On-Site and Visual Detection of the H5 Subtype Avian Influenza Virus Based on RT-RPA and CRISPR/Cas12a.

Avian influenza viruses (AIVs) of the H5 subtype rank among the most serious pathogens, leading to significant economic losses in the global poultry industry and posing risks to human health. Therefore, rapid and accurate virus detection is crucial for the prevention and control of H5 AIVs. In this study, we established a novel detection method for H5 viruses by utilizing the precision of CRISPR/Cas12a and the efficiency of RT-RPA technologies. This assay facilitates the direct visualization of detection results through blue light and lateral flow strips, accurately identifying H5 viruses with high specificity and without cross-reactivity against other AIV subtypes, NDV, IBV, and IBDV. With detection thresholds of 1.9 copies/µL (blue light) and 1.9 × 103 copies/µL (lateral flow strips), our method not only competes with but also slightly surpasses RT-qPCR, demonstrating an 80.70% positive detection rate across 81 clinical samples. The RT-RPA/CRISPR-based detection method is characterized by high sensitivity, specificity, and independence from specialized equipment. The immediate field applicability of the RT-RPA/CRISPR approach underscores its importance as an effective tool for the early detection and management of outbreaks caused by the H5 subtype of AIVs.

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.

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.

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.

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.

Rapid detection of avian influenza virus based on CRISPR-Cas12a.

BACKGROUND: Avian influenza (AI) is a disease caused by the avian influenza virus (AIV). These viruses spread naturally among wild aquatic birds worldwide and infect domestic poultry, other birds, and other animal species. Currently, real-time reverse transcription polymerase chain reaction (rRT-PCR) is mainly used to detect the presence of pathogens and has good sensitivity and specificity. However, the diagnosis requires sophisticated instruments under laboratory conditions, which significantly limits point-of-care testing (POCT). Rapid, reliable, non-lab-equipment-reliant, sensitive, and specific diagnostic tests are urgently needed for rapid clinical detection and diagnosis. Our study aimed to develop a reverse transcription recombinase polymerase amplification (RT-RPA)/CRISPR method which improves on these limitations. METHODS: The Cas12a protein was purified by affinity chromatography with Ni-agarose resin and observed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Specific CRISPR RNA (crRNA) and primers targeting the M and NP genes of the AIV were designed and screened. By combining RT-RPA with the Cas12a/crRNA trans-cleavage system, a detection system that uses fluorescence readouts under blue light or lateral flow strips was established. Sensitivity assays were performed using a tenfold dilution series of plasmids and RNA of the M and NP genes as templates. The specificity of this method was determined using H1-H16 subtype AIVs and other avian pathogens, such as newcastle disease virus (NDV), infectious bursal disease virus (IBDV), and infectious bronchitis virus (IBV). RESULTS: The results showed that the method was able to detect AIV and that the detection limit can reach 6.7 copies/µL and 12 copies/µL for the M and NP gene, respectively. In addition, this assay showed no cross-reactivity with other avian-derived RNA viruses such as NDV, IBDV, and IBV. Moreover, the detection system presented 97.5% consistency and agreement with rRT-PCR and virus isolation for detecting samples from poultry. This portable and accurate method has great potential for AIV detection in the field. CONCLUSION: An RT-RPA/CRISPR method was developed for rapid, sensitive detection of AIV. The new system presents a good potential as an accurate, user-friendly, and inexpensive platform for point-of-care testing applications.

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 Tough and Reliable Poly(Amic Acid) with Ballistic Impact-Resistance through Modulation of Hydrogen Bonding Interactions.

Poly(amic acid) (PAA) materials as the precursor of polyimide generally show remarkably poor mechanical properties, thus limiting their application as the engineering plastics. In this study, it is demonstrated that the mechanical properties of PAA materials can be improved significantly for tens of folds with breaking strength >50 MPa, Young's modulus >400 MPa, and elongation at break >300% by incorporation of 20% (mol%) poly(propylene glycol) (PPO) soft segments. The optimization for suitable hard-soft composition with 20% PPO and the existence of various hydrogen bonds with different binding energies can dissipate energies efficiently, which simultaneously improve the material strength and toughness. In addition, PAA82 films exhibit excellent tolerance toward cyclic stretch, and have the capability to resist various harsh conditions including solar radiation testing (1 sun), heat (85 °C), alkalinity (pH 10), and acidity (pH 4) over one month. Noted that PAA82 films can be laminated with Kapton films, which show excellent resistance to ultrahigh (200 °C) and ultralow temperature (-196 °C). The laminated film also exhibits bulletproof property with a thickness of 6 mm. The strategy via modulation of hard-soft compositions and hydrogen bonds in PAA materials shows great potentials to improve the mechanical properties of polymeric materials.

Genetic analysis and biological characterization of H10N3 influenza A viruses isolated in China from 2014 to 2021.

The H10 subtypes of avian influenza viruses pose a continual threat to the poultry industry and human health. The sporadic spillover of H10 subtypes viruses from poultry to humans is represented by the H10N8 human cases in 2013 and the recent H10N3 human infection in 2021. However, the genesis and characteristics of the recent reassortment H10N3 viruses have not been systemically investigated. In this study, we characterized 20 H10N3 viruses isolated in live poultry markets during routine nationwide surveillance in China from 2014 to 2021. The viruses in the recent reassortant genotype acquired their hemagglutinin (HA) and neuraminidase (NA) genes from the duck H10 viruses and H7N3 viruses, respectively, whereas the internal genes were derived from chicken H9N2 viruses as early as 2019. Receptor-binding analysis indicated that two of the tested H10N3 viruses had a higher affinity for human-type receptors than for avian-type receptors, highlighting the potential risk of avian-to-human transmission. Animal studies showed that only viruses belonging to the recent reassortant genotype were pathogenic in mice; two tested viruses transmitted via direct contact and one virus transmitted by respiratory droplets in guinea pigs, though with limited efficiency. These findings emphasize the need for enhanced surveillance of H10N3 viruses.

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.

ARNT Inhibits H5N1 Influenza A Virus Replication by Interacting with the PA Protein.

Increasing evidence suggests that the polymerase acidic (PA) protein of influenza A viruses plays an important role in viral replication and pathogenicity. However, information regarding the interaction(s) of host factors with PA is scarce. By using a yeast two-hybrid screen, we identified a novel host factor, aryl hydrocarbon receptor nuclear translocator (ARNT), that interacts with the PA protein of the H5N1 virus. The interaction between PA and human ARNT was confirmed by co-immunoprecipitation and immunofluorescence microscopy. Moreover, overexpression of ARNT downregulated the polymerase activity and inhibited virus propagation, whereas knockdown of ARNT significantly increased the polymerase activity and virus replication. Mechanistically, overexpression of ARNT resulted in the accumulation of PA protein in the nucleus and inhibited both the replication and transcription of the viral genome. Interaction domain mapping revealed that the bHLH/PAS domain of ARNT mainly interacted with the C-terminal domain of PA. Together, our results demonstrate that ARNT inhibits the replication of the H5N1 virus and could be a target for the development of therapeutic strategies against H5N1 influenza viruses.

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.

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.