Airborne transmission of human-isolated avian H3N8 influenza virus between ferrets.

H3N8 avian influenza viruses (AIVs) in China caused two confirmed human infections in 2022, followed by a fatal case reported in 2023. H3N8 viruses are widespread in chicken flocks; however, the zoonotic features of H3N8 viruses are poorly understood. Here, we demonstrate that H3N8 viruses were able to infect and replicate efficiently in organotypic normal human bronchial epithelial (NHBE) cells and lung epithelial (Calu-3) cells. Human isolates of H3N8 virus were more virulent and caused severe pathology in mice and ferrets, relative to chicken isolates. Importantly, H3N8 virus isolated from a patient with severe pneumonia was transmissible between ferrets through respiratory droplets; it had acquired human-receptor-binding preference and amino acid substitution PB2-E627K necessary for airborne transmission. Human populations, even when vaccinated against human H3N2 virus, appear immunologically naive to emerging mammalian-adapted H3N8 AIVs and could be vulnerable to infection at epidemic or pandemic proportion.

BAG6 inhibits influenza A virus replication by inducing viral polymerase subunit PB2 degradation and perturbing RdRp complex assembly.

The interaction between influenza A virus (IAV) and host proteins is an important process that greatly influences viral replication and pathogenicity. PB2 protein is a subunit of viral ribonucleoprotein (vRNP) complex playing distinct roles in viral transcription and replication. BAG6 (BCL2-associated athanogene 6) as a multifunctional host protein participates in physiological and pathological processes. Here, we identify BAG6 as a new restriction factor for IAV replication through targeting PB2. For both avian and human influenza viruses, overexpression of BAG6 reduced viral protein expression and virus titers, whereas deletion of BAG6 significantly enhanced virus replication. Moreover, BAG6-knockdown mice developed more severe clinical symptoms and higher viral loads upon IAV infection. Mechanistically, BAG6 restricted IAV transcription and replication by inhibiting the activity of viral RNA-dependent RNA polymerase (RdRp). The co-immunoprecipitation assays showed BAG6 specifically interacted with the N-terminus of PB2 and competed with PB1 for RdRp complex assembly. The ubiquitination assay indicated that BAG6 promoted PB2 ubiquitination at K189 residue and targeted PB2 for K48-linked ubiquitination degradation. The antiviral effect of BAG6 necessitated its N-terminal region containing a ubiquitin-like (UBL) domain (17-92aa) and a PB2-binding domain (124-186aa), which are synergistically responsible for viral polymerase subunit PB2 degradation and perturbing RdRp complex assembly. These findings unravel a novel antiviral mechanism via the interaction of viral PB2 and host protein BAG6 during avian or human influenza virus infection and highlight a potential application of BAG6 for antiviral drug development.

Recombinant parainfluenza virus 5 expressing clade 2.3.4.4b H5 hemagglutinin protein confers broad protection against H5Ny influenza viruses.

The global circulation of clade 2.3.4.4b H5Ny highly pathogenic avian influenza viruses (HPAIVs) in poultry and wild birds, increasing mammal infections, continues to pose a public health threat and may even form a pandemic. An efficacious vaccine against H5Ny HPAIVs is crucial for emergency use and pandemic preparedness. In this study, we developed a parainfluenza virus 5 (PIV5)-based vaccine candidate expressing hemagglutinin (HA) protein of clade 2.3.4.4b H5 HPAIV, termed rPIV5-H5, and evaluated its safety and efficacy in mice and ferrets. Our results demonstrated that intranasal immunization with a single dose of rPIV5-H5 could stimulate H5-specific antibody responses, moreover, a prime-boost regimen using rPIV5-H5 stimulated robust humoral, cellular, and mucosal immune responses in mice. Challenge study showed that rPIV5-H5 prime-boost regimen provided sterile immunity against lethal clade 2.3.4.4b H5N1 virus infection in mice and ferrets. Notably, rPIV5-H5 prime-boost regimen provided protection in mice against challenge with lethal doses of heterologous clades 2.2, 2.3.2, and 2.3.4 H5N1, and clade 2.3.4.4h H5N6 viruses. These results revealed that rPIV5-H5 can elicit protective immunity against a diverse clade of highly pathogenic H5Ny virus infection in mammals, highlighting the potential of rPIV5-H5 as a pan-H5 influenza vaccine candidate for emergency use.IMPORTANCEClade 2.3.4.4b H5Ny highly pathogenic avian influenza viruses (HPAIVs) have been widely circulating in wild birds and domestic poultry all over the world, leading to infections in mammals, including humans. Here, we developed a recombinant PIV5-vectored vaccine candidate expressing the HA protein of clade 2.3.4.4b H5 virus. Intranasal immunization with rPIV5-H5 in mice induced airway mucosal IgA responses, high levels of antibodies, and robust T-cell responses. Importantly, rPIV5-H5 conferred complete protection in mice and ferrets against clade 2.3.4.4b H5N1 virus challenge, the protective immunity was extended against heterologous H5Ny viruses. Taken together, our data demonstrate that rPIV5-H5 is a promising vaccine candidate against diverse H5Ny influenza viruses in mammals.

