Influenza A virus (IAV) remains a major threat to human and animal health, highlighting the need for efficient approaches to identify host factors and antivirals. Reporter viruses are critical tools for these efforts, but incorporating large reporter genes often compromises viral fitness and genetic stability. Here, we developed a replication competent reporter IAV by fusing an 11-amino acid HiBiT tag into the nonstructural protein 1 (NS1) of the A/WSN/1933 (H1N1) backbone. The modified virus retained parental virion morphology, comparable replication kinetics, and in vivo tissue tropism, while enabling the highly sensitive, rapid, and quantitative detection of viral replication. Using this reporter virus, we established a robust high-throughput screening (HTS) platform with excellent assay quality and validated its capability to identify host regulators of IAV infection. Application of this platform to host-encoded micropeptides (miPEPs) led to the identification of cytokine-inhibitory micropeptide 53 (CIM53) as a novel regulator of IAV replication. Mechanistically, CIM53 promoted IAV replication by intrinsically suppressing antiviral innate immune responses. Furthermore, although CIM53 enhanced viral replication, its immunomodulatory activity contributed to reduced lung injury and improved survival when combined with Oseltamivir (OSV) treatment in infected mice. Our study establishes a highly practical screening platform for virological research and highlights CIM53 as a potential host-directed adjunct therapy for severe IAV Infection.

The H12 subtypes of avian influenza viruses (AIVs) are globally prevalent in wild birds, occasionally spilling over into poultry. In this study, we isolated an H12N8 virus from ducks in a live poultry market. Full genomic analysis revealed that the virus bears a single basic amino acid in the cleavage site of the hemagglutinin gene. Phylogenetic analysis revealed that the eight gene segments of the H12N8 virus belong to the Eurasian lineage and the HA gene was clustered with wild bird-originated H12 viruses, with its NP gene showing the highest nucleotide similarity to 2013-like H7N9 viruses. The H12N8 virus replicated effectively in both mammalian and avian cells without prior adaptation. Moreover, the H12N8 virus could infect and replicate in the upper respiratory tract of BALB/c mice without prior adaptation. The H12N8 virus replicated and transmitted inefficiently in both ducks and chickens and hardly triggered high hemagglutination inhibition (HI) antibody titers in the inoculated and contact animals. These results suggest that the wild bird-origin H12N8 virus has reassorted with viruses circulating in domestic poultry, but it inefficiently replicates and transmits in avian hosts. Our findings demonstrate that H12N8 AIV has emerged in domestic poultry, emphasizing the importance of active surveillance of AIVs in both wild and domestic birds.

H3N3 avian influenza viruses (AIVs) are less prevalent in poultry than H3N8 viruses. However, although relatively rare, reassortant H3N3 viruses have been known to appear in both domestic poultry and wild birds. In this study, we isolated the H3N3 virus in chickens sourced from a live poultry market in China. A comprehensive genomic analysis revealed that the virus possessed a single basic amino acid in the cleavage site of the hemagglutinin (HA) gene. Phylogenetic analysis indicated that eight genes in the H3N3 virus belong to the Eurasian lineage. Specifically, the HA and NA genes were clustered with H3N2 and H11N3, respectively, while the internal genes were closely related to the H3N8 and H9N2 viruses. Furthermore, the H3N3 virus exhibited high and moderate stability in thermal and acidic conditions and efficient replication capabilities in mammalian cells. The H3N3 virus demonstrated that it could infect and replicate in the upper and lower respiratory tract of BALB/c mice without prior adaptation, triggering hemagglutination inhibition (HI) antibody titres ranging from 80 to 160; notably, the H3N3 virus replicated vigorously within the chicken respiratory and digestive tracts. The virus also transmitted efficiently and swiftly among chickens through direct contact, leading to higher levels of HI antibodies in both the inoculated and contact birds. These findings suggest that the H3N3 virus may be a novel reassortant originating from viruses circulating in domestic poultry, thus demonstrating an increased pathogenicity and transmissibility in chickens. Our study determines that H3N3 AIV potentially threatens the poultry industry and public health, highlighting the importance of active surveillance of AIVs.

Influenza A virus (IAV) poses a significant threat due to its rapid evolution through gene mutations and reassortments. Understanding host-virus protein interactions is vital for developing countermeasures. In this study, we developed a live-cell screening platform using the NanoBiT system for rapid discovery of host-virus protein-protein interactions (PPIs). Novel interactions between the host factor SNAPIN and the viral M1, M2 and NS2 were identified using this system. We confirmed the platform's reliability by validating the SNAPIN-M1 interaction using independent methods including co-immunoprecipitation (Co-IP) and glutathione S-transferase (GST) pull-down assays. These results demonstrate the robustness of the PPI screening system and provide a basis for studying the role of SNAPIN in regulating IAV replication.

