Influenza virus uses mGluR2 as an endocytic receptor to enter cells.

Influenza virus infection is initiated by the attachment of the viral haemagglutinin (HA) protein to sialic acid receptors on the host cell surface. Most virus particles enter cells through clathrin-mediated endocytosis (CME). However, it is unclear how viral binding signals are transmitted through the plasma membrane triggering CME. Here we found that metabotropic glutamate receptor subtype 2 (mGluR2) and potassium calcium-activated channel subfamily M alpha 1 (KCa1.1) are involved in the initiation and completion of CME of influenza virus using an siRNA screen approach. Influenza virus HA directly interacted with mGluR2 and used it as an endocytic receptor to initiate CME. mGluR2 interacted and activated KCa1.1, leading to polymerization of F-actin, maturation of clathrin-coated pits and completion of the CME of influenza virus. Importantly, mGluR2-knockout mice were significantly more resistant to different influenza subtypes than the wild type. Therefore, blocking HA and mGluR2 interaction could be a promising host-directed antiviral strategy.

Evolution of H7N9 highly pathogenic avian influenza virus in the context of vaccination.

Human infections with the H7N9 influenza virus have been eliminated in China through vaccination of poultry; however, the H7N9 virus has not yet been eradicated from poultry. Carefully analysis of H7N9 viruses in poultry that have sub-optimal immunity may provide a unique opportunity to witness the evolution of highly pathogenic avian influenza virus in the context of vaccination. Between January 2020 and June 2023, we isolated 16 H7N9 viruses from samples we collected during surveillance and samples that were sent to us for disease diagnosis. Genetic analysis indicated that these viruses belonged to a single genotype previously detected in poultry. Antigenic analysis indicated that 12 of the 16 viruses were antigenically close to the H7-Re4 vaccine virus that has been used since January 2022, and the other four viruses showed reduced reactivity with the vaccine. Animal studies indicated that all 16 viruses were nonlethal in mice, and four of six viruses showed reduced virulence in chickens upon intranasally inoculation. Importantly, the H7N9 viruses detected in this study exclusively bound to the avian-type receptors, having lost the capacity to bind to human-type receptors. Our study shows that vaccination slows the evolution of H7N9 virus by preventing its reassortment with other viruses and eliminates a harmful characteristic of H7N9 virus, namely its ability to bind to human-type receptors.

Recombinant duck enteritis virus bearing the hemagglutinin genes of H5 and H7 influenza viruses is an ideal multivalent live vaccine in ducks.

Due to the fact that many avian influenza viruses that kill chickens are not lethal to ducks, farmers are reluctant to use avian influenza inactivated vaccines on ducks. Large numbers of unvaccinated ducks play an important role in the transmission of avian influenza viruses from wild birds to domestic poultry, creating a substantial challenge to vaccination strategies for avian influenza control. To solve this problem, we constructed a recombinant duck enteritis virus (DEV), rDEV-dH5/H7, using a live attenuated DEV vaccine strain (vDEV) as a vector. rDEV-dH5/H7 carries the hemagglutinin gene of two H5 viruses [GZ/S4184/17 (H5N6) (clade 2.3.4.4 h) and LN/SD007/17 (H5N1) (clade 2.3.2.1d)] and an H7 virus [GX/SD098/17 (H7N9)]. These three hemagglutinin genes were stably inherited in rDEV-dH5/H7 and expressed in rDEV-dH5/H7-infected cells. Animal studies revealed that rDEV-dH5/H7 and vDEV induced similar neutralizing antibody responses and protection against lethal DEV challenge. Importantly, rDEV-dH5/H7 induced strong and long-lasting hemagglutinin inhibition antibodies against different H5 and H7 viruses and provided complete protection against challenges with homologous and heterologous highly pathogenic H5 and H7 influenza viruses in ducks. Our study shows that rDEV-dH5/H7 could serve as an ideal live attenuated vaccine to protect ducks against infection with lethal DEV and highly pathogenic avian influenza viruses.