Spatiotemporal genotype replacement of H5N8 avian influenza viruses contributed to H5N1 emergence in 2021/2022 panzootic.

Since 2020, clade 2.3.4.4b highly pathogenic avian influenza H5N8 and H5N1 viruses have swept through continents, posing serious threats to the world. Through comprehensive analyses of epidemiological, genetic, and bird migration data, we found that the dominant genotype replacement of the H5N8 viruses in 2020 contributed to the H5N1 outbreak in the 2021/2022 wave. The 2020 outbreak of the H5N8 G1 genotype instead of the G0 genotype produced reassortment opportunities and led to the emergence of a new H5N1 virus with G1's HA and MP genes. Despite extensive reassortments in the 2021/2022 wave, the H5N1 virus retained the HA and MP genes, causing a significant outbreak in Europe and North America. Furtherly, through the wild bird migration flyways investigation, we found that the temporal-spatial coincidence between the outbreak of the H5N8 G1 virus and the bird autumn migration may have expanded the H5 viral spread, which may be one of the main drivers of the emergence of the 2020-2022 H5 panzootic.IMPORTANCESince 2020, highly pathogenic avian influenza (HPAI) H5 subtype variants of clade 2.3.4.4b have spread across continents, posing unprecedented threats globally. However, the factors promoting the genesis and spread of H5 HPAI viruses remain unclear. Here, we found that the spatiotemporal genotype replacement of H5N8 HPAI viruses contributed to the emergence of the H5N1 variant that caused the 2021/2022 panzootic, and the viral evolution in poultry of Egypt and surrounding area and autumn bird migration from the Russia-Kazakhstan region to Europe are important drivers of the emergence of the 2020-2022 H5 panzootic. These findings provide important targets for early warning and could help control the current and future HPAI epidemics.

A new chromosome-scale duck genome shows a major histocompatibility complex with several expanded multigene families.

BACKGROUND: The duck (Anas platyrhynchos) is one of the principal natural hosts of influenza A virus (IAV), harbors almost all subtypes of IAVs and resists to many IAVs which cause extreme virulence in chicken and human. However, the response of duck's adaptive immune system to IAV infection is poorly characterized due to lack of a detailed gene map of the major histocompatibility complex (MHC). RESULTS: We herein reported a chromosome-scale Beijing duck assembly by integrating Nanopore, Bionano, and Hi-C data. This new reference genome SKLA1.0 covers 40 chromosomes, improves the contig N50 of the previous duck assembly with highest contiguity (ZJU1.0) of more than a 5.79-fold, surpasses the chicken and zebra finch references in sequence contiguity and contains a complete genomic map of the MHC. Our 3D MHC genomic map demonstrated that gene family arrangement in this region was primordial; however, families such as AnplMHCI, AnplMHCIIß, AnplDMB, NKRL (NK cell receptor-like genes) and BTN underwent gene expansion events making this area complex. These gene families are distributed in two TADs and genes sharing the same TAD may work in a co-regulated model. CONCLUSIONS: These observations supported the hypothesis that duck's adaptive immunity had been optimized with expanded and diversified key immune genes which might help duck to combat influenza virus. This work provided a high-quality Beijing duck genome for biological research and shed light on new strategies for AIV control.

