predominate genotypes in the lungs containing surface genes of highly pathogenic avian influenza H5N1 virus in the backbone of pandemic H1N1 2009 (pH1N1). Compared to parental strains, these reassortants exhibited increased growth and virulence in vitro and in mice but failed to be transmitted indirectly to naive contact ferrets. Thus, this demonstrates a possible natural reassortment following coinfection as well as the pathogenicity of the potential reassortants.

Mouse adaptation of influenza B virus increases replication in the upper respiratory tract and results in droplet transmissibility in ferrets.

To investigate the molecular changes that allow influenza B viruses to adapt to new mammalian hosts, influenza B/Florida/04/2006 was serially passaged in BALB/c mice until highly virulent. The viral factors underlying this transition were then investigated in mice and ferrets. Five viruses, including the wild-type virus (P0), three intermediate viruses (P5, P9, and P12), and a lethal mouse-adapted virus (P17 (MA)), harbored one to five amino acid substitutions in the hemagglutinin, M, NP, and PA segments suggesting that these mutations enhance virulence. The P17 (MA) virus replicated significantly more efficiently than the P0 virus both in vitro and in vivo (P < 0.0001), and was highly virulent (MLD50: 10(5.25)TCID50) while the P0, P5, and P9 viruses did not kill any infected mice (MLD50 > 10(6.0)TCID50). Furthermore, the P17 (MA) virus grew to greater titers in the ferret upper respiratory tract compared with the P0 and intermediate viruses, and only the P17 (MA) virus was transmissible between ferrets via both direct and aerosol contact. To our knowledge, this is the first study to demonstrate ferret-to-ferret transmission of influenza B virus and to delineate factors that may affect its transmission.