Evolution and biological characterization of H5N1 influenza viruses bearing the clade 2.3.2.1 hemagglutinin gene.

H5N1 avian influenza viruses bearing the clade 2.3.2.1 hemagglutinin (HA) gene have been widely detected in birds and poultry in several countries. During our routine surveillance, we isolated 28 H5N1 viruses between January 2017 and October 2020. To investigate the genetic relationship of the globally circulating H5N1 viruses and the biological properties of those detected in China, we performed a detailed phylogenic analysis of 274 representative H5N1 strains and analyzed the antigenic properties, receptor-binding preference, and virulence in mice of the H5N1 viruses isolated in China. The phylogenic analysis indicated that the HA genes of the 274 viruses belonged to six subclades, namely clades 2.3.2.1a to 2.3.2.1f; these viruses acquired gene mutations and underwent complicated reassortment to form 58 genotypes, with G43 being the dominant genotype detected in eight Asian and African countries. The 28 H5N1 viruses detected in this study carried the HA of clade 2.3.2.1c (two strains), 2.3.2.1d (three strains), or 2.3.2.1f (23 strains), and formed eight genotypes. These viruses were antigenically well-matched with the H5-Re12 vaccine strain used in China. Animal studies showed that the pathogenicity of the H5N1 viruses ranged from non-lethal to highly lethal in mice. Moreover, the viruses exclusively bound to avian-type receptors and have not acquired the ability to bind to human-type receptors. Our study reveals the overall picture of the evolution of clade 2.3.2.1 H5N1 viruses and provides insights into the control of these viruses.

M6PR interacts with the HA2 subunit of influenza A virus to facilitate the fusion of viral and endosomal membranes.

Influenza A virus (IAV) commandeers numerous host cellular factors for successful replication. However, very few host factors have been revealed to be involved in the fusion of viral envelope and late endosomal membranes. In this study, we identified cation-dependent mannose-6-phosphate receptor (M6PR) as a crucial host factor for the replication of IAV. We found that siRNA knockdown of M6PR expression significantly reduced the growth titers of different subtypes of IAV, and that the inhibitory effect of M6PR siRNA treatment on IAV growth was overcome by the complement of exogenously expressed M6PR. When A549 cells were treated with siRNA targeting M6PR, the nuclear accumulation of viral nucleoprotein (NP) was dramatically inhibited at early timepoints post-infection, indicating that M6PR engages in the early stage of the IAV replication cycle. By investigating the role of M6PR in the individual entry and post-entry steps of IAV replication, we found that the downregulation of M6PR expression had no effect on attachment, internalization, early endosome trafficking, or late endosome acidification. However, we found that M6PR expression was critical for the fusion of viral envelope and late endosomal membranes. Of note, M6PR interacted with the hemagglutinin (HA) protein of IAV, and further studies showed that the lumenal domain of M6PR and the ectodomain of HA2 mediated the interaction and directly promoted the fusion of the viral and late endosomal membranes, thereby facilitating IAV replication. Together, our findings highlight the importance of the M6PR-HA interaction in the fusion of viral and late endosomal membranes during IAV replication.

Key Amino Acid Residues That Determine the Antigenic Properties of Highly Pathogenic H5 Influenza Viruses Bearing the Clade 2.3.4.4 Hemagglutinin Gene.

The H5 subtype highly pathogenic avian influenza viruses bearing the clade 2.3.4.4 HA gene have been pervasive among domestic poultry and wild birds worldwide since 2014, presenting substantial risks to human and animal health. Continued circulation of clade 2.3.4.4 viruses has resulted in the emergence of eight subclades (2.3.4.4a-h) and multiple distinct antigenic groups. However, the key antigenic substitutions responsible for the antigenic change of these viruses remain unknown. In this study, we analyzed the HA gene sequences of 5713 clade 2.3.4.4 viruses obtained from a public database and found that 23 amino acid residues were highly variable among these strains. We then generated a series of single-amino-acid mutants based on the H5-Re8 (a vaccine seed virus) background and tested their reactivity with a panel of eight monoclonal antibodies (mAbs). Six mutants bearing amino acid substitutions at positions 120, 126, 141, 156, 185, or 189 (H5 numbering) led to reduced or lost reactivity to these mAbs. Further antigenic cartography analysis revealed that the amino acid residues at positions 126, 156, and 189 acted as immunodominant epitopes of H5 viruses. Collectively, our findings offer valuable guidance for the surveillance and early detection of emerging antigenic variants.