Enhanced stability of M1 protein mediated by a phospho-resistant mutation promotes the replication of prevailing avian influenza virus in mammals.

Avian influenza virus (AIV) can evolve multiple strategies to combat host antiviral defenses and establish efficient infectivity in mammals, including humans. H9N2 AIV and its reassortants (such as H5N6 and H7N9 viruses) pose an increasing threat to human health; however, the mechanisms involved in their increased virulence remain poorly understood. We previously reported that the M1 mutation T37A has become predominant among chicken H9N2 isolates in China. Here, we report that, since 2010, this mutation has also been found in the majority of human isolates of H9N2 AIV and its emerging reassortants. The T37A mutation of M1 protein enhances the replication of H9N2 AIVs in mice and in human cells. Interestingly, having A37 instead of T37 increases the M1 protein stability and resistance to proteasomal degradation. Moreover, T37 of the H9N2 M1 protein is phosphorylated by protein kinase G (PKG), and this phosphorylation induces the rapid degradation of M1 and reduces viral replication. Similar effects are also observed in the novel H5N6 virus. Additionally, ubiquitination at K187 contributes to M1-37T degradation and decreased replication of the virus harboring T37 in the M1 protein. The prevailing AIVs thereby evolve a phospho-resistant mutation in the M1 protein to avoid viral protein degradation by host factors, which is advantageous in terms of replication in mammalian hosts.

p21 restricts influenza A virus by perturbing the viral polymerase complex and upregulating type I interferon signaling.

Many cellular genes and networks induced in human lung epithelial cells infected with the influenza virus remain uncharacterized. Here, we find that p21 levels are elevated in response to influenza A virus (IAV) infection, which is independent of p53. Silencing, pharmacological inhibition or deletion of p21 promotes virus replication in vitro and in vivo, indicating that p21 is an influenza restriction factor. Mechanistically, p21 binds to the C-terminus of IAV polymerase subunit PA and competes with PB1 to limit IAV polymerase activity. Besides, p21 promotes IRF3 activation by blocking K48-linked ubiquitination degradation of HO-1 to enhance type I interferons expression. Furthermore, a synthetic p21 peptide (amino acids 36 to 43) significantly inhibits IAV replication in vitro and in vivo. Collectively, our findings reveal that p21 restricts IAV by perturbing the viral polymerase complex and activating the host innate immune response, which may aid the design of desperately needed new antiviral therapeutics.

Novel Avian Influenza Virus (H5N1) Clade 2.3.4.4b Reassortants in Migratory Birds, China.

Two novel reassortant highly pathogenic avian influenza viruses (H5N1) clade 2.3.4.4b.2 were identified in dead migratory birds in China in November 2021. The viruses probably evolved among wild birds through different flyways connecting Europe and Asia. Their low antigenic reaction to vaccine antiserum indicates high risks to poultry and to public health.

H9N2 virus-derived M1 protein promotes H5N6 virus release in mammalian cells: Mechanism of avian influenza virus inter-species infection in humans.

H5N6 highly pathogenic avian influenza virus (HPAIV) clade 2.3.4.4 not only exhibits unprecedented intercontinental spread in poultry, but can also cause serious infection in humans, posing a public health threat. Phylogenetic analyses show that 40% (8/20) of H5N6 viruses that infected humans carried H9N2 virus-derived internal genes. However, the precise contribution of H9N2 virus-derived internal genes to H5N6 virus infection in humans is unclear. Here, we report on the functional contribution of the H9N2 virus-derived matrix protein 1 (M1) to enhanced H5N6 virus replication capacity in mammalian cells. Unlike H5N1 virus-derived M1 protein, H9N2 virus-derived M1 protein showed high binding affinity for H5N6 hemagglutinin (HA) protein and increased viral progeny particle release in different mammalian cell lines. Human host factor, G protein subunit beta 1 (GNB1), exhibited strong binding to H9N2 virus-derived M1 protein to facilitate M1 transport to budding sites at the cell membrane. GNB1 knockdown inhibited the interaction between H9N2 virus-derived M1 and HA protein, and reduced influenza virus-like particles (VLPs) release. Our findings indicate that H9N2 virus-derived M1 protein promotes avian H5N6 influenza virus release from mammalian, in particular human cells, which could be a major viral factor for H5N6 virus cross-species infection.

