Epistasis mediates the evolution of the receptor binding mode in recent human H3N2 hemagglutinin.

The receptor-binding site of influenza A virus hemagglutinin partially overlaps with major antigenic sites and constantly evolves. In this study, we observe that mutations G186D and D190N in the hemagglutinin receptor-binding site have coevolved in two recent human H3N2 clades. X-ray crystallography results show that these mutations coordinately drive the evolution of the hemagglutinin receptor binding mode. Epistasis between G186D and D190N is further demonstrated by glycan binding and thermostability analyses. Immunization and neutralization experiments using mouse and human samples indicate that the evolution of receptor binding mode is accompanied by a change in antigenicity. Besides, combinatorial mutagenesis reveals that G186D and D190N, along with other natural mutations in recent H3N2 strains, alter the compatibility with a common egg-adaptive mutation in seasonal influenza vaccines. Overall, our findings elucidate the role of epistasis in shaping the recent evolution of human H3N2 hemagglutinin and substantiate the high evolvability of its receptor-binding mode.

Exploration of influenza A virus PA protein-associated cellular proteins discloses its impact on mitochondrial function.

Influenza A virus can infect respiratory tracts and may cause severe illness in humans. Proteins encoded by influenza A virus can interact with cellular factors and dysregulate host biological processes to support viral replication and cause pathogenicity. The influenza viral PA protein is not only a subunit of influenza viral polymerase but also a virulence factor involved in pathogenicity during infection. To explore the role of the influenza virus PA protein in regulating host biological processes, we performed immunoprecipitation and LC‒MS/MS to globally identify cellular factors that interact with the PA proteins of the influenza A H1N1, 2009 pandemic H1N1, and H3N2 viruses. The results demonstrated that proteins located in the mitochondrion, proteasome, and nucleus are associated with the PA protein. We further discovered that the PA protein is partly located in mitochondria by immunofluorescence and mitochondrial fractionation and that overexpression of the PA protein reduces mitochondrial respiration. In addition, our results revealed the interaction between PA and the mitochondrial matrix protein PYCR2 and the antiviral role of PYCR2 during influenza A virus replication. Moreover, we found that the PA protein could also trigger autophagy and disrupt mitochondrial homeostasis. Overall, our research revealed the impacts of the influenza A virus PA protein on mitochondrial function and autophagy.

Evolution of human H3N2 influenza virus receptor specificity has substantially expanded the receptor-binding domain site.

Hemagglutinins (HAs) from human influenza viruses descend from avian progenitors that bind α2-3-linked sialosides and must adapt to glycans with α2-6-linked sialic acids on human airway cells to transmit within the human population. Since their introduction during the 1968 pandemic, H3N2 viruses have evolved over the past five decades to preferentially recognize human α2-6-sialoside receptors that are elongated through addition of poly-LacNAc. We show that more recent H3N2 viruses now make increasingly complex interactions with elongated receptors while continuously selecting for strains maintaining this phenotype. This change in receptor engagement is accompanied by an extension of the traditional receptor-binding site to include residues in key antigenic sites on the surface of HA trimers. These results help explain the propensity for selection of antigenic variants, leading to vaccine mismatching, when H3N2 viruses are propagated in chicken eggs or cells that do not contain such receptors.

Heteromultivalent Ligand Display on Reversible Self-Assembled Monolayers (rSAMs): A Fluidic Platform for Tunable Influenza Virus Recognition.

We report on the design of heteromultivalent influenza A virus (IAV) receptors based on reversible self-assembled monolayers (SAMs) featuring two distinct mobile ligands. The principal layer building blocks consist of α-(4-amidinophenoxy)alkanes decorated at the ω-position with sialic acid (SA) and the neuraminidase inhibitor Zanamivir (Zan), acting as two mobile ligands binding to the complementary receptors hemagglutinin (HA) and neuraminidase (NA) on the virus surface. From ternary amphiphile mixtures comprising these ligands, the amidines spontaneously self-assemble on top of carboxylic acid-terminated SAMs to form reversible mixed monolayers (rSAMs) that are easily tunable with respect to the ligand ratio. We show that this results in the ability to construct surfaces featuring a very strong affinity for the surface proteins and specific virus subtypes. Hence, an rSAM prepared from solutions containing 15% SA and 10% Zan showed an exceptionally high affinity and selectivity for the avian IAV H7N9 (Kd = 11 fM) that strongly exceeded the affinity for other subtypes (H3N2, H5N1, H1N1). Changing the SA/Zan ratio resulted in changes in the relative preference between the four tested subtypes, suggesting this to be a key parameter for rapid adjustments of both virus affinity and selectivity.