ABTB1 facilitates the replication of influenza A virus by counteracting TRIM4-mediated degradation of viral NP protein.

Influenza A viruses (IAVs) continue to cause tremendous economic losses to the global animal industry and respiratory diseases and deaths among humans. The nuclear import of the vRNP complex, composed of polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2), polymerase acidic protein (PA), nucleoprotein (NP), and viral RNA, is essential for the efficient replication of IAV. Host factors involved in this process can be targeted for the development of countermeasures against IAV infection. Here, we found that Ankyrin Repeat and BTB Domain Containing 1 (ABTB1) promotes the replication of IAV, and positively regulates the nuclear import of the vRNP complex. ABTB1 did not interact directly with NP, indicating that ABTB1 plays an indirect role in facilitating the nuclear import of the vRNP complex. Immunoprecipitation and mass spectrometry revealed that Tripartite Motif Containing 4 (TRIM4) interacts with ABTB1. We found that TRIM4 relies on its E3 ubiquitin ligase activity to inhibit the replication of IAV by targeting and degrading NP within the incoming vRNP complex as well as the newly synthesized NP. ABTB1 interacted with TRIM4, leading to TRIM4 degradation through the proteasome system. Notably, ABTB1-mediated degradation of TRIM4 blocked the effect of TRIM4 on NP stability, and largely counteracted the inhibitory effect of TRIM4 on IAV replication. Our findings define a novel role for ABTB1 in aiding the nuclear import of the vRNP complex of IAV by counteracting the destabilizing effect of TRIM4 on the viral NP protein.

Analysis of avian influenza A (H3N8) viruses in poultry and their zoonotic potential, China, September 2021 to May 2022.

BackgroundTwo human cases of avian influenza A (H3N8) virus infection were reported in China in 2022.AimTo characterise H3N8 viruses circulating in China in September 2021-May 2022.MethodsWe sampled poultry and poultry-related environments in 25 Chinese provinces. After isolating H3N8 viruses, whole genome sequences were obtained for molecular and phylogenetic analyses. The specificity of H3N8 viruses towards human or avian receptors was assessed in vitro. Their ability to replicate in chicken and mice, and to transmit between guinea pigs was also investigated.ResultsIn total, 98 H3N8 avian influenza virus isolates were retrieved from 38,639 samples; genetic analysis of 31 representative isolates revealed 17 genotypes. Viruses belonging to 10 of these genotypes had six internal genes originating from influenza A (H9N2) viruses. These reassorted viruses could be found in live poultry markets and comprised the strains responsible for the two human infections. A subset of nine H3N8 viruses (including six reassorted) that replicated efficiently in mice bound to both avian-type and human-type receptors in vitro. Three reassorted viruses were shed by chickens for up to 9 days, replicating efficiently in their upper respiratory tract. Five reassorted viruses tested on guinea pigs were transmissible among these by respiratory droplets.ConclusionAvian H3N8 viruses with H9N2 virus internal genes, causing two human infections, occurred in live poultry markets in China. The low pathogenicity of H3N8 viruses in poultry allows their continuous circulation with potential for reassortment. Careful monitoring of spill-over infections in humans is important to strengthen early-warning systems and maintain influenza pandemic preparedness.

Highly Pathogenic Avian Influenza Virus (H5N1) Clade 2.3.4.4b Introduced by Wild Birds, China, 2021.