Prevalent Eurasian avian-like H1N1 swine influenza virus with 2009 pandemic viral genes facilitating human infection.

Pigs are considered as important hosts or "mixing vessels" for the generation of pandemic influenza viruses. Systematic surveillance of influenza viruses in pigs is essential for early warning and preparedness for the next potential pandemic. Here, we report on an influenza virus surveillance of pigs from 2011 to 2018 in China, and identify a recently emerged genotype 4 (G4) reassortant Eurasian avian-like (EA) H1N1 virus, which bears 2009 pandemic (pdm/09) and triple-reassortant (TR)-derived internal genes and has been predominant in swine populations since 2016. Similar to pdm/09 virus, G4 viruses bind to human-type receptors, produce much higher progeny virus in human airway epithelial cells, and show efficient infectivity and aerosol transmission in ferrets. Moreover, low antigenic cross-reactivity of human influenza vaccine strains with G4 reassortant EA H1N1 virus indicates that preexisting population immunity does not provide protection against G4 viruses. Further serological surveillance among occupational exposure population showed that 10.4% (35/338) of swine workers were positive for G4 EA H1N1 virus, especially for participants 18 y to 35 y old, who had 20.5% (9/44) seropositive rates, indicating that the predominant G4 EA H1N1 virus has acquired increased human infectivity. Such infectivity greatly enhances the opportunity for virus adaptation in humans and raises concerns for the possible generation of pandemic viruses.

Insight into sludge dewatering by periodate driven directly with Fe(â ¡): Extracellular polymeric substances solubilization and mineralization.

The production of waste activated sludge is expanding in tandem with the significant growth in the global population. It is important to explore sludge pretreatment technology to achieve sludge reduction. In this study, deep sludge dewatering was achieved by using Fe2+-catalyzed periodate (Fe2+/PI) conditioning. The result showed that capillary suction time was reduced by 48.27% under the optimum Fe2+ and PI dosages. ·OH, FeⅣ, O2·-, 1O2, and IO3· generated from the reaction between Fe2+ and PI, while ·OH (49.79%) and FeⅣ (47.76%) contributed significantly to sludge dewatering. Investigations of the mechanism revealed that the synergistic action of radical species oxidation and iron species flocculation in Fe2+/PI conditioning led to the mineralization and aggregation of hydrophilic substances in extracellular polymeric substances. The hydrophobic groups on the protein surface were more exposed to soluble extracellular polymeric substances and reduced protein-water interaction. The variations in zeta potential and particle size also verified the presence of a synergistic effect of oxidation and flocculation. The morphology observations revealed that the increased frictional forces generated when water flowed over the raw sludge (RS) surface prevented the rapid passage of internal water. In addition, the hydrophobic and electrostatic interactions in the sludge samples were essential influences that promoted flocculation and sedimentation of the sludge. This research aids engineers by providing a new option to better optimize sludge management while also deepening understanding of the Fe2+/PI conditioning involved in sludge dewatering.

Neurovirulence of avian influenza virus is dependent on the interaction of viral NP protein with host factor FMRP in the murine brain.

