Avian Influenza Virus Infection Research Articles

Role of miRNA in Highly Pathogenic H5 Avian Influenza Virus Infection: An Emphasis on Cellular and Chicken Models

The avian influenza virus, particularly the H5N1 strain, poses a significant and ongoing threat to both human and animal health. Recent outbreaks have affected domestic and wild birds on a massive scale, raising concerns about the virus’ spread to mammals. This review focuses on the critical role of microRNAs (miRNAs) in modulating pro-inflammatory signaling pathways during the pathogenesis of influenza A virus (IAV), with an emphasis on highly pathogenic avian influenza (HPAI) H5 viral infections. Current research indicates that miRNAs play a significant role in HPAI H5 infections, influencing various aspects of the disease process. This review aims to synthesize recent findings on the impact of different miRNAs on immune function, viral cytopathogenicity, and respiratory viral replication. Understanding these mechanisms is essential for developing new therapeutic strategies to combat avian influenza and mitigate its effects on both human and animal populations.

Role of miRNA in Highly Pathogenic H5 Avian Influenza Virus Infection: An Emphasis on Cellular and Chicken Models

The avian influenza virus, particularly the H5N1 strain, poses a significant and ongoing threat to both human and animal health. Recent outbreaks have affected domestic and wild birds on a massive scale, raising concerns about the virus’ spread to mammals. This review focuses on the critical role of microRNAs (miRNAs) in modulating pro-inflammatory signaling pathways during the pathogenesis of influenza A virus (IAV), with an emphasis on highly pathogenic avian influenza (HPAI) H5 viral infections. Current research indicates that miRNAs play a significant role in HPAI H5 infections, influencing various aspects of the disease process. This review aims to synthesize recent findings on the impact of different miRNAs on immune function, viral cytopathogenicity, and respiratory viral replication. Understanding these mechanisms is essential for developing new therapeutic strategies to combat avian influenza and mitigate its effects on both human and animal populations.

A(H2N2) and A(H3N2) influenza pandemics elicited durable cross-reactive and protective antibodies against avian N2 neuraminidases

Human cases of avian influenza virus (AIV) infections are associated with an age-specific disease burden. As the influenza virus N2 neuraminidase (NA) gene was introduced from avian sources during the 1957 pandemic, we investigate the reactivity of N2 antibodies against A(H9N2) AIVs. Serosurvey of healthy individuals reveal the highest rates of AIV N2 antibodies in individuals aged ≥65 years. Exposure to the 1968 pandemic N2, but not recent N2, protected against A(H9N2) AIV challenge in female mice. In some older adults, infection with contemporary A(H3N2) virus could recall cross-reactive AIV NA antibodies, showing discernable human- or avian-NA type reactivity. Individuals born before 1957 have higher anti-AIV N2 titers compared to those born between 1957 and 1968. The anti-AIV N2 antibodies titers correlate with antibody titers to the 1957 N2, suggesting that exposure to the A(H2N2) virus contribute to this reactivity. These findings underscore the critical role of neuraminidase immunity in zoonotic and pandemic influenza risk assessment.

Characterization of highly pathogenic avian influenza virus in retail dairy products in the US.

In March 2024, clade 2.3.4.4b H5N1 highly pathogenic avian influenza virus (HPAIV) was detected in dairy cattle in the US, and it was discovered that the virus could be detected in raw milk. Although affected cow's milk is diverted from human consumption and current pasteurization requirements are expected to reduce or eliminate infectious HPAIV from the milk supply, a study was conducted to characterize whether the virus could be detected by quantitative real-time RT-PCR (qrRT-PCR) in pasteurized retail dairy products and, if detected, to determine whether the virus was viable. From 18 April to 22 April 2024, a total of 297 samples of Grade A pasteurized retail milk products (23 product types) were collected from 17 US states that represented products from 132 processors in 38 states. Viral RNA was detected in 60 samples (20.2%), with qrRT-PCR-based quantity estimates (non-infectious) of up to 5.4log1050% egg infectious doses per mL, with a mean and median of 3.0log10/mL and 2.9log10/mL, respectively. Samples that were positive for type A influenza by qrRT-PCR were confirmed to be clade 2.3.4.4 H5 HPAIV by qrRT-PCR. No infectious virus was detected in any of the qrRT-PCR-positive samples in embryonating chicken eggs. Further studies are needed to monitor the milk supply, but these results provide evidence that the infectious virus did not enter the US pasteurized milk supply before control measures for HPAIV were implemented in dairy cattle.IMPORTANCEHighly pathogenic avian influenza virus (HPAIV) infections in US dairy cattle were first confirmed in March 2024. Because the virus could be detected in raw milk, a study was conducted to determine whether it had entered the retail food supply. Pasteurized dairy products were collected from 17 states in April 2024. Viral RNA was detected in one in five samples, but infectious virus was not detected. This provides a snapshot of HPAIV in milk products early in the event and reinforces that with current safety measures, infectious viruses in milk are unlikely to enter the food supply.