Highly pathogenic avian influenza (HPAI) subtype H5N1 clade 2.3.4.4b virus has spread globally, causing unprecedented large-scale avian influenza outbreaks since 2020. In 2021, we isolated 17 highly pathogenic avian influenza H5N1 viruses from wild birds in China. To determine virus origin, we genetically analyzed 1,529 clade 2.3.4.4b H5N1 viruses reported globally since October 2020 and found that they formed 35 genotypes. The 17 viruses belonged to genotypes G07, which originated from eastern Asia, and G10, which originated from Russia. The viruses were moderately pathogenic in mice but were highly lethal in ducks. The viruses were in the same antigenic cluster as the current vaccine strain (H5-Re14) used in China. In chickens, the H5/H7 trivalent vaccine provided complete protection against clade 2.3.4.4b H5N1 virus challenge. Our data indicate that vaccination is an effective strategy for preventing and controlling the globally prevalent clade 2.3.4.4b H5N1 virus.

H3N8 subtype avian influenza virus originated from wild birds exhibited dual receptor-binding profiles.

We present the phylogeny, receptor binding property, growth in mammal cells and pathogenicity in mammal model of H3N8 viruses, which were isolated from wild birds in China. The human receptor preference and efficient replication in mice without prior adaption highlight that the H3N8 virus possesses the public threat potential.

Alarming situation of emerging H5 and H7 avian influenza and effective control strategies.

Avian influenza viruses continue to present challenges to animal and human health. Viruses bearing the hemagglutinin (HA) gene of the H5 subtype and H7 subtype have caused 2634 human cases around the world, including more than 1000 deaths. These viruses have caused numerous disease outbreaks in wild birds and domestic poultry, and are responsible for the loss of at least 422 million domestic birds since 2005. The H5 influenza viruses are spread by migratory wild birds and have caused three waves of influenza outbreaks across multiple continents, and the third wave that started in 2020 is ongoing. Many countries in Europe and North America control highly pathogenic avian influenza by culling alone, whereas some countries, including China, have adopted a "cull plus vaccination" strategy. As the largest poultry-producing country in the world, China lost relatively few poultry during the three waves of global H5 avian influenza outbreaks, and nearly eliminated the pervasive H7N9 viruses that emerged in 2013. In this review, we briefly summarize the damages the H5 and H7 influenza viruses have caused to the global poultry industry and public health, analyze the origin, evolution, and spread of the H5 viruses that caused the waves, and discuss how and why the vaccination strategy in China has been a success. Given that the H5N1 viruses are widely circulating in wild birds and causing problems in domestic poultry around the world, we recommend that any unnecessary obstacles to vaccination strategies should be removed immediately and forever.

Influenza A virus use of BinCARD1 to facilitate the binding of viral NP to importin α7 is counteracted by TBK1-p62 axis-mediated autophagy.

As a major component of the viral ribonucleoprotein (vRNP) complex in influenza A virus (IAV), nucleoprotein (NP) interacts with isoforms of importin α family members, leading to the import of itself  and vRNP complex into the nucleus, a process pivotal in the replication cycle of IAV. In this study, we found that BinCARD1, an isoform of Bcl10-interacting protein with CARD (BinCARD), was leveraged by IAV for efficient viral replication. BinCARD1 promoted the nuclear import of the vRNP complex and newly synthesized NP and thus enhanced vRNP complex activity. Moreover, we found that BinCARD1 interacted with NP to promote NP binding to importin α7, an adaptor in the host nuclear import pathway. However, we also found that BinCARD1 promoted RIG-I-mediated innate immune signaling by mediating Lys63-linked polyubiquitination of TRAF3, and that TBK1 appeared to degrade BinCARD1. We showed that BinCARD1 was polyubiquitinated at residue K103 through a Lys63 linkage, which was recognized by the TBK1-p62 axis for autophagic degradation. Overall, our data demonstrate that IAV leverages BinCARD1 as an important host factor that promotes viral replication, and two mechanisms in the host defense system are triggered-innate immune signaling and autophagic degradation-to mitigate the promoting effect of BinCARD1 on the life cycle of IAV.