Avian influenza viruses (AIVs) are zoonotic viruses that exhibit a range infectivity and severity in the human host. Severe human cases of AIVs infection are often accompanied by neurological symptoms, however, the factors involved in the infection of the central nervous system (CNS) are not well known. In this study, we discovered that avian-like sialic acid (SA)-α2, 3 Gal receptor is highly presented in mammalian (human and mouse) brains. In the generation of a mouse-adapted neurotropic H9N2 AIV (SD16-MA virus) in BALB/c mice, we identified key adaptive mutations in its hemagglutinin (HA) and polymerase basic protein 2 (PB2) genes that conferred viral replication ability in mice brain. The SD16-MA virus showed binding affinity for avian-like SA-α2, 3 Gal receptor, enhanced viral RNP polymerase activity, increased viral protein production and transport that culminated in elevated progeny virus production and severe pathogenicity. We further established that host Fragile X Mental Retardation Protein (FMRP), a highly expressed protein in the brain that physically associated with viral nucleocapsid protein (NP) to facilitate RNP assembly and export, was an essential host factor for the neuronal replication of neurotropic AIVs (H9N2, H5N1 and H10N7 viruses). Our study identified a mechanistic process for AIVs to acquire neurovirulence in mice.IMPORTANCE Infection of the CNS is a serious complication of human cases of AIVs infection. The viral and host factors associated with neurovirulence of AIVs infection are not well understood. We identified and functionally characterized specific changes in the viral HA and PB2 genes of a mouse-adapted neurotropic avian H9N2 virus responsible for enhanced virus replication in neuronal cells and pathogenicity in mice. Importantly, we showed that host FMRP was a crucial host factor that was necessary for neurotropic AIVs (H9N2, H5N1 and H10N7 viruses) to replicate in neuronal cells. Our findings have provided insights into the pathogenesis of neurovirulence of AIV infection.

Reassortment with dominant chicken H9N2 influenza virus contributed to the fifth H7N9 virus human epidemic.

H9N2 Avian influenza virus (AIV) is regarded as a principal donor of viral genes through reassortment to co-circulating influenza viruses that can result in zoonotic reassortants. Whether H9N2 virus can maintain sustained evolutionary impact on such reassortants is unclear. Since 2013, avian H7N9 virus had caused five sequential human epidemics in China; the fifth wave in 2016-2017 was by far the largest but the mechanistic explanation behind the scale of infection is not clear. Here, we found that, just prior to the fifth H7N9 virus epidemic, H9N2 viruses had phylogenetically mutated into new sub-clades, changed antigenicity and increased its prevalence in chickens vaccinated with existing H9N2 vaccines. In turn, the new H9N2 virus sub-clades of PB2 and PA genes, housing mammalian adaptive mutations, were reassorted into co-circulating H7N9 virus to create a novel dominant H7N9 virus genotype that was responsible for the fifth H7N9 virus epidemic. H9N2-derived PB2 and PA genes in H7N9 virus conferred enhanced polymerase activity in human cells at 33°C and 37°C, and increased viral replication in the upper and lower respiratory tracts of infected mice which could account for the sharp increase in human cases of H7N9 virus infection in the 2016-2017 epidemic. The role of H9N2 virus in the continual mutation of H7N9 virus highlights the public health significance of H9N2 virus in the generation of variant reassortants of increasing zoonotic potential.IMPORTANCEAvian H9N2 influenza virus, although primarily restricted to chicken populations, is a major threat to human public health by acting as a donor of variant viral genes through reassortment to co-circulating influenza viruses. We established that the high prevalence of evolving H9N2 virus in vaccinated flocks played a key role, as donor of new sub-clade PB2 and PA genes in the generation of a dominant H7N9 virus genotype (G72) with enhanced infectivity in humans during the 2016-2017 N7N9 virus epidemic. Our findings emphasize that the ongoing evolution of prevalent H9N2 virus in chickens is an important source, via reassortment, of mammalian adaptive genes for other influenza virus subtypes. Thus, close monitoring of prevalence and variants of H9N2 virus in chicken flocks is necessary in the detection of zoonotic mutations.

Correction: Induction of PGRN by influenza virus inhibits the antiviral immune responses through downregulation of type I interferons signaling.

[This corrects the article DOI: 10.1371/journal.ppat.1008062.].

Comparison of curative effect between different retrograde filling materials in young permanent molar root canal therapy.

