PB2 residue 473 contributes to the mammalian virulence of H7N9 avian influenza virus by modulating viral polymerase activity via ANP32A.

Since the first human infection reported in 2013, H7N9 avian influenza virus (AIV) has been regarded as a serious threat to human health. In this study, we sought to identify the virulence determinant of the H7N9 virus in mammalian hosts. By comparing the virulence of the SH/4664 H7N9 virus, a non-virulent H9N2 virus, and various H7N9-H9N2 hybrid viruses in infected mice, we first pinpointed PB2 as the primary viral factor accounting for the difference between H7N9 and H9N2 in mammalian virulence. We further analyzed the in vivo effects of individually mutating H7N9 PB2 residues different from the closely related H9N2 virus and consequently found residue 473, alongside the well-known residue 627, to be critical for the virulence of the H7N9 virus in mice and the activity of its reconstituted viral polymerase in mammalian cells. The importance of PB2-473 was further strengthened by studying reverse H7N9 substitutions in the H9N2 background. Finally, we surprisingly found that species-specific usage of ANP32A, a family member of host factors connecting with the PB2-627 polymorphism, mediates the contribution of PB2 473 residue to the mammalian adaption of AIV polymerase, as the attenuating effect of PB2 M473T on the viral polymerase activity and viral growth of the H7N9 virus could be efficiently complemented by co-expression of chicken ANP32A but not mouse ANP32A and ANP32B. Together, our studies uncovered the PB2 473 residue as a novel viral host range determinant of AIVs via species-specific co-opting of the ANP32 host factor to support viral polymerase activity.IMPORTANCEThe H7N9 avian influenza virus has been considered to have the potential to cause the next pandemic since the first case of human infection reported in 2013. In this study, we identified PB2 residue 473 as a new determinant of mouse virulence and mammalian adaptation of the viral polymerase of the H7N9 virus and its non-pathogenic H9N2 counterparts. We further demonstrated that the variation in PB2-473 is functionally linked to differential co-opting of the host ANP32A protein in supporting viral polymerase activity, which is analogous to the well-known PB2-627 polymorphism, albeit the two PB2 positions are spatially distant. By providing new mechanistic insight into the PB2-mediated host range determination of influenza A viruses, our study implicated the potential existence of multiple PB2-ANP32 interfaces that could be targets for developing new antivirals against the H7N9 virus as well as other mammalian-adapted influenza viruses.

Differential interferon responses to influenza A and B viruses in primary ferret respiratory epithelial cells.

Influenza B viruses (IBV) cocirculate with influenza A viruses (IAV) and cause periodic epidemics of disease, yet antibody and cellular responses following IBV infection are less well understood. Using the ferret model for antisera generation for influenza surveillance purposes, IAV resulted in robust antibody responses following infection, whereas IBV required an additional booster dose, over 85% of the time, to generate equivalent antibody titers. In this study, we utilized primary differentiated ferret nasal epithelial cells (FNECs) which were inoculated with IAV and IBV to study differences in innate immune responses which may result in differences in adaptive immune responses in the host. FNECs were inoculated with IAV (H1N1pdm09 and H3N2 subtypes) or IBV (B/Victoria and B/Yamagata lineages) and assessed for 72 h. Cells were analyzed for gene expression by quantitative real-time PCR, and apical and basolateral supernatants were assessed for virus kinetics and interferon (IFN), respectively. Similar virus kinetics were observed with IAV and IBV in FNECs. A comparison of gene expression and protein secretion profiles demonstrated that IBV-inoculated FNEC expressed delayed type-I/II IFN responses and reduced type-III IFN secretion compared to IAV-inoculated cells. Concurrently, gene expression of Thymic Stromal Lymphopoietin (TSLP), a type-III IFN-induced gene that enhances adaptive immune responses, was significantly downregulated in IBV-inoculated FNECs. Significant differences in other proinflammatory and adaptive genes were suppressed and delayed following IBV inoculation. Following IBV infection, ex vivo cell cultures derived from the ferret upper respiratory tract exhibited reduced and delayed innate responses which may contribute to reduced antibody responses in vivo.IMPORTANCEInfluenza B viruses (IBV) represent nearly one-quarter of all human influenza cases and are responsible for significant clinical and socioeconomic impacts but do not pose the same pandemic risks as influenza A viruses (IAV) and have thus received much less attention. IBV accounts for greater severity and deaths in children, and vaccine efficacy remains low. The ferret can be readily infected with human clinical isolates and demonstrates a similar course of disease and immune responses. IBV, however, generates lower antibodies in ferrets than IAV following the challenge. To determine whether differences in initial innate responses following infection may affect the development of robust adaptive immune responses, ferret respiratory tract cells were isolated, infected with IAV/IBV, and compared. Understanding the differences in the initial innate immune responses to IAV and IBV may be important in the development of more effective vaccines and interventions to generate more robust protective immune responses.