A Eurasian avian-like H1N1 swine influenza reassortant virus became pathogenic and highly transmissible due to mutations in its PA gene.

Previous studies have shown that the Eurasian avian-like H1N1 (EA H1N1) swine influenza viruses circulated widely in pigs around the world and formed multiple genotypes by acquiring non-hemagglutinin and neuraminidase segments derived from other swine influenza viruses. Swine influenza control is not a priority for the pig industry in many countries, and it is worrisome that some strains may become more pathogenic and/or transmissible during their circulation in nature. Our routine surveillance indicated that the EA H1N1 viruses obtained different internal genes from different swine influenza viruses and formed various new genotypes. In this study, we found that a naturally isolated swine influenza reassortant, A/swine/Liaoning/265/2017 (LN265), a representative strain of one of the predominant genotypes in recent years, is lethal in mice and transmissible in ferrets. LN265 contains the hemagglutinin, neuraminidase, and matrix of the EA H1N1 virus; the basic polymerase 2, basic polymerase 1, acidic polymerase (PA), and nucleoprotein of the 2009 H1N1 pandemic virus; and the nonstructural protein of the North American triple-reassortment H1N2 virus. By generating and testing a series of reassortants and mutants, we found that four gradually accumulated mutations in PA are responsible for the increased pathogenicity and transmissibility of LN265. We further revealed that these mutations increase the messenger RNA transcription of viral proteins by enhancing the endonuclease cleavage activity and viral RNA-binding ability of the PA protein. Our study demonstrates that EA H1N1 swine influenza virus became pathogenic and transmissible in ferrets by acquiring key mutations in PA and provides important insights for monitoring field strains with pandemic potential.

Global dissemination of H5N1 influenza viruses bearing the clade 2.3.4.4b HA gene and biologic analysis of the ones detected in China.

H5N1 avian influenza viruses bearing the clade 2.3.4.4b hemagglutinin gene have been widely circulating in wild birds and are responsible for the loss of over 70 million domestic poultry in Europe, Africa, Asia, and North America since October 2020. During our routine surveillance, 13 H5N1 viruses were isolated from 26,767 wild bird and poultry samples that were collected between September 2021 and March 2022 in China. To investigate the origin of these Chinese isolates and understand their genetic relationship with the globally circulating H5N1 viruses, we performed a detailed phylogenic analysis of 233 representative H5N1 strains that were isolated from 28 countries. We found that, after they emerged in the Netherlands, the H5N1 viruses encountered complicated gene exchange with different viruses circulating in wild birds and formed 16 genotypes. Genotype one (G1) was predominant, being detected in 22 countries, whereas all other genotypes were only detected in one or two continents. H5N1 viruses of four genotypes (G1, G7, G9, and G10) were detected in China; three of these genotypes have been previously reported in other countries. The H5N1 viruses detected in China replicated in mice, with pathogenicity varying among strains; the G1 virus was highly lethal in mice. Moreover, we found that these viruses were antigenically similar to and well matched with the H5-Re14 vaccine strain currently used in China. Our study reveals the overall picture of H5N1 virus evolution and provides insights for the control of these viruses.

Novel H7N7 avian influenza viruses detected in migratory wild birds in eastern China between 2018 and 2020.

Wild birds are the natural reservoirs of avian influenza viruses, and surveillance and assessment of these viruses in wild birds provide valuable information for early warning and control of animal diseases. In this study, we isolated 19 H7N7 avian influenza viruses from wild bird between 2018 and 2020. Full genomic analysis revealed that these viruses bear a single basic amino acid in the cleavage site of their hemagglutinin gene, and formed four different genotypes by actively reassorting other avian influenza viruses circulating in wild birds and ducks. The H7N7 viruses bound to both avian-type and human-type receptors, although their affinity for human-type receptors was markedly lower than that for avian-type receptors. Moreover, we found that the H7N7 viruses could replicate efficiently in the upper respiratory tract and caecum of domestic ducks, and that the H5/H7 inactivated vaccine used in poultry in China provided complete protection against H7N7 wild bird virus challenge in ducks. Our findings demonstrate that wild bird H7N7 viruses pose a substantial threat to the poultry industry across the East Asian-Australian migratory flyway, emphasize the importance of influenza virus surveillance in both wild and domestic birds, and support the development of active control strategies against H7N7 virus.