The first mandible and maxilla permanent molars are the first permanent teeth that grow next to the deciduous teeth and may decay due to carelessness. Their caries can spread to the pulpal tentacles and cause pulpal and periapical diseases. In the current study, we tried to compare the curative effect of different retrograde filling materials, i.e. white MTA, gray MTA, Portland cement, and IRM, in young permanent molar root canal therapy. Because IL-1ß stimulates bone degradation by osteoclasts, IL-1ß gene expression was also measured for further evaluation. For this purpose, 400 students (240 boys and 160 girls) aged 8 to 11 years referred to the Pediatric Dental Center for first permanent molar root canal therapy were selected during two years. After recording the demographic characteristics of each patient, the first permanent molar teeth were examined by a general dentist with Abslang and decayed teeth were considered to have both discolorations in their grooves and apparent opacity. The patients, who need root canal therapy, were divided into four groups. The first group was treated with gray MTA. The second group was treated with white MTA. The third group received Portland cement for root canal therapy. The fourth group was treated with IRM. Also, IL-1ß gene expression was evaluated by the real-time PCR technique. Relative changes in gene expression in PBMC cells were performed using One Way ANOVA. SPSS 18 software was used to determine the correlation of gene expression in PBMCs. The results showed that there was no significant difference between the groups in terms of age (p = 0.12) and gender (p = 0.24). Also, the need for endodontic treatment in the mandible (n = 278) was higher than the maxilla (n = 85) and both jaws (n = 37). But there was no significant difference between the groups in terms of the need for endodontic treatment (p = 0.32). The results of Pearson correlation coefficients between studied groups in terms of IL-1ß gene expression showed that gray MTA and white MTA were not statistically different, but MTAs were generally different from Portland cement and IRM, with higher IL-1ß gene expression. In general, the results showed that the teeth in the vicinity of gray MTA and white MTA showed a more appropriate response than Portland cement and IRM, so the use of MTA and its preference over other materials is recommended. In the case of Portland cement, more studies are needed to reach a conclusion comparing this material with MTA.

A novel pre-magnetized ZVI/PS pretreatment for improving sludge dewaterability: The role of EPS fractions.

The dewaterability of waste-activated sludge (WAS) has been extensively examined using zero-valent iron (ZVI)-based advanced oxidation processes (AOPs). However, the high dosage and low utilization efficiencies of ZVI cast doubt on the dependability and viability of ZVI-based AOPs. In this study, we successfully demonstrated pre-magnetization as an efficient, chemical-free, and ecological method for improving the efficiency of sludge dewatering by ZVI/persulfate (PS) process, in which the reduction ratios of capillary suction time (CST) and specific resistance to filtration (SRF) increased by 8.67% and 11.06% under optimal conditions, respectively. The highly active Fe2+ released during ZVI corrosion may be more essential than ZVI itself during PS activation, which could be strengthened by pre-magnetization. Both homogeneous and heterogeneous Fe2+ could react with PS to produce aqueous hydroxyl radicals (∙OH) and sulfate radicals (SO4-∙) as well as surface-bound ∙OH and SO4-∙, further decomposing bound-extracellular polymeric substances fractions, broking hydrophilic functional groups and compounds, altering protein secondary structure to expose more hydrophobic sites, and releasing abundant EPS-bound water. Due to the protection of tightly-bound extracellular polymeric substances (TB-EPS) and the competitive oxidation of organics released during the early disintegration stage, radical oxidation primarily occurs at extracellular levels, releasing a bit of intracellular water. Besides, polysaccharides in TB-EPS may function a more significant role in flocculation than proteins, and a porous structure favorable to drainage will be formed after the pre-magnetized ZVI/PS treatment. The cost-benefit analysis further reveals that the Pre-ZVI/PS process presents high reusability and utilization, making it potential for particle application in sludge dewatering.

Truncation of PA-X Contributes to Virulence and Transmission of H3N8 and H3N2 Canine Influenza Viruses in Dogs.