Inactivation of Avian Influenza Virus Inoculated into Ground Beef Patties Cooked on a Commercial Open-Flame Gas Grill

With the emergence of clade 2.3.4.4b H5N1 highly pathogenic avian influenza virus (AIV) infection of dairy cattle and its subsequent detection in raw milk, coupled with recent AIV infections affecting dairy farm workers, experiments were conducted to affirm the safety of cooked ground beef related to AIV because such meat is often derived from cull dairy cows. Specifically, retail ground beef (percent lean:fat = ca. 80:20) was inoculated with a low pathogenic AIV (LPAIV) isolate to an initial level of 5.6 log10 EID50 per 300 g patty. The inoculated meat was pressed into patties (ca. 2.54 cm thick, ca. 300 g each) and then held at 4 °C for up to 60 min. In each of the two trials, two patties for each of the following three treatments were cooked on a commercial open-flame gas grill to internal instantaneous temperatures of 48.9 °C (120°F), 62.8 °C (145°F), or 71.1 °C (160°F), but without any dwell time. Cooking inoculated ground beef patties to 48.9 °C (ave. cooking time of ca. 15 min) resulted in a mean reduction of ≥2.5 ± 0.9 log10 50% egg infectious doses (EID50) per 300 g of ground beef as assessed via quantification of virus in embryonating chicken eggs (ECEs). Likewise, cooking patties on a gas grill to 62.8 °C (ave. cooking time of ca. 21 min) or to the USDA FSIS recommended minimum internal temperature for ground beef of 71.1 °C (ave. cooking time of ca. 24 min) resulted in a reduction to nondetectable levels from initial levels of ≥5.6 log10 EID50 per 300 g. These data establish that levels of infectious AIV are substantially reduced within inoculated ground beef patties (20% fat) using recommended cooking procedures.

RNF216 Inhibits the Replication of H5N1 Avian Influenza Virus and Regulates the RIG-I Signaling Pathway in Ducks.

The RING finger (RNF) family, a group of E3 ubiquitin ligases, plays multiple essential roles in the regulation of innate immunity and resistance to viral infection in mammals. However, it is still unclear whether RNF proteins affect the production of IFN-I and the replication of avian influenza virus (AIV) in ducks. In this article, we found that duck RNF216 (duRNF216) inhibited the duRIG-I signaling pathway. Conversely, duRNF216 deficiency enhanced innate immune responses in duck embryonic fibroblasts. duRNF216 did not interacted with duRIG-I, duMDA5, duMAVS, duSTING, duTBK1, or duIRF7 in the duck RIG-I pathway. However, duRNF216 targeted duTRAF3 and inhibited duMAVS in the recruitment of duTRAF3 in a dose-dependent manner. duRNF216 catalyzed K48-linked polyubiquitination of duck TRAF3, which was degraded by the proteasome pathway. Additionally, AIV PB1 protein competed with duTRAF3 for binding to duRNF216 to reduce degradation of TRAF3 by proteasomes in the cytoplasm, thereby slightly weakening duRNF216-mediated downregulation of IFN-I. Moreover, although duRNF216 downregulated the IFN-β expression during virus infection, the expression level of IFN-β in AIV-infected duck embryonic fibroblasts overexpressing duRNF216 was still higher than that in uninfected cells, which would hinder the viral replication. During AIV infection, duRNF216 protein targeted the core protein PB1 of viral polymerase to hinder viral polymerase activity and viral RNA synthesis in the nucleus, ultimately strongly restricting viral replication. Thus, our study reveals a new mechanism by which duRNF216 downregulates innate immunity and inhibits AIV replication in ducks. These findings broaden our understanding of the mechanisms by which the duRNF216 protein affects AIV replication in ducks.