Molecular identification of neuraminidase gene mutations in influenza A/H1N1 and A/H3N2 isolates of Mazandaran province, north of Iran.

OBJECTIVES: The neuraminidase (NA) mutations causing resistance to NA inhibitors (NAIs) mostly compromise the fitness of influenza viruses. Considering the importance of these mutations, constant monitoring of the effectiveness of available drugs is critical. This study aimed to identify NA mutations in the influenza A/H1N1 and A/H3N2 subtypes in the samples of Mazandaran, Iran from 2016 to 2020. METHODS: In this cross-sectional study, 20 influenza A/H1N1 and 20 influenza A/H3N2 samples were included in the study. After design of appropriate primers for NA gene, all samples subjected to RT-PCR and electrophoresis. Then the PCR product was sequenced to determine the mutations. RESULTS: In the present study, no oseltamivir resistance-related mutations were detected. Still, NA gene showed variations compared to the vaccine strains. In A/H1N1, a total of 43 mutations were detected. Similarly, in A/H3N2, a total of 66 mutations were observed. In all isolates of H1N1, N200S, N248D and I321V mutations were detected in the antigenic site of NA protein, which can affect vaccine incompatibility and virus escape from the host's immune system. Also, H150R mutation was observed in the NA active site of H3N2, which is the cause of agglutination by NA protein. Also, S245N mutation was identified as a new N-Glycosylation site of H3N2 subtype. CONCLUSIONS: The study of NA gene sequences revealed no oseltamivir resistance mutations. In H1N1 isolates, ca. 97% identities and in the H3N2 subtype, 96% identities were observed compared to reference isolate of 2009, which indicates the importance of constant monitoring of the emergence of the drug resistance mutations.

Isolation by multistep chromatography improves the consistency of secreted recombinant influenza neuraminidase antigens.

Recombinant protein-based approaches are ideally suited for producing vaccine antigens that are not overly abundant in viruses, such as influenza neuraminidase (NA). However, obtaining sufficient quantities of recombinant viral surface antigens remains challenging, often resulting in the use of chimeric proteins with affinity tags that can invariably impact the antigen's properties. Here, we developed multistep chromatography approaches for purifying secreted recombinant NA (rNA) antigens that are derived from recent H1N1 and H3N2 viruses and produced using insect cells. Analytical analyses showed that these isolation procedures yielded homogenous tetrameric rNA preparations with consistent specific activities that were not possible from a common immobilized metal affinity chromatography purification procedure. The use of classical chromatography improved the rNA tetramer homogeneity by removing the requirement of the N-terminal poly-histidine affinity tag that was shown to promote higher order rNA oligomer formation. In addition, these procedures reduced the specific activity variation by eliminating the exposure to Ni2+ ions and imidazole, with the latter showing pH and NA subtype dependent effects. Together, these results demonstrate that purification by multistep chromatography improves the homogeneity of secreted rNAs and eliminates the need for affinity tag-based approaches that can potentially alter the properties of these recombinant antigens.

Genomic Analysis of Influenza A and B Viruses Carrying Baloxavir Resistance-Associated Substitutions Serially Passaged in Human Epithelial Cells.

Baloxavir marboxil (baloxavir) is an FDA-approved inhibitor of the influenza virus polymerase acidic (PA) protein. Here, we used next-generation sequencing to compare the genomic mutational profiles of IAV H1N1 and H3N2, and IBV wild type (WT) and mutants (MUT) viruses carrying baloxavir resistance-associated substitutions (H1N1-PA I38L, I38T, and E199D; H3N2-PA I38T; and IBV-PA I38T) during passaging in normal human bronchial epithelial (NHBE) cells. We determined the ratio of nonsynonymous to synonymous nucleotide mutations (dN/dS) and identified the location and type of amino acid (AA) substitutions that occurred at a frequency of ≥30%. We observed that IAV H1N1 WT and MUT viruses remained relatively stable during passaging. While the mutational profiles for IAV H1N1 I38L, I38T, and E199D, and IBV I38T MUTs were relatively similar after each passage compared to the respective WTs, the mutational profile of the IAV H3N2 I38T MUT was significantly different for most genes compared to H3N2 WT. Our work provides insight into how baloxavir resistance-associated substitutions may impact influenza virus evolution in natural settings. Further characterization of the potentially adaptive mutations identified in this study is needed.