Antibody inhibition of influenza A virus assembly and release.

Antibodies are frontline defenders against influenza virus infection, providing protection through multiple complementary mechanisms. Although a subset of monoclonal antibodies (mAbs) has been shown to restrict replication at the level of virus assembly and release, it remains unclear how potent and pervasive this mechanism of protection is, due in part to the challenge of separating this effect from other aspects of antibody function. To address this question, we developed imaging-based assays to determine how effectively a broad range of mAbs against the IAV surface proteins can specifically restrict viral egress. We find that classically neutralizing antibodies against hemagglutinin are broadly multifunctional, inhibiting virus assembly and release at concentrations 1-20-fold higher than the concentrations at which they inhibit viral entry. These antibodies are also capable of altering the morphological features of shed virions, reducing the proportion of filamentous particles. We find that antibodies against neuraminidase and M2 also restrict viral egress and that inhibition by anti-neuraminidase mAbs is only partly attributable to a loss in enzymatic activity. In all cases, antigen crosslinking-either on the surface of the infected cell, between the viral and cell membrane, or both-plays a critical role in inhibition, and we are able to distinguish between these modes experimentally and through a structure-based computational model. Together, these results provide a framework for dissecting antibody multifunctionality that could help guide the development of improved therapeutic antibodies or vaccines and that can be extended to other viral families and antibody isotypes.IMPORTANCEAntibodies against influenza A virus provide multifaceted protection against infection. Although sensitive and quantitative assays are widely used to measure inhibition of viral attachment and entry, the ability of diverse antibodies to inhibit viral egress is less clear. We address this challenge by developing an imaging-based approach to measure antibody inhibition of virus release across a panel of monoclonal antibodies targeting the influenza A virus surface proteins. Using this approach, we find that inhibition of viral egress is common and can have similar potency to the ability of an antibody to inhibit viral entry. Insights into this understudied aspect of antibody function may help guide the development of improved countermeasures.

Exploring associations between viral titer measurements and disease outcomes in ferrets inoculated with 125 contemporary influenza A viruses.

As use of the ferret model to study influenza A virus (IAV) pathogenicity increases, periodic assessment of data generated in this model is warranted, to identify features associated with virus replication throughout the respiratory tract and to refine future analyses. However, protocol-specific differences present between independent laboratories limit easy aggregation of virological data. We compiled viral titer and clinical data from >1,000 ferrets inoculated with 125 contemporary IAV under a consistent experimental protocol (including high- and low-pathogenicity avian, swine-origin, and human viruses, spanning H1, H2, H3, H5, H7, and H9 subtypes) and examined which meaningful and statistically supported associations were present among numerous quantitative measurements. Viral titers correlated positively between ferret nasal turbinate tissue, lung tissue, and nasal wash specimens, though the strength of the associations varied, notably regarding the particular nasal wash summary measure employed and properties of the virus itself. Use of correlation coefficients and mediation analyses further supported the interconnectedness of viral titer measurements taken at different sites throughout the respiratory tract. IAV possessing mammalian host adaptation markers in the HA and PB2 exhibited more rapid growth in the ferret upper respiratory tract early after infection, supported by quantities derived from infectious titer data to capture infection progression, compared with viruses bearing hallmarks of avian IAV. Collectively, this work identifies summary metrics most closely linked with virological and phenotypic outcomes in ferrets, supporting continued refinement of data analyzed from in vivo experimentation, notably from studies conducted to evaluate the public health risk posed by novel and emerging IAV.IMPORTANCEFerrets are frequently employed to study the pandemic potential of novel and emerging influenza A viruses. However, systematic retrospective analyses of data generated from these experiments are rarely performed, limiting our ability to identify trends in this data and explore how analyses can be refined. Using logarithmic viral titer and clinical data aggregated from one research group over 20 years, we assessed which meaningful and statistically supported associations were present among numerous quantitative measurements obtained from influenza A virus (IAV)-infected ferrets, including those capturing viral titers, infection progression, and disease severity. We identified numerous linear correlations between parameters assessing virus replication at discrete sites in vivo, including parameters capturing infection progression not frequently employed in the field, and sought to investigate the interconnected nature of these associations. This work supports continued refinement of data analyzed from in vivo experimentation, notably from studies which evaluate the public health risk posed by IAV.

VarEPS-Influ:an risk evaluation system of occurred and virtual variations of influenza virus genomes.