Effect of Nonpharmaceutical Interventions in Preventing COVID-19 on the Circulation of Avian Influenza Virus in Wuhan, Hubei Province, China

Background. In late 2019, several medical institutions in Wuhan, Hubei Province, China, reported cases of unexplained pneumonia. A novel coronavirus was isolated from human airway epithelial cells causing coronavirus disease-2019 (COVID-19). In recent years, many nonpharmaceutical interventions (NPIs) have been implemented to stop COVID-19 epidemic. This study aimed to explore the effect of NPIs on the circulation of avian influenza virus (AIV) in Wuhan. Materials and Methods. External environmental samples were collected and subjected to viral RNA extraction. Real-time quantitative polymerase chain reaction was used to detect the H5, H7, and H9 subtypes of AIV. Statistical analyses were performed using the chi-square test and binary logistic regression in SPSS 20.0 software. Results. A total of 2,451 external environmental samples were collected from seven districts from 2018 to 2022 in Wuhan, comprising 1,041 samples collected before COVID-19 and 1,410 samples after COVID-19. After COVID-19, the positive rate of AIV decreased significantly with the implementation of NPIs. The dominant subtype was the H9 subtype, followed by the H5 subtype. The positive rates of AIV in live poultry markets and poultry free-range sites were reduced significantly through the implementation of NPIs. Among the different sample types, higher positive rates of AIV were found in chopping boards, sewage, and cages. The positive rate of AIV was higher in trafficked source samples than that in autotrophic source samples. Conclusions. This study identified the characteristics of AIV in terms of different districts, surveillance sites, sample types, and bird sources in Wuhan. This study conducted a multifactorial analysis of the factors affecting AIV infection and provided a theoretical basis and guidance for the future prevention and control of AIV in Wuhan.

Revealing novel and conservative T-cell epitopes with MHC B2 restriction on H9N2 avian influenza virus (AIV)

B2 haplotype major histocompatibility complex (MHC) has been extensively reported to confer resistance to various avian diseases. But its peptide-binding motif is unknown, and the presenting peptide is rarely identified. Here, we identified its peptide-binding motif (X-A/V/I/L/P/S/G-X-X-X-X-X-X-V/I/L) invitro using Random Peptide Library-based MHC I LC-MS/MS analysis. To further clarify the structure basis of motif, we determined the crystal structure of the BF2∗02:01-PB2552-560 complex at 1.9Å resolution. We found that BF2∗02:01 had a relatively wide antigen-binding groove, and the structural characterization of pockets was consistent with the characterization of peptide-binding motif. The wider features of the peptide-binding motif and increased number of peptides bound by BF2∗02:01 than BF2∗04:01 might resolve the puzzles for the presence of potential H9N2 resistance in B2 chickens. Afterward, we explored the H9N2 avian influenza virus (AIV)-induced cellular immune response in B2 haplotype chickens invivo. We found that ratio of CD8+ T cell and kinetic expression of cytotoxicity genes including Granzyme K, interferon-γ, NK lysin, and poly-(ADP-ribose) polymerase in peripheral blood mononuclear cells were significantly increased in defending against H9N2 AIV infection. Especially, we selected 425 epitopes as candidate epitopes based on the peptide-binding motif and further identified four CD8+ T-cell epitopes on H9N2 AIV including NS198-106, PB2552-560, NP182-190, and NP455-463 via ELI-spot interferon-γ detections after stimulating memory lymphocytes with peptides. More importantly, these epitopes were found to be conserved in H7N9 AIV and H9N2 AIV. These findings provide direction for developing effective T cell epitope vaccines using well-conserved internal viral antigens in chickens.