Emergence, Evolution, and Biological Characteristics of H10N4 and H10N8 Avian Influenza Viruses in Migratory Wild Birds Detected in Eastern China in 2020.

H10Nx influenza viruses have caused increasing public concern due to their occasional infection of humans. However, the genesis and biological characteristics of H10 viruses in migratory wild birds are largely unknown. In this study, we conducted active surveillance to monitor circulation of avian influenza viruses in eastern China and isolated five H10N4 and two H10N8 viruses from migratory birds in 2020. Genetic analysis indicated that the hemagglutinin (HA) genes of the seven H10 viruses were clustered into the North American lineage and established as a novel Eurasian branch in wild birds in South Korea, Bangladesh, and China. The neuraminidase (NA) genes of the H10N4 and H10N8 viruses originated from the circulating HxN4 and H5N8 viruses in migratory birds in Eurasia. We further revealed that some of the novel H10N4 and H10N8 viruses acquired the ability to bind human-like receptors. Animal studies indicated that these H10 viruses can replicate in mice, chickens, and ducks. Importantly, we found that the H10N4 and H10N8 viruses can transmit efficiently among chickens and ducks but induce lower HA inhibition (HI) antibody titers in ducks. These findings emphasized that annual surveillance in migratory waterfowl should be strengthened to monitor the introduction of wild-bird H10N4 and H10N8 reassortants into poultry. IMPORTANCE The emerging avian influenza reassortants and mutants in birds pose an increasing threat to poultry and public health. H10 avian influenza viruses are widely prevalent in wild birds, poultry, seals, and minks and pose an increasing threat to human health. The occasional human infections with H10N8 and H10N3 viruses in China have significantly increased public concern about the potential pandemic risk posed by H10 viruses. In this study, we found that the North American H10 viruses have been successfully introduced to Asia by migratory birds and further reassorted with other subtypes to generate novel H10N4 and H10N8 viruses in eastern China. These emerging H10 reassortants have a high potential to threaten the poultry industry and human health due to their efficient replication and transmission in chickens, ducks, and mice.

Novel H5N6 reassortants bearing the clade 2.3.4.4b HA gene of H5N8 virus have been detected in poultry and caused multiple human infections in China.

The globally circulating H5N8 avian influenza viruses bearing the clade 2.3.4.4b hemagglutinin (HA) gene are responsible for the loss of more than 33 million domestic poultry since January 2020. Moreover, the H5N8 viruses have reassorted with other avian influenza viruses and formed H5N1, H5N2, H5N3, H5N4, and H5N5 viruses in Europe, Africa, and North America. In this study, we analyzed 15 H5N6 viruses isolated from poultry and seven H5N6 viruses isolated from humans, and found these viruses formed seven different genotypes by deriving the clade 2.3.4.4b HA gene of H5N8 viruses, the neuraminidase of domestic duck H5N6 viruses, and internal genes of different viruses that previously circulated in domestic ducks and wild birds in China. Two of these genotypes (genotype 3 and genotype 6) have caused human infections in multiple provinces. The H5N6 viruses isolated from poultry have distinct pathotypes in mice; some of them replicate systemically and are highly lethal in mice. Although these viruses exclusively bind to avian-type receptors, it is worrisome that they may obtain key mutations that would increase their affinity for human-type receptors during replication in humans. Our study indicates that the novel H5N6 reassortants bearing the clade 2.3.4.4b HA gene of H5N8 viruses were generated through reassortment in domestic ducks and may have spread across a wide area of China, thereby posing a new challenge to the poultry industry and human health. Our findings emphasize the importance of careful monitoring, evaluation, and control of the H5N6 viruses circulating in nature.