Influenza viruses undergo frequent genomic mutations, leading to potential cross-species transmission, phenotypic changes, and challenges in diagnostic reagents and vaccines. Accurately evaluating and predicting the risk of such variations remain significant challenges. To address this, we developed the VarEPS-Influ database, an influenza virus variations risk evaluation system (VarEPS-Influ). This database employs a 'multi-dimensional evaluation of mutations' strategy, utilizing various tools to assess the physical and chemical properties, primary, secondary, and tertiary structures, receptor affinity, antibody binding capacity, antigen epitopes, and other aspects of the variation's impact. Additionally, we consider space-time distribution, host species distribution, pedigree analysis, and frequency of mutations to provide a comprehensive risk evaluation of mutations and viruses. The VarEPS-Influ database evaluates both observed variations and virtual variations (variations that have not yet occurred), thereby addressing the time-lag issue in risk predictions. Our current one-stop evaluation system for influenza virus genomic variation integrates 1065290 sequences from 224 927 Influenza A, B and C isolates retrieved from public resources. Researchers can freely access the data at https://nmdc.cn/influvar/.

Palmitoylation of the hemagglutinin of influenza B virus by ER-localized DHHC enzymes 1, 2, 4, and 6 is required for efficient virus replication.

IMPORTANCE: Influenza viruses are a public health concern since they cause seasonal outbreaks and occasionally pandemics. Our study investigates the importance of a protein modification called "palmitoylation" in the replication of influenza B virus. Palmitoylation involves attaching fatty acids to the viral protein hemagglutinin and has previously been studied for influenza A virus. We found that this modification is important for the influenza B virus to replicate, as mutating the sites where palmitate is attached prevented the virus from generating viable particles. Our experiments also showed that this modification occurs in the endoplasmic reticulum. We identified the specific enzymes responsible for this modification, which are different from those involved in palmitoylation of HA of influenza A virus. Overall, our research illuminates the similarities and differences in fatty acid attachment to HA of influenza A and B viruses and identifies the responsible enzymes, which might be promising targets for anti-viral therapy.

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.

Vigilance Urged Against Bird Flu Amid Ongoing Outbreaks in Mammals.

This Medical News article discusses the risk to humans from the currently circulating highly pathogenic avian influenza A(H5N1) virus.

Impact of Baloxavir Resistance-Associated Substitutions on Influenza Virus Growth and Drug Susceptibility.

Baloxavir marboxil (baloxavir) is a recently FDA-approved influenza virus polymerase acidic (PA) endonuclease inhibitor. Several PA substitutions have been demonstrated to confer reduced susceptibility to baloxavir; however, their impacts on measurements of antiviral drug susceptibility and replication capacity when present as a fraction of the viral population have not been established. We generated recombinant A/California/04/09 (H1N1)-like viruses (IAV) with PA I38L, I38T, or E199D substitutions and B/Victoria/504/2000-like virus (IBV) with PA I38T. These substitutions reduced baloxavir susceptibility by 15.3-, 72.3-, 5.4-, and 54.5-fold, respectively, when tested in normal human bronchial epithelial (NHBE) cells. We then assessed the replication kinetics, polymerase activity, and baloxavir susceptibility of the wild-type:mutant (WT:MUT) virus mixtures in NHBE cells. The percentage of MUT relative to WT virus necessary to detect reduced baloxavir susceptibility in phenotypic assays ranged from 10% (IBV I38T) to 92% (IAV E199D). While I38T did not alter IAV replication kinetics or polymerase activity, IAV PA I38L and E199D MUTs and the IBV PA I38T MUT exhibited reduced replication levels and significantly altered polymerase activity. Differences in replication were detectable when the MUTs comprised ≥90%, ≥90%, or ≥75% of the population, respectively. Droplet digital PCR (ddPCR) and next-generation sequencing (NGS) analyses showed that WT viruses generally outcompeted the respective MUTs after multiple replication cycles and serial passaging in NHBE cells when initial mixtures contained ≥50% of the WT viruses; however, we also identified potential compensatory substitutions (IAV PA D394N and IBV PA E329G) that emerged and appeared to improve the replication capacity of baloxavir-resistant virus in cell culture. IMPORTANCE Baloxavir marboxil, an influenza virus polymerase acidic endonuclease inhibitor, represents a recently approved new class of influenza antivirals. Treatment-emergent resistance to baloxavir has been observed in clinical trials, and the potential spread of resistant variants could diminish baloxavir effectiveness. Here, we report the impact of the proportion of drug-resistant subpopulations on the ability to detect resistance in clinical isolates and the impact of substitutions on viral replication of mixtures containing both drug-sensitive and drug-resistant variants. We also show that ddPCR and NGS methods can be successfully used for detection of resistant subpopulations in clinical isolates and to quantify their relative abundance. Taken together, our data shed light on the potential impact of baloxavir-resistant I38T/L and E199D substitutions on baloxavir susceptibility and other biological properties of influenza virus and the ability to detect resistance in phenotypic and genotypic assays.