Inhibitory effects of Belamcanda extract on inflammatory response and antiviral mechanism in H9N2 Avian influenza virus: insights from in vitro and in vivo studies

Avian influenza, particularly the H9N2 subtype, presents significant challenges to poultry health, underscoring the need for effective antiviral interventions. This study explores the antiviral capabilities of Belamcanda extract, a traditional Chinese medicinal herb, against H9N2 Avian Influenza Virus (AIV) in Specific Pathogen-Free (SPF) chicks. Through a comprehensive approach, we evaluated the impact of the extract on cytokine modulation and crucial immunological signaling pathways, essential for understanding the host-virus interaction. Our findings demonstrate that Belamcanda extract significantly modulates the expression of key inflammatory cytokines, including Tumor Necrosis Factor Alpha (TNF-α), Interleukin-1 (IL-1), Interleukin-2 (IL-2), and Interleukin-6 (IL-6), which are pivotal to the host's response to H9N2 AIV infection. Western blot analysis further revealed that the extract markedly reduces the expression of critical immune signaling molecules such as Toll-Like Receptor 3 (TLR3), TIR-domain-containing adapter-inducing interferon-β (TRIF), and Nuclear Factor Kappa B (NF-κB). These insights into the mechanisms by which Belamcanda extract influences host immune responses and hinders viral replication highlight its potential as an innovative antiviral agent for poultry health management. The study advances our comprehension of natural compounds' antiviral mechanisms and lays the groundwork for developing strategies to manage viral infections in poultry. The demonstrated ability of Belamcanda extract to modulate immune responses and inhibit viral replication establishes it as a promising candidate for future antiviral therapy development, especially in light of the need for effective treatments against evolving influenza virus strains and the critical demand for enhanced poultry health management strategies.

Ptičja influenca u divljih kanida

The risk of cross-species transmission of infectious diseases and zoonoses has increased due to the adaptive evolution of pathogens and anthropogenic landscape changes. A current example is the avian influenza (AI) virus, which can infect not only avian species but also mammals. In these species, infections are often associated with severe neurological symptoms. AI viruses are primarily not well adapted to mammalian hosts and are not efficiently transmitted among them. To accomplish this, AI viruses must acquire adaptations or mutations that contribute to replication efficiency and virulence in mammals. The exact combination and interaction of mutations that result in optimal adaptation to mammals is still unknown and may vary between host species and virus subtypes. The globally increasing number of infected birds with highly pathogenic AI (HPAI) increases the likelihood of contact between birds and mammals, especially wildlife. One reason for the adaptation of HPAI to mammals is theexposure of wild mammals to infected birds or their carcasses. According to the current data, wild canids, such as red foxes, are among the most infected mammals. However, infections caused by neurotropic viruses, as well as HPAI, have been scarcely studied or not at all in jackals. Since jackals are scavengers that have spread rapidly in Europe and Croatia in recent years, surveillance of AI virus infection in these canids is of the utmost importance. Furthermore, interactions between wild carnivores and domestic animals are of concern, as infections with HPAI pose a public health threat due to the increased risk of mammalian adaptation.

Molecular Characterization of Non-H5 and Non-H7 Avian Influenza Viruses from Non-Mallard Migratory Waterbirds of the North American Flyways, 2006-2011.

The surveillance of migratory waterbirds (MWs) for avian influenza virus (AIV) is indispensable for the early detection of a potential AIV incursion into poultry. Surveying AIV infections and virus subtypes in understudied MW species could elucidate their role in AIV ecology. Oropharyngeal-cloacal (OPC) swabs were collected from non-mallard MWs between 2006 and 2011. OPC swabs (n = 1158) that molecularly tested positive for AIV (Cts ≤ 32) but tested negative for H5 and H7 subtypes were selected for virus isolation (VI). The selected samples evenly represented birds from all four North American flyways (Pacific, Central, Mississippi, and Atlantic). Eighty-seven low pathogenic AIV isolates, representing 31 sites in 17 states, were recovered from the samples. All isolates belonged to the North American lineage. The samples representing birds from the Central Flyway had the highest VI positive rate (57.5%) compared to those from the other flyways (10.3-17.2%), suggesting that future surveillance can focus on the Central Flyway. Of the isolates, 43.7%, 12.6%, and 10.3% were obtained from blue-winged teal, American wigeon, and American black duck species, respectively. Hatch-year MWs represented the majority of the isolates (70.1%). The most common H and N combinations were H3N8 (23.0%), H4N6 (18.4%), and H4N8 (18.4%). The HA gene between non-mallard and mallard MW isolates during the same time period shared 85.5-99.5% H3 identity and 89.3-99.7% H4 identity. Comparisons between MW (mallard and non-mallard) and poultry H3 and H4 isolates also revealed high similarity (79.0-99.0% and 88.7-98.4%), emphasizing the need for continued AIV surveillance in MWs.