Influenza A viruses (IAVs) are highly contagious pathogens that cause zoonotic disease with limited availability of antiviral therapies, presenting ongoing challenges to both public health and the livestock industry. Unveiling host proteins that are crucial to the IAV life cycle can help clarify mechanisms of viral replication and identify potential targets for developing alternative host-directed therapies. Using a four-dimensional (4D), label-free methodology coupled with bioinformatics analysis, we analyzed the expression patterns of cellular proteins that changed following H9N2 virus infection. Compared to the control group, the H9N2 infected group displayed 732 differentially expressed proteins (DEPs), with 298 proteins showing upregulation and 434 proteins showing downregulation. Gene Ontology (GO) functional analysis showed that DEPs were catalog in 11 biological processes, three cellular components, and eight molecular functions. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that DEPs were involved in processes including cytokine signaling pathways induced by virus infection and protein digestion and absorption. Proteins including TP53, DDX58, and STAT3 were among the top hub proteins in the protein-protein interaction (PPI) analysis, suggesting that these signaling cascades could be essential for the propagation of IAVs. Furthermore, the host protein SNAPIN was chosen to ascertain the accuracy of expression changes identified through a proteomic analysis. The results indicated that SNAPIN was downregulated following infection with IAVs both in vitro and in vivo, which is consistent with the proteomics results, suggesting that SNAPIN may serve as a key regulatory factor in the viral life cycle of IAVs. Our research delineates an extensive interaction map of IAV infection within the A549 cells, facilitating the discovery of pivotal proteins that contribute to the virus's propagation, potentially offering target candidates to screen for antiviral therapeutics.

The H3N3 avian influenza viruses (AIVs) are not as prevalent as H3N8 viruses in poultry. Yet, reassortant H3N3 viruses are occasionally detected in domestic poultry and wild birds. In this study, we isolated an H3N3 virus in chickens from a live poultry market in China. Full genomic analysis revealed that the virus had a single basic amino acid in the cleavage site of the hemagglutinin (HA) gene. Phylogenetic analysis revealed that eight genes of the H3N3 virus belong to the Eurasian lineage and the HA and NA genes were clustered with H3N2 and H11N3, respectively, while the internal genes were closely related to H3N8 and H9N2 viruses. The H3N3 virus demonstrated high and moderate stability in thermal and acid conditions, respectively, along with efficient replication capabilities in mammalian cells. Moreover, the H3N3 virus could infect and replicate in the upper and lower respiratory tract of BALB/c mice without prior adaptation, triggering hemagglutination inhibition (HI) antibody titers ranging from 80 to 160. The H3N3 virus replicated vigorously within the chicken respiratory and digestive tracts, transmitted efficiently and swiftly among chickens via direct contact, and resulted in elevated HI antibody titers in both the inoculated and contact birds. These findings indicate that the H3N3 virus might be a novel reassortant originating from viruses circulating in domestic poultry, demonstrating increased pathogenicity and transmissibility in chickens. Our findings demonstrate that H3N3 AIV may pose a potential threat to poultry industry and public health, thereby highlighting the importance of active surveillance of AIVs.

The continued evolution and emergence of novel influenza viruses in wild and domestic animals poses an increasing public health risk. Two human cases of H3N8 avian influenza virus infection in China in 2022 have caused public concern regarding the risk of transmission between birds and humans. However, the prevalence of H3N8 avian influenza viruses in their natural reservoirs and their biological characteristics are largely unknown. To elucidate the potential threat of H3N8 viruses, we analyzed five years of surveillance data obtained from an important wetland region in eastern China and evaluated the evolutionary and biological characteristics of 21 H3N8 viruses isolated from 15,899 migratory bird samples between 2017 and 2021. Genetic and phylogenetic analyses showed that the H3N8 viruses circulating in migratory birds and ducks have evolved into different branches and have undergone complicated reassortment with viruses in waterfowl. The 21 viruses belonged to 12 genotypes, and some strains induced body weight loss and pneumonia in mice. All the tested H3N8 viruses preferentially bind to avian-type receptors, although they have acquired the ability to bind human-type receptors. Infection studies in ducks, chickens and pigeons demonstrated that the currently circulating H3N8 viruses in migratory birds have a high possibility of infecting domestic waterfowl and a low possibility of infecting chickens and pigeons. Our findings imply that circulating H3N8 viruses in migratory birds continue to evolve and pose a high infection risk in domestic ducks. These results further emphasize the importance of avian influenza surveillance at the wild bird and poultry interface.