Bioinformatics and Structural Analysis of Antigenic Variation in the Hemagglutinin Gene of the Influenza A(H1N1)pdm09 Virus Circulating in Shiraz (2013 to 2015).

Circulating influenza A virus provided an excellent opportunity to study the adaptation of the influenza A(H1N1)pdm09 virus to the human host. Particularly, due to the availability of sequences taken from isolates, we could monitor amino acid changes and the stability of mutations that occurred in hemagglutinin (HA). HA is crucial to viral infection because it binds to ciliated cell receptors and mediates the fusion of cells and viral membranes; because antibodies that bind to HA may block virus entry to the cell, this protein is subjected to high selective pressure. In this study, the locations of mutations in the structures of mutant HA were analyzed and the three-dimensional (3D) structures of these mutations were modeled in I-TASSER. Also, the location of these mutations was visualized and studied using Swiss PDB Viewer software and the PyMOL Molecular Graphics System. The crystal structure of the HA from A/California/07/2009 (3LZG) was used for further analysis. The new noncovalent bond formations in mutant luciferases were analyzed via WHAT IF and PIC, and protein stability was evaluated in the iStable server. We identified 33 and 23 mutations in A/Shiraz/106/2015 and A/California/07/2009 isolates, respectively; some mutations are located on the antigenic sites of Sa, Sb, Ca1, Ca2, and Cb HA1 and the fusion peptide of HA2. The results show that with the mutation some interactions are lost and new interactions are formed with other amino acids. The results of the free-energy analysis suggested that these new interactions have a destabilizing effect, which needs confirmation experimentally. IMPORTANCE Due to the fact that the mutations that occurred in the influenza virus HA cause the instability of the protein produced by the virus and antigenic changes and the escape of the virus from the immune system, the mutations that occurred in A/Shiraz/1/2013 were investigated in terms of energy level and stability. The mutations located in a globular portion of the HA are S188T, Q191H, S270P, K285Q, and P299L. On the other hand, the E374K, E46K-B, S124N-B, and I321V mutations are located in the stem portion of the HA (HA2). The change V252L mutation eliminates interactions with Ala181, Phe147, Leu151, and Trp153 and forms new interactions with Gly195, Asn264, Phe161, Met244, Tyr246, Leu165, and Trp167 which can change the stability of the HA structure. The K166Q mutation, which is located within the antigenic site Sa, causes the virus to escape from the immune response.

BTN3A3 evasion promotes the zoonotic potential of influenza A viruses.

Spillover events of avian influenza A viruses (IAVs) to humans could represent the first step in a future pandemic1. Several factors that limit the transmission and replication of avian IAVs in mammals have been identified. There are several gaps in our understanding to predict which virus lineages are more likely to cross the species barrier and cause disease in humans1. Here, we identified human BTN3A3 (butyrophilin subfamily 3 member A3)2 as a potent inhibitor of avian IAVs but not human IAVs. We determined that BTN3A3 is expressed in human airways and its antiviral activity evolved in primates. We show that BTN3A3 restriction acts primarily at the early stages of the virus life cycle by inhibiting avian IAV RNA replication. We identified residue 313 in the viral nucleoprotein (NP) as the genetic determinant of BTN3A3 sensitivity (313F or, rarely, 313L in avian viruses) or evasion (313Y or 313V in human viruses). However, avian IAV serotypes, such as H7 and H9, that spilled over into humans also evade BTN3A3 restriction. In these cases, BTN3A3 evasion is due to substitutions (N, H or Q) in NP residue 52 that is adjacent to residue 313 in the NP structure3. Thus, sensitivity or resistance to BTN3A3 is another factor to consider in the risk assessment of the zoonotic potential of avian influenza viruses.

Sequential disruption of SPLASH-identified vRNA-vRNA interactions challenges their role in influenza A virus genome packaging.

A fundamental step in the influenza A virus (IAV) replication cycle is the coordinated packaging of eight distinct genomic RNA segments (i.e. vRNAs) into a viral particle. Although this process is thought to be controlled by specific vRNA-vRNA interactions between the genome segments, few functional interactions have been validated. Recently, a large number of potentially functional vRNA-vRNA interactions have been detected in purified virions using the RNA interactome capture method SPLASH. However, their functional significance in coordinated genome packaging remains largely unclear. Here, we show by systematic mutational analysis that mutant A/SC35M (H7N7) viruses lacking several prominent SPLASH-identified vRNA-vRNA interactions involving the HA segment package the eight genome segments as efficiently as the wild-type virus. We therefore propose that the vRNA-vRNA interactions identified by SPLASH in IAV particles are not necessarily critical for the genome packaging process, leaving the underlying molecular mechanism elusive.

From bad to worse.

How the avian flu must change before it can trigger a human pandemic.