Automated quantification of avian influenza virus antigen in different organs.

As immunohistochemistry is valuable for determining tissue and cell tropism of avian influenza viruses (AIV), but time-consuming, an artificial intelligence-based workflow was developed to automate the AIV antigen quantification. Organ samples from experimental AIV infections including brain, heart, lung and spleen on one slide, and liver and kidney on another slide were stained for influenza A-matrixprotein and analyzed with QuPath: Random trees algorithms were trained to identify the organs on each slide, followed by threshold-based quantification of the immunoreactive area. The algorithms were trained and tested on two different slide sets, then retrained on both and validated on a third set. Except for the kidney, the best algorithms for organ selection correctly identified the largest proportion of the organ area. For most organs, the immunoreactive area assessed following organ selection was significantly and positively correlated to a manually assessed semiquantitative score. In the validation set, intravenously infected chickens showed a generally higher percentage of immunoreactive area than chickens infected oculonasally. Variability between the slide sets and a similar tissue texture of some organs limited the ability of the algorithms to select certain organs. Generally, suitable correlations of the immunoreactivity data results were achieved, facilitating high-throughput analysis of AIV tissue tropism.

Cecropin AD reduces viral load and inflammatory response against H9N2 avian influenza virus in chickens.

This study focuses on evaluating the therapeutic efficacy of cecropin AD, an antimicrobial peptide, against H9N2 avian influenza virus (AIV) in chickens. Given the global impact of H9N2 AIV on poultry health, identifying effective treatments is crucial. To assess the impact of cecropin AD, we conducted in vivo experiments involving 108 5-week-old chickens divided into control, infected, and various treatment groups based on cecropin AD dosage levels (high, medium, and low). The methodologies included hemagglutination (HA) tests for viral titers, histopathological examination and toluidine blue (TB) staining for lung pathology, real-time PCR for viral detection, and enzyme-linked immunosorbent assays for measuring serum levels of inflammatory markers. The findings revealed that cecropin AD substantially reduced lung pathology and viral load, especially at higher dosages, comparing favorably with the effects seen from conventional treatments. Moreover, cecropin AD effectively modulated mast cell activity and the levels of inflammatory markers such as IL-6, TNF-α, IFN-γ, and 5-HT, indicating its potential to diminish inflammation and viral spread. Cecropin AD presents a significant potential as an alternative treatment for H9N2 AIV in chickens, as evidenced by its ability to lessen lung damage, decrease viral presence, and adjust immune responses. This positions cecropin AD as a promising candidate for further exploration in the management of H9N2 AIV infections in poultry.

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.

TRIM21 Promotes Oxidative Stress and Ferroptosis through the SQSTM1-NRF2-KEAP1 Axis to Increase the Titers of H5N1 Highly Pathogenic Avian Influenza Virus.

Tripartite motif-containing protein 21 (TRIM21) is involved in signal transduction and antiviral responses through the ubiquitination of protein targets. TRIM21 was reported to be related to the imbalance of host cell homeostasis caused by viral infection. Our studies indicated that H5N1 highly pathogenic avian influenza virus (HPAIV) infection up-regulated TRIM21 expression in A549 cells. Western blot and qPCR results showed that knockdown of TRIM21 alleviated oxidative stress and ferroptosis induced by H5N1 HPAIV and promoted the activation of antioxidant pathways. Co-IP results showed that TRIM21 promoted oxidative stress and ferroptosis by regulating the SQSTM1-NRF2-KEAP1 axis by increasing SQSTM1 K63-linked polyubiquitination under the condition of HPAIV infection. In addition, TRIM21 attenuated the inhibitory effect of antioxidant NAC on HPAIV titers and enhanced the promoting effect of ferroptosis agonist Erastin on HPAIV titers. Our findings provide new insight into the role of TRIM21 in oxidative stress and ferroptosis induced by viral infection.

Chicken UFL1 Restricts Avian Influenza Virus Replication by Disrupting the Viral Polymerase Complex and Facilitating Type I IFN Production.