H16 avian influenza viruses mainly circulate in wild migratory gulls worldwide, and the infection risks in poultry and mammals remain largely unknown. In this study, we isolated a novel H16N3 virus from migratory gulls in eastern China in 2021. Genetic analysis indicated that the H16N3 virus originated from the H16 and H13 viruses that circulated in wild birds. This H16N3 virus has not adapted to replicate in chickens, ducks, or mice, although it can be transmitted between inoculated and contacted birds. The circulation of H16Nx viruses in the Northern Hemisphere indicates that we should strengthen active surveillance to monitor their prevalence and evolution in migratory gulls and their introduction into other migratory and domestic waterfowl. IMPORTANCE Migratory wild birds are natural reservoirs of H16 viruses and play a key role in the global prevalence of these viruses. Here, we found that H16 viruses predominantly circulate in migratory gulls and that the gull H16N3 virus cannot replicate efficiently in chickens, ducks, or mice without prior adaptation. These findings contribute to our understanding of the ecology, evolution, and biological properties of H16 viruses and will guide avian influenza surveillance in birds.

H4N6亚型禽流感病毒(AIV)在全球多个地区的多种动物和鸟类中存在,为了解其对动物和人类健康的潜在威胁,本研究对H4N6亚型AIV A/chicken/Shanxi/S 1452/2018(H4N6)(CK/SX/S1452/2018)株进行了 病毒全基因组测序和遗传进化分析,结果显示该病毒具有明显的遗传多样性,其内部基因与鸭或水鸟体内分离的H2N8、H3N3、H3N8、H4N8或H7N3等亚型AIV的相关基因高度同源.HA蛋白存在T160A突变,并且碱性裂解位点基序仅有一个碱性氨基酸,符合低致病性AIV分子特征.通过固相结合ELISA分析CK/SX/S1452/2018病毒株的受体结合特性,结果显示该病毒具有结合禽型α-2,3和人型α-2,6两种唾液酸受体的能力.以106 EID50/50 μL剂量经鼻腔感染小鼠进行小鼠感染性评估实验,结果显示该病毒株可未经预先适应即可直接感染小鼠,并在小鼠鼻甲和肺脏中有效复制.以上研究结果对全面了解H4N6亚型AIV的生物学特性及其对公共卫生风险有重要意义,为对该亚型AIV流行病学的持续监测和对其感染防控措施的拟定提供了数据支持.

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

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.

>Dear Editor,In the last century, H1N1, H2N2, and H3N2 influenza viruses caused pandemics in 1918, 1957, and 1968, respectively. In 2009, a novel H1N1 reassortant jumped from pigs to humans and caused a fourth influenza pandemic. Different lineages of H3N2 influenza viruses are commonly found in animal reservoirs. If a different lineage of H3N2 virus jumps to humans,

The H5N8 avian influenza viruses have been widely circulating in wild birds and are responsible for the loss of over 33 million domestic poultry in Europe, Russia, Middle East, and Asia since January 2020. To monitor the invasion and spread of the H5N8 virus in China, we performed active surveillance by analyzing 317 wild bird samples and swab samples collected from 41,172 poultry all over the country. We isolated 22 H5N8 viruses from wild birds and 14 H5N8 viruses from waterfowls. Genetic analysis indicated that the 36 viruses formed two different genotypes: one genotype viruses were widely detected from different wild birds and domestic waterfowls; the other genotype was isolated from a whopper swan. We further revealed the origin and spatiotemporal spread of these two distinct H5N8 virus genotypes in 2020 and 2021. Animal studies indicated that the H5N8 isolates are highly pathogenic to chickens, mildly pathogenic in ducks, but have distinct pathotypes in mice. Moreover, we found that vaccinated poultry in China could be completely protected against H5N8 virus challenge. Given that the H5N8 viruses are likely to continue to spread in wild birds, vaccination of poultry is highly recommended in high-risk countries to prevent H5N8 avian influenza.

H9N2 avian influenza viruses are widely prevalent in birds and pose an increasing threat to humans because of their enhanced virulence and transmissibility in mammals. Active surveillance on the prevalence and evolution of H9N2 viruses in different avian hosts will help develop eradication measures. We isolated 16 H9N2 viruses from chickens, green peafowls, and wild birds in eastern China from 2017 to 2019 and characterized their comparative genetic evolution, receptor-binding specificity, antigenic diversity, replication, and transmission in chickens and mice. The phylogenetic analysis indicated that the green peafowl viruses and swan reassortant shared the same ancestor with the poultry H9N2 viruses prevalent in eastern China, while the seven wild bird viruses belonged to wild bird lineage. The chicken, peafowl, and swan H9N2 viruses that belonged to the poultry lineage preferentially recognized α-2, 6-linked sialic acids (human-like receptor), but the wild bird lineage viruses can bind both α-2, 3 (avian-like receptor) and human-like receptor similarly. Interestingly, the H9N2 viruses of poultry lineage replicated well and transmitted efficiently, but the viruses of wild bird lineage replicated and transmitted with low efficiency. Importantly, the H9N2 viruses of poultry lineage replicated in higher titer in mammal cells and mice than the viruses of wild birds lineage. Altogether, our study indicates that co-circulation of the H9N2 viruses in poultry, wild birds, and ornamental birds increased their cross-transmission risk in different birds because of their widespread dissemination.