PIAS1-mediated SUMOylation of influenza A virus PB2 restricts viral replication and virulence.

Host defense systems employ posttranslational modifications to protect against invading pathogens. Here, we found that protein inhibitor of activated STAT 1 (PIAS1) interacts with the nucleoprotein (NP), polymerase basic protein 1 (PB1), and polymerase basic protein 2 (PB2) of influenza A virus (IAV). Lentiviral-mediated stable overexpression of PIAS1 dramatically suppressed the replication of IAV, whereas siRNA knockdown or CRISPR/Cas9 knockout of PIAS1 expression significantly increased virus growth. The expression of PIAS1 was significantly induced upon IAV infection in both cell culture and mice, and PIAS1 was involved in the overall increase in cellular SUMOylation induced by IAV infection. We found that PIAS1 inhibited the activity of the viral RNP complex, whereas the C351S or W372A mutant of PIAS1, which lacks the SUMO E3 ligase activity, lost the ability to suppress the activity of the viral RNP complex. Notably, the SUMO E3 ligase activity of PIAS1 catalyzed robust SUMOylation of PB2, but had no role in PB1 SUMOylation and a minimal role in NP SUMOylation. Moreover, PIAS1-mediated SUMOylation remarkably reduced the stability of IAV PB2. When tested in vivo, we found that the downregulation of Pias1 expression in mice enhanced the growth and virulence of IAV. Together, our findings define PIAS1 as a restriction factor for the replication and pathogenesis of IAV.

H7N9 virus infection triggers lethal cytokine storm by activating gasdermin E-mediated pyroptosis of lung alveolar epithelial cells.

The H7N9 influenza virus emerged in China in 2013, causing more than 1560 human infections, 39% of which were fatal. A 'cytokine storm' in the lungs of H7N9 patients has been linked to a poor prognosis and death; however, the underlying mechanism that triggers the cytokine storm is unknown. Here, we found that efficient replication of the H7N9 virus in mouse lungs activates gasdermin E (GSDME)-mediated pyroptosis in alveolar epithelial cells, and that the released cytosolic contents then trigger a cytokine storm. Knockout of Gsdme switched the manner of death of A549 and human primary alveolar epithelial cells from pyroptosis to apoptosis upon H7N9 virus infection, and Gsdme knockout mice survived H7N9 virus lethal infection. Our findings reveal that GSDME activation is a key and unique mechanism for the pulmonary cytokine storm and lethal outcome of H7N9 virus infection and thus opens a new door for the development of antivirals against the H7N9 virus.

Continued evolution of H6 avian influenza viruses isolated from farms in China between 2014 and 2018.

H6 avian influenza virus (AIV) is one of the most prevalent AIV subtypes in the world. Our previous studies have demonstrated that H6 AIVs isolated from live poultry markets pose a potential threat to human health. In recent years, increasing number of H6 AIVs has been constantly isolated from poultry farms. In order to understand the biological characteristics of H6 AIVs in the context of farms, here, we analyzed the phylogenetic relationships, antigenicity, replication in mice and receptor binding properties of H6 AIVs isolated from farms in China between 2014 and 2018. Phylogenetic analysis showed that 19 different genotypes were formed among 20 representative H6 viruses. Notably, the internal genes of these H6 viruses exhibited complicated relationships with different subtypes of AIVs worldwide, indicating that these viruses are the products of complex and frequent reassortment events. Antigenic analysis revealed that 13 viruses tested were divided into three antigenic groups. 10 viruses examined could all replicate in the respiratory organs of infected mice without prior adaptation. Receptor binding analysis demonstrated that some of the H6 AIVs bound to both α-2, 3-linked glycans (avian-type receptor) and α-2, 6-linked glycans (human-type receptor), thereby posing a potential threat to human health. Together, these findings revealed the prevalence, complicated genetic evolution, diverse antigenicity, and dual receptor binding specificity of H6 AIVs in the settings of poultry farms, which emphasize the importance to continuously monitor the evolution and biological properties of H6 AIVs in nature.