Equine-origin H3N8 and avian-origin H3N2 canine influenza viruses (CIVs) prevalent in dogs are thought to pose a public health threat arising from intimate contact between dogs and humans. However, our understanding of CIV virulence is still limited. Influenza A virus PA-X is a fusion protein encoded in part by a +1 frameshifted open reading frame (X-ORF) in segment 3. The X-ORF can be translated in full-length (61-amino-acid) or truncated (41-amino-acid) form. Genetic analysis indicated that the X-ORFs of equine H3N8 and avian H3N2 influenza viruses encoded 61 amino acids but were truncated after introduction into dogs. To determine the effect of PA-X truncation on the biological characteristics of CIVs, we constructed four recombinant viruses on H3N8 and H3N2 CIV backgrounds bearing truncated or full-length PA-Xs. We observed that truncation of PA-X increased growth of both H3N8 and H3N2 CIVs in MDCK cells and suppressed expression from cotransfected plasmids in MDCK cells. Furthermore, truncation of PA-X enhanced viral pathogenicity in dogs, as shown by aggravated clinical symptoms and histopathological changes, increased viral replication in the respiratory system, and prolonged virus shedding. Additionally, CIVs with truncated PA-Xs were transmitted more efficiently in dogs. Global gene expression profiling of the lungs of infected dogs revealed that differentially expressed genes were mainly associated with inflammatory responses, which might contribute to the pathogenicity of PA-X-truncated CIVs. Our findings revealed that truncation of PA-X might be important for the adaptation of influenza viruses to dogs.IMPORTANCE Epidemics of equine-origin H3N8 and avian-origin H3N2 influenza viruses in canine populations are examples of successful cross-species transmission of influenza A viruses. Genetic analysis showed that the PA-X genes of equine H3N8 or avian H3N2 influenza viruses were full-length, with X-ORFs encoding 61 amino acids; however, those of equine-origin H3N8 or avian-origin H3N2 CIVs were truncated, suggesting that PA-X truncation occurred after transmission to dogs. In this study, we extended the PA-X genes of H3N8 and H3N2 CIVs and compared the biological characteristics of CIVs bearing different lengths of PA-X. We demonstrated that for both H3N8 and H3N2 viruses, truncation of PA-X increased virus yields in MDCK cells and enhanced viral replication, pathogenicity, and transmission in dogs. These results might reflect enhanced suppression of host gene expression and upregulation of genes related to inflammatory responses. Collectively, our data partially explain the conservation of truncated PA-X in CIVs.

Swine MicroRNAs ssc-miR-221-3p and ssc-miR-222 Restrict the Cross-Species Infection of Avian Influenza Virus.

Avian influenza virus (AIV) can cross species barriers to infect humans and other mammals. However, these species-cross transmissions are most often dead-end infections due to host restriction. Current research about host restriction focuses mainly on the barriers of cell membrane, nuclear envelope, and host proteins; whether microRNAs (miRNAs) play a role in host restriction is largely unknown. In this study, we used porcine alveolar macrophage (PAM) cells as a model to elucidate the role of miRNAs in host range restriction. During AIV infection, 40 dysregulation expressed miRNAs were selected in PAM cells. Among them, two Sus scrofa (ssc; swine) miRNAs, ssc-miR-221-3p and ssc-miR-222, could inhibit the infection and replication of AIV in PAM cells by directly targeting viral genome and inducing cell apoptosis via inhibiting the expression of anti-apoptotic protein HMBOX1. Avian but not swine influenza virus caused upregulated expressions of ssc-miR-221-3p and ssc-miR-222 in PAM cells. We further found that NF-κB P65 was more effectively phosphorylated upon AIV infection and that P65 functioned as a transcription activator to regulate the AIV-induced expression of miR-221-3p/222 Importantly, we found that ssc-miR-221-3p and ssc-miR-222 could also be specifically upregulated upon AIV infection in newborn pig tracheal epithelial (NPTr) cells and also exerted anti-AIV function. In summary, our study indicated that miRNAs act as a host barrier during cross-species infection of influenza A virus.IMPORTANCE The host range of an influenza A virus is determined by species-specific interactions between virus and host cell factors. Host miRNAs can regulate influenza A virus replication; however, the role of miRNAs in host species specificity is unclear. Here, we show that the induced expression of ssc-miR-221-3p and ssc-miR-222 in swine cells is modulated by NF-κB P65 phosphorylation in response to AIV infection but not swine influenza virus infection. ssc-miR-221-3p and ssc-miR-222 exerted antiviral function via targeting viral RNAs and causing apoptosis by inhibiting the expression of HMBOX1 in host cells. These findings uncover miRNAs as a host range restriction factor that limits cross-species infection of influenza A virus.