A novel N-heterocycles substituted oseltamivir derivatives as potent inhibitors of influenza virus neuraminidase: discovery, synthesis and biological evaluation.

Our previous studies have shown that the introduction of structurally diverse benzyl side chains at the C5-NH2 position of oseltamivir to occupy 150-cavity contributes to the binding affinity with neuraminidase and anti-influenza activity. To obtain broad-spectrum neuraminidase inhibitors, we designed and synthesised a series of novel oseltamivir derivatives bearing different N-heterocycles substituents that have been proved to induce opening of the 150-loop of group-2 neuraminidases. Among them, compound 6k bearing 4-((r)-2-methylpyrrolidin-1-yl) benzyl group exhibited antiviral activities similar to or weaker than those of oseltamivir carboxylate against H1N1, H3N2, H5N1, H5N6 and H5N1-H274Y mutant neuraminidases. More encouragingly, 6k displayed nearly 3-fold activity enhancement against H3N2 virus over oseltamivir carboxylate and 2-fold activity enhancement over zanamivir. Molecular docking studies provided insights into the explanation of its broad-spectrum potency against wild-type neuraminidases. Overall, as a promising lead compound, 6k deserves further optimisation by fully considering the ligand induced flexibility of the 150-loop.

Transcriptomics profile of human bronchial epithelial cells exposed to ambient fine particles and influenza virus (H3N2).

Fine particulate matter (PM2.5) pollution remains a major threat to public health. As the physical barrier against inhaled air pollutants, airway epithelium is a primary target for PM2.5 and influenza viruses, two major environmental insults. Recent studies have shown that PM2.5 and influenza viruses may interact to aggravate airway inflammation, an essential event in the pathogenesis of diverse pulmonary diseases. Airway epithelium plays a critical role in lung health and disorders. Thus far, the mechanisms for the interactive effect of PM2.5 and the influenza virus on gene transcription of airway epithelial cells have not been fully uncovered. In this present pilot study, the transcriptome sequencing approach was introduced to identify responsive genes following individual and co-exposure to PM2.5 and influenza A (H3N2) viruses in a human bronchial epithelial cell line (BEAS-2B). Enrichment analysis revealed the function of differentially expressed genes (DEGs). Specifically, the DEGs enriched in the xenobiotic metabolism by the cytochrome P450 pathway were linked to PM2.5 exposure. In contrast, the DEGs enriched in environmental information processing and human diseases, such as viral protein interaction with cytokines and cytokine receptors and epithelial cell signaling in bacterial infection, were significantly related to H3N2 exposure. Meanwhile, co-exposure to PM2.5 and H3N2 affected G protein-coupled receptors on the cell surface. Thus, the results from this study provides insights into PM2.5- and influenza virus-induced airway inflammation and potential mechanisms.

Proteomic and genetic analyses of influenza A viruses identify pan-viral host targets.

Influenza A Virus (IAV) is a recurring respiratory virus with limited availability of antiviral therapies. Understanding host proteins essential for IAV infection can identify targets for alternative host-directed therapies (HDTs). Using affinity purification-mass spectrometry and global phosphoproteomic and protein abundance analyses using three IAV strains (pH1N1, H3N2, H5N1) in three human cell types (A549, NHBE, THP-1), we map 332 IAV-human protein-protein interactions and identify 13 IAV-modulated kinases. Whole exome sequencing of patients who experienced severe influenza reveals several genes, including scaffold protein AHNAK, with predicted loss-of-function variants that are also identified in our proteomic analyses. Of our identified host factors, 54 significantly alter IAV infection upon siRNA knockdown, and two factors, AHNAK and coatomer subunit COPB1, are also essential for productive infection by SARS-CoV-2. Finally, 16 compounds targeting our identified host factors suppress IAV replication, with two targeting CDK2 and FLT3 showing pan-antiviral activity across influenza and coronavirus families. This study provides a comprehensive network model of IAV infection in human cells, identifying functional host targets for pan-viral HDT.

Contemporary human H3N2 influenza A viruses require a low threshold of suitable glycan receptors for efficient infection.