During avian influenza virus (AIV) infection, host defensive proteins promote antiviral innate immunity or antagonize viral components to limit viral replication. UFM1-specific ligase 1 (UFL1) is involved in regulating innate immunity and DNA virus replication in mammals, but the molecular mechanism by which chicken (ch)UFL1 regulates AIV replication is unclear. In this study, we first identified chUFL1 as a negative regulator of AIV replication by enhancing innate immunity and disrupting the assembly of the viral polymerase complex. Mechanistically, chUFL1 interacted with chicken stimulator of IFN genes (chSTING) and contributed to chSTING dimerization and the formation of the STING-TBK1-IRF7 complex. We further demonstrated that chUFL1 promoted K63-linked polyubiquitination of chSTING at K308 to facilitate chSTING-mediated type I IFN production independent of UFMylation. Additionally, chUFL1 expression was upregulated in response to AIV infection. Importantly, chUFL1 also interacted with the AIV PA protein to inhibit viral polymerase activity. Furthermore, chUFL1 impeded the nuclear import of the AIV PA protein and the assembly of the viral polymerase complex to suppress AIV replication. Collectively, these findings demonstrate that chUFL1 restricts AIV replication by disrupting the viral polymerase complex and facilitating type I IFN production, which provides new insights into the regulation of AIV replication in chickens.

Analysis of H5N8 influenza virus infection in chicken with mApple reporter genes in vivo and in vitro

H5N8 highly pathogenic avian influenza virus (HPAIV) has caused huge losses to the global poultry industry and critically threatens public health. Chickens are the important host for the transmission. However, the distribution of H5N8 avian influenza virus (AIV) in chicken and the infected cell types are limitedly studied. Therefore, in this study, we detected viral replication and infection by generating recombinant H5N8 AIV expressing an easily tracked mApple fluorescent reporter. The results showed that recombinant viruses passaged four times in chicken embryos successfully expressed mApple proteins detected by fluorescence microscopy and WB, which verified that the constructed recombinant viruses were stable. Compared to parental virus, although recombinant virus attenuated for replication in MDCK cells, it can still replicate effectively, and form visible plaques. Importantly, the experiments on infection of chicken PBMCs in vitro showed a strong correlation between mApple positivity rate and NP positivity rate (r = 0.7594, P =0.0176), demonstrating that mApple reporter could be used as an indicator to accurately reflect AIV infection. Then we infected monocytes/macrophages in PBMCs in vitro and detected the mApple positive percentage was 55.1%-80.4%, which confirmed the chicken primary monocytic/macrophages are important target cells for avian influenza virus infection. In chicken, compared with parental virus, the recombinant virus-infected chickens had lower viral titers in oropharyngeal cloacal and organs, but it can cause significant pathogenicity in chicken and the mortality rate was approximately 66%. In addition, the results of bioluminescent imaging showed that the fluorescence in the lungs was strongest at 5 days post-infection (DPI). Finally, we discovered the mApple positive expression in chicken lung immune cells (CD45+ cells), especially some T cells (CD4 and CD8 T cells) also carrying mApple, which indicates that the H5N8 AIV showed a tropism for immune cells including chicken T cells causing potentially aggressive against cellular immunity. We have provided a simple visualization for further exploration of H5N8 AIV infected chicken immune cells, which contributes to further understanding pathogenic mechanism of H5N8 AIV infection in chicken.

Potential use of poultry farms by wild waterfowl in California's Central Valley varies across space, times of day, and species: implications for influenza transmission risk

Interactions between wildlife and livestock can lead to cross‐species disease transmission, which incurs economic costs and threatens wildlife conservation. Wild waterfowl are natural hosts of avian influenza viruses (AIVs), are often abundant near poultry farms, and have been linked to outbreaks of AIVs in poultry. Interspecific and seasonal variation in waterfowl movement and habitat use means that the risk of disease transmission between wild birds and poultry inevitably varies across species, space, and time. Here, we used GPS telemetry data from 10 waterfowl species in and near California's Central Valley, a region where both wild waterfowl and domestic poultry are abundant, to study selection of poultry farms by waterfowl across diel, seasonal, and annual cycles. We found that waterfowl selected for wetlands, open water, protected areas, and croplands, which meant that they generally avoided habitats that were likely to be used for poultry farming. These selection patterns were linked to species' ecology and diel behavioral patterns, such that avoidance of poultry habitats was stronger for local or partial migrants than for long‐distance migrants, and stronger during daytime than at night. We then combined these habitat selection results with data on poultry farm locations to map risk of waterfowl–poultry contact across the Central Valley. Average selection strength at poultry farms was low, suggesting that current placement of poultry farms is generally effective for limiting risk of contact with wild birds. When we combined these habitat selection results with data on species' abundances and AIV infection prevalence, we found dramatic variation in potential AIV transmission risk among species. These results could be used to prioritize surveillance and biosecurity efforts for regions and times of relatively high risk. More generally, these results highlight that fine‐scale movement data can help identify interspecific, seasonal, and diel patterns in animal behaviors that affect wildlife and poultry health.