An R195K Mutation in the PA-X Protein Increases the Virulence and Transmission of Influenza A Virus in Mammalian Hosts.

In the 21st century, the emergence of H7N9 and H1N1/2009 influenza viruses, originating from animals and causing severe human infections, has prompted investigations into the genetic alterations required for cross-species transmission. We previously found that replacement of the human-origin PA gene segment in avian influenza virus (AIV) could overcome barriers to cross-species transmission. Recently, it was reported that the PA gene segment encodes both the PA protein and a second protein, PA-X. Here, we investigated the role of PA-X. We found that an H9N2 avian influenza reassortant virus bearing a human-origin H1N1/2009 PA gene was attenuated in mice after the loss of PA-X. Reverse genetics analyses of PA-X substitutions conserved in human influenza viruses indicated that R195K, K206R, and P210L substitutions conferred significantly increased replication and pathogenicity on H9N2 virus in mice and ferrets. PA-X R195K was present in all human H7N9 and H1N1/2009 viruses and predominated in human H5N6 viruses. Compared with PA-X 195R, H7N9 influenza viruses bearing PA-X 195K showed increased replication and transmission in ferrets. We further showed that PA-X 195K enhanced lung inflammatory responses, potentially due to decreased host shutoff function. A competitive transmission study in ferrets indicated that 195K provides a replicative advantage over 195R in H1N1/2009 viruses. In contrast, PA-X 195K did not influence the virulence of H9N2 AIV in chickens, suggesting that the effects of the substitution were mammal specific. Therefore, future surveillance efforts should scrutinize this region of PA-X because of its potential impact on cross-species transmission of influenza viruses.IMPORTANCE Four influenza pandemics in humans (the Spanish flu of 1918 [H1N1], the Asian flu of 1957 [H2N2], the Hong Kong flu of 1968 [H3N2], and the swine origin flu of 2009 [H1N1]) are all proposed to have been caused by avian or swine influenza viruses that acquired virulence factors through adaptive mutation or reassortment with circulating human viruses. Currently, influenza viruses circulating in animals are repeatedly transmitted to humans, posing a significant threat to public health. However, the molecular properties accounting for interspecies transmission of influenza viruses remain unclear. In the present study, we demonstrated that PA-X plays an important role in cross-species transmission of influenza viruses. At least three human-specific amino acid substitutions in PA-X dramatically enhanced the adaptation of animal influenza viruses in mammals. In particular, PA-X 195K might have contributed to cross-species transmission of H7N9, H5N6, and H1N1/2009 viruses from animal reservoirs to humans.

Induction of PGRN by influenza virus inhibits the antiviral immune responses through downregulation of type I interferons signaling.

Type I interferons (IFNs) play a critical role in host defense against influenza virus infection, and the mechanism of influenza virus to evade type I IFNs responses remains to be fully understood. Here, we found that progranulin (PGRN) was significantly increased both in vitro and in vivo during influenza virus infection. Using a PGRN knockdown assay and PGRN-deficient mice model, we demonstrated that influenza virus-inducing PGRN negatively regulated type I IFNs production by inhibiting the activation of NF-κB and IRF3 signaling. Furthermore, we showed that PGRN directly interacted with NF-κB essential modulator (NEMO) via its Grn CDE domains. We also verified that PGRN recruited A20 to deubiquitinate K63-linked polyubiquitin chains on NEMO at K264. In addition, we found that macrophage played a major source of PGRN during influenza virus infection, and PGRN neutralizing antibodies could protect against influenza virus-induced lethality in mice. Our data identify a PGRN-mediated IFN evasion pathway exploited by influenza virus with implication in antiviral applications. These findings also provide insights into the functions and crosstalk of PGRN in innate immunity.