Recent human H3N2 influenza A viruses have evolved to employ elongated glycans terminating in α2,6-linked sialic acid as their receptors. These glycans are displayed in low abundancies by (humanized) Madin-Darby Canine Kidney cells, which are commonly employed to propagate influenza A virus, resulting in low or no viral propagation. Here, we examined whether the overexpression of the glycosyltransferases ß-1,3-N-acetylglucosaminyltransferase and ß-1,4-galactosyltransferase 1, which are responsible for the elongation of poly-N-acetyllactosamines (LacNAcs), would result in improved A/H3N2 propagation. Stable overexpression of ß-1,3-N-acetylglucosaminyltransferase and ß-1,4-galactosyltransferase 1 in Madin-Darby Canine Kidney and "humanized" Madin-Darby Canine Kidney cells was achieved by lentiviral integration and subsequent antibiotic selection and confirmed by qPCR and protein mass spectrometry experiments. Flow cytometry and glycan mass spectrometry experiments using the ß-1,3-N-acetylglucosaminyltransferase and/or ß-1,4-galactosyltransferase 1 knock-in cells demonstrated increased binding of viral hemagglutinins and the presence of a larger number of LacNAc repeating units, especially on "humanized" Madin-Darby Canine Kidney-ß-1,3-N-acetylglucosaminyltransferase cells. An increase in the number of glycan receptors did, however, not result in a greater infection efficiency of recent human H3N2 viruses. Based on these results, we propose that H3N2 influenza A viruses require a low number of suitable glycan receptors to infect cells and that an increase in the glycan receptor display above this threshold does not result in improved infection efficiency.

Synthesis of multivalent sialyllactose-conjugated PAMAM dendrimers: Binding to SARS-CoV-2 spike protein and influenza hemagglutinin.

Severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2) and influenza viruses have spread around the world at an unprecedented rate. Despite multiple vaccines, new variants of SARS-CoV-2 and influenza have caused a remarkable level of pathogenesis. The development of effective antiviral drugs to treat SARS-CoV-2 and influenza remains a high priority. Inhibiting viral cell surface attachment represents an early and efficient means to block virus infection. Sialyl glycoconjugates, on the surface of human cell membranes, play an important role as host cell receptors for influenza A virus and 9-O-acetyl-sialylated glycoconjugates are receptors for MERS, HKU1 and bovine coronaviruses. We designed and synthesized multivalent 6'-sialyllactose-counjugated polyamidoamine dendrimers through click chemistry at room temperature concisely. These dendrimer derivatives have good solubility and stability in aqueous solutions. SPR, a real-time analysis quantitative method for of biomolecular interactions, was used to study the binding affinities of our dendrimer derivatives by utilizing only 200 micrograms of each dendrimer. Three SARS-CoV-2 S-protein receptor binding domain (wild type and two Omicron mutants) bound to multivalent 9-O-acetyl-6'-sialyllactose-counjugated and 6'-sialyllactose-counjugated dendrimers bound to a single H3N2 influenza A virus's HA protein (A/Hong Kong/1/1968), the SPR study results suggest their potential anti-viral activities.

Sodium taurocholate hydrate inhibits influenza virus replication and suppresses influenza a Virus-triggered inflammation in vitro and in vivo.

Influenza A virus is an important respiratory pathogen that poses serious threats to human health. Owing to the high mutation rate of viral genes, weaker cross-protection of vaccines, and rapid emergence of drug resistance, there is an urgent need to develop new antiviral drugs against influenza viruses. Taurocholic acid is a primary bile acid that promotes digestion, absorption, and excretion of dietary lipids. Here, we demonstrate that sodium taurocholate hydrate (STH) exhibits broad-spectrum antiviral activity against influenza strains H5N6, H1N1, H3N2, H5N1, and H9N2 in vitro. STH significantly inhibited the early stages of influenza A virus replication. The levels of influenza virus viral RNA (vRNA), complementary RNA (cRNA), and mRNA were specifically reduced in virus-infected cells following STH treatment. In vivo, STH treatment of infected mice alleviated clinical signs and reduced weight loss and mortality. STH also reduced TNF-α, IL-1ß, and IL-6 overexpression. STH significantly inhibited the upregulation of TLR4 and the NF-kB family member p65, both in vivo and in vitro. These results suggest that STH exerts a protective effect against influenza infection via suppression of the NF-kB pathway, highlighting the potential use of STH as a drug for treating influenza infection.

Detecting early-warning signals for influenza by dysregulated dynamic network biomarkers.

As a dynamical system, complex disease always has a sudden state transition at the tipping point, which is the result of the long-term accumulation of abnormal regulations. This paper proposes a novel approach to detect the early-warning signals of influenza A (H3N2 and H1N1) outbreaks by dysregulated dynamic network biomarkers (dysregulated DNBs) for individuals. The results of cross-validation show that our approach can detect early-warning signals before the symptom appears successfully. Unlike the traditional DNBs, our dysregulated DNBs are anchored and very few, which is essential for disease early diagnosis in clinical practice. Moreover, the genes of dysregulated DNBs are significantly enriched in the influenza-related pathways. The source code of this paper can be freely downloaded from https://github.com/YanhaoHuo/dysregulated-DNBs.git.