The H9N2 avian influenza virus increases APEC adhesion to oviduct epithelia by viral NS1 protein-mediated activation of the TGF-β pathway.

H9N2 avian influenza is a low-pathogenic avian influenza circulating in poultry and wild birds worldwide and frequently contributes to chicken salpingitis that is caused by avian pathogenic Escherichia coli (APEC), leading to huge economic losses and risks for food safety. Currently, how the H9N2 virus contributes to APEC infection and facilitates salpingitis remains elusive. In this study, in vitro chicken oviduct epithelial cell (COEC) model and in vivo studies were performed to investigate the role of H9N2 viruses on secondary APEC infection, and we identified that H9N2 virus enhances APEC infection both in vitro and in vivo. To understand the mechanisms behind this phenomenon, adhesive molecules on the cell surface facilitating APEC adhesion were checked, and we found that H9N2 virus could upregulate the expression of fibronectin, which promotes APEC adhesion onto COECs. We further investigated how fibronectin expression is regulated by H9N2 virus infection and revealed that transforming growth factor beta (TGF-β) signaling pathway is activated by the NS1 protein of the virus, thus regulating the expression of adhesive molecules. These new findings revealed the role of H9N2 virus in salpingitis co-infected with APEC and discovered the molecular mechanisms by which the H9N2 virus facilitates APEC infection, offering new insights to the etiology of salpingitis with viral-bacterial co-infections.IMPORTANCEH9N2 avian influenza virus (AIV) widely infects poultry and is sporadically reported in human infections. The infection in birds frequently causes secondary bacterial infections, resulting in severe symptoms like pneumonia and salpingitis. Currently, the mechanism that influenza A virus contributes to secondary bacterial infection remains elusive. Here we discovered that H9N2 virus infection promotes APEC infection and further explored the underlying molecular mechanisms. We found that fibronectin protein on the cell surface is vital for APEC adhesion and also showed that H9N2 viral protein NS1 increased the expression of fibronectin by activating the TGF-β signaling pathway. Our findings offer new information on how AIV infection promotes APEC secondary infection, providing potential targets for mitigating severe APEC infections induced by H9N2 avian influenza, and also give new insights on the mechanisms on how viruses promote secondary bacterial infections in animal and human diseases.

Constitutive immunity is influenced by avian influenza virus-induced modification of gut microbiota in Eurasian teal (Anas crecca)

Understanding the dynamics of migrant birds' gut microbial communities is essential for evaluating their ecological interactions, since these birds act as vectors for zoonotic viruses and their gut microbiome may have exceptional relationship with zoonotic viral infection. The Eurasian teal duck Anas crecca traverses continents during migration, combining and providing intercontinental links for avian influenza viruses (AIV) of different origins. The present study aimed to investigate how the AIV infection affects gut microbial composition and evaluate the consequent physiological stress and constitutive immunity of teal birds. Samples were collected from 2 flocks during their migratory stopover in northern Egypt. An important shift in gut microbiota of AIV-infected individuals has been detected by RT-PCR. In healthy teal, firmicutes dominated followed by proteobacteria, while the structure was reversed in infected birds. Infection with AIV significantly increased the stress hormone corticosterone, accompanied by a significant increase in both oxidative stress markers and antioxidants. Constitutive immunity, measured by plasma bactericidal effect against E. coli, the nonspecific natural antibodies, and the mediated complement activation, was reduced in AIV-infected teal birds. Constitutive immunity parameters were proportionally correlated to the firmicutes and inversely to the proteobacteria abundances, but not to the viral positivity. In conclusion, the present study provides initial evidence of the alteration of the gut microbiome in the Eurasian teal Anas crecca by AIV infection and demonstrates that the AIV-induced reduction in constitutive immunity is a consequence of the shift in microbiome composition rather than the virus infection itself or its induced stress.