为了解山东省活禽市场上分离的H9N2亚型禽流感病毒(AIV)的分子特征及遗传进化规律,试验选取本实验室分离鉴定的3株H9N2 AIV进行遗传进化分析,并对数据库中历年分离的H9N2毒株血凝素(HA)的关键氨基酸位点残基组成、变异趋势,以及分离株与疫苗株HA、NA基因的核苷酸差异性进行统计分析.结果显示:3株分离株的基因同源性依次为PB2 96.4％～96.8％、PB1 94.8％～97.6％、PA 96.3％～96.9％、HA 95.6％～98.0％、NP 97.3％～98.7％、NA97.3％～97.6％、M94.2％～97.8％、NS 97.4％～98.3％.PB1、NP、PA基因属于F98分支,M基因属于G1分支,PB2、NS 基因与早年分离的代表性流感病毒毒株的遗传进化关系比较远,形成单独的Unknown Avian分支.H9N2 AIV的HA蛋白第183位(对应H3亚型位置)由早年的H逐渐向N转化,第190位点主要比例由A向T转化,第226位氨基酸由早年的Q突变为L.本试验说明H9N2 AIV不断变异,与疫苗株的差异性逐渐增大,需对其关键位点变异及跨种间传播进行持续监测.

为优化H10亚型禽流感病毒快速检测方法,按照鸡偏嗜性密码子将A/Jiangxi/IPB13/2013(H10N8)的血凝素(HA)基因序列优化后人工合成,克隆至真核表达载体pCAGGs.将重组质粒双酶切后测序证明该质粒构建成功.用200μg的表达载体免疫6周龄SPF鸡,分别在第一次免疫后的第30天和第60天用相同剂量实施第二次和第三次免疫,末次免疫后的第10天心脏采血,分离血清,制备单因子血清.间接免疫荧光试验和western blot试验证明HA蛋白成功表达,单因子血清制备成功.血凝抑制试验结果表明抗体效价≥128,单因子血清特异性强,与其他亚型禽流感病毒无交叉反应.该研究为H10亚型流感病毒快速鉴定和研究奠定了基础.

禽流感(AI)是由禽流感病毒(AIV)引起的一种高度接触性传染病,主要感染家禽和野禽,1966年首次分离得到H9N2.H9N2是AIV的一个亚型,经过长达数十年的遗传变异,已成为当前流行的禽流感病毒主要亚型之一.除了感染鸡、鸭等禽类,H9N2亚型AIV还可感染猪、水貂等哺乳类动物,甚至还可以感染人.虽然H9N2亚型AIV是低致病性的,但它能与其他病毒或细菌引起共感染,还能与其他亚型流感病毒进行重排,形成新型重组病毒,对公共卫生安全造成了不容忽视的潜在危害.文章就H9N2亚型AIV的遗传变异、重组、致病力和跨种传播等方面进行了简要论述.

Wild and domestic aquatic birds are the natural reservoirs of avian influenza viruses (AIVs). All subtypes of AIVs, including 16 hemagglutinin (HA) and nine neuraminidase (NA), have been isolated from the waterfowls. The H5 viruses in wild birds display distinct biological differences from their highly pathogenic H5 counterparts. Here, we isolated seven H5N3 AIVs including three from wild birds and four from domestic ducks in China from 2015 to 2018. The isolation sites of all the seven viruses were located in the region of the East Asian-Australasian Migratory Flyway. Phylogenetic analysis indicated that the surface genes of these viruses originated from the wild bird H5 HA subtype and the N3 Eurasian lineage. The internal genes of the seven H5N3 isolates are derived from the five gene donors isolated from the wild birds or ducks in Eastern-Asia region. They were also divided into five genotypes according to their surface genes and internal gene combinations. Interestingly, two of the seven H5N3 viruses contributed their partial internal gene segments (PB1, M and NS) to the newly emerged H7N4 reassortants, which have caused first human H7N4 infection in China in 2018. Moreover, we found that the H5N3 virus used in this study react with the anti-serum of the H5 subtype vaccine isolate (Re-11 and Re-12) and reacted well with the Re-12 anti-serum. Our findings suggest that worldwide intensive surveillance and the H5 vaccination (Re-11 and Re-12) in domestic ducks are needed to monitor the emergence of novel H5N3 reassortants in wild birds and domestic ducks and to prevent H5N3 viruses transmission from the apparently healthy wild birds and domestic ducks to chickens.