Single Particle Analysis of H3N2 Influenza Entry Differentiates the Impact of the Sialic Acids (Neu5Ac and Neu5Gc) on Virus Binding and Membrane Fusion.

Entry of influenza A viruses (IAVs) into host cells is initiated by binding to sialic acids (Sias), their primary host cell receptor, followed by endocytosis and membrane fusion to release the viral genome into the cytoplasm of the host cell. Host tropism is affected by these entry processes, with a primary factor being receptor specificity. Sias exist in several different chemical forms, including the hydroxylated N-glycolylneuraminic acid (Neu5Gc), which is found in many hosts; however, it has not been clear how modified Sias affect viral binding and entry. Neu5Gc is commonly found in many natural influenza hosts, including pigs and horses, but not in humans or ferrets. Here, we engineered HEK293 cells to express the hydoxylase gene (CMAH) that converts Neu5Ac to Neu5Gc, or knocked out the Sia-CMP transport gene (SLC35A1), resulting in cells that express 95% Neu5Gc or minimal level of Sias, respectively. H3N2 (X-31) showed significantly reduced infectivity in Neu5Gc-rich cells compared to wild-type HEK293 (>95% Neu5Ac). To determine the effects on binding and fusion, we generated supported lipid bilayers (SLBs) derived from the plasma membranes of these cells and carried out single particle microscopy. H3N2 (X-31) exhibited decreased binding to Neu5Gc-containing SLBs, but no significant difference in H3N2 (X-31)'s fusion kinetics to either SLB type, suggesting that reduced receptor binding does not affect subsequent membrane fusion. This finding suggests that for this virus to adapt to host cells rich in Neu5Gc, only receptor affinity changes are required without further adaptation of virus fusion machinery. IMPORTANCE Influenza A virus (IAV) infections continue to threaten human health, causing over 300,000 deaths yearly. IAV infection is initiated by the binding of influenza glycoprotein hemagglutinin (HA) to host cell sialic acids (Sias) and the subsequent viral-host membrane fusion. Generally, human IAVs preferentially bind to the Sia N-acetylneuraminic acid (Neu5Ac). Yet, other mammalian hosts, including pigs, express diverse nonhuman Sias, including N-glycolylneuraminic acid (Neu5Gc). The role of Neu5Gc in human IAV infections in those hosts is not well-understood, and the variant form may play a role in incidents of cross-species transmission and emergence of new epidemic variants. Therefore, it is important to investigate how human IAVs interact with Neu5Ac and Neu5Gc. Here, we use membrane platforms that mimic the host cell surface to examine receptor binding and membrane fusion events of human IAV H3N2. Our findings improve the understanding of viral entry mechanisms that can affect host tropism and virus evolution.

Development of an HiBiT-tagged reporter H3N2 influenza A virus and its utility as an antiviral screening platform.

The balance of the segmented genome derived from naturally occurring influenza A viruses (IAVs) is delicate and vulnerable to foreign insertions, thus most reporter IAVs up to date are generated using the backbone of the laboratory-adapted strains. In this study, we constructed a reporter influenza A/H3N2 virus (A/NY-HiBiT) which was derived from a clinical isolate, by placing a minimized HiBiT tag to the N-terminus of the viral nuclear-export protein (NEP). Here, we show that this 11-amino acid HiBiT tag did not adversely impact the viral genome balance, and the recombinant A/NY-HiBiT virus maintains its relative stability. Moreover, the replication profile of the HiBiT-tagged virus can be measured by a simple Nano-Glo assay, providing a robust high-throughput screening (THS) platform. We used this platform to evaluate a collection of the pre-purified fractions which were derived from rare Chinese medicinal materials, and we identified three fractions, including wild Trametes robiniophila (50% methanol fraction), Ganoderma (water fraction), and wild Phellinus igniarius (ethyl acetate fraction), as potent anti-IAV actives. Our results demonstrate that this IAV reporter can be used as a powerful HTS platform for antiviral development.

Sialosides Containing 7-N-Acetyl Sialic Acid Are Selective Substrates for Neuraminidases from Influenza A Viruses.

Sialidases or neuraminidases are sialic-acid-cleaving enzymes that are expressed by a broad spectrum of organisms, including pathogens. In nature, sialic acids are monosaccharides with diverse structural variations, but the lack of novel probes has made it difficult to determine how sialic acid modifications impact the recognition by sialidases. Here, we used a chemoenzymatic synthon strategy to generate a set of α2-3- and α2-6-linked sialoside probes that contain 7-N-acetyl or 7,9-di-N-acetyl sialic acid as structure mimics for those containing the less stable naturally occurring 7-O-acetyl- or 7,9-di-O-acetyl modifications. These probes were used to compare the substrate specificity of several sialidases from different origins. Our results show that 7-N-acetyl sialic acid was readily cleaved by neuraminidases from H1N1 and H3N2 influenza A viruses, but not by sialidases of human or bacterial origin, thereby indicating that the influenza enzymes possess a distinctive and more promiscuous substrate binding pocket.

Antibodies targeting the neuraminidase active site inhibit influenza H3N2 viruses with an S245N glycosylation site.

Contemporary influenza A H3N2 viruses circulating since 2016 have acquired a glycosylation site in the neuraminidase in close proximity to the enzymatic active site. Here, we investigate if this S245N glycosylation site, as a result of antigenic evolution, can impact binding and function of human monoclonal antibodies that target the conserved active site. While we find that a reduction in the inhibitory ability of neuraminidase active site binders is measurable, this class of broadly reactive monoclonal antibodies maintains protective efficacy in vivo.

Low prevalence of influenza A strains with resistance markers in Brazil during 2017-2019 seasons.

The influenza A virus (IAV) is of a major public health concern as it causes annual epidemics and has the potential to cause pandemics. At present, the neuraminidase inhibitors (NAIs) are the most widely used anti-influenza drugs, but, more recently, the drug baloxavir marboxil (BXM), a polymerase inhibitor, has also been licensed in some countries. Mutations in the viral genes that encode the antiviral targets can lead to treatment resistance. Worldwide, a low prevalence of antiviral resistant strains has been reported. Despite that, this situation can change rapidly, and resistant strain surveillance is a priority. Thus, the aim of this was to evaluate Brazilian IAVs antiviral resistance from 2017 to 2019 through the identification of viral mutations associated with reduced inhibition of the drugs and by testing the susceptibility of IAV isolates to oseltamivir (OST), the most widely used NAI drug in the country. Initially, we analyzed 282 influenza A(H1N1)pdm09 and 455 A(H3N2) genetic sequences available on GISAID. The amino acid substitution (AAS) NA:S247N was detected in one A(H1N1)pdm09 strain. We also identified NA:I222V (n = 6) and NA:N329K (n = 1) in A(H3N2) strains. In addition, we performed a molecular screening for NA:H275Y in 437 A(H1N1)pdm09 samples, by pyrosequencing, which revealed a single virus harboring this mutation. Furthermore, the determination of OST IC50 values for 222 A(H1N1)pdm09 and 83 A(H3N2) isolates revealed that all isolates presented a normal susceptibility profile to the drug. Interestingly, we detected one A(H3N2) virus presenting with PA:E119D AAS. Moreover, the majority of the IAV sequences had the M2:S31N adamantanes resistant marker. In conclusion, we show a low prevalence of Brazilian IAV strains with NAI resistance markers, in accordance with what is reported worldwide, indicating that NAIs still remain an option for the treatment of influenza infections in Brazil. However, surveillance of influenza resistance should be strengthened in the country for improving the representativeness of investigated viruses and the robustness of the analysis.

H3N2 canine influenza virus NS1 protein inhibits canine NLRP3 inflammasome activation.

Inflammation is an innate immune response of the body against pathogens and other irritants. The NLRP3 (NACHT, LRR and PYD domains-containing protein 3) inflammasome is a major player in the inflammatory response against pathogenic microorganisms. In this study, we analyzed the relationship between the NLRP3 inflammasome and the influenza virus NS1 protein, which is involved in host immune escape. The canine influenza virus NS1 protein transcriptionally attenuated proinflammatory cytokines by inhibiting the nuclear factor-κB (NF-κB) activator. NS1 also directly interacted with NLRP3 and blocked ASC (Apoptosis-associated speck-like protein containing CARD) oligomerization, which deactivated the NLRP3 inflammasome. In addition, NS1 inhibited pro-caspase 1 cleavage into caspase-1, which prevents maturation of IL-1ß and IL-18 from their respective precursors, eventually reducing the inflammatory response. Taken together, the influenza NS1 protein evades host immunity, and our findings provide a theoretical basis for the prevention and treatment of canine influenza.