H5Nx highly pathogenic avian influenza viruses pose persistent threats to poultry, wildlife, and public health. Over the past two decades, their geographic and host ranges have expanded across migratory networks whose epidemiological connectivity has become increasingly apparent through recent surveillance and genomic analyses. To elucidate these dynamics, we conduct long-term nationwide wild-bird surveillance in China, integrating active and passive monitoring. Our analyses reveal the maintenance, reassortment, and transmission of H5Nx viruses in wild birds, highlighting the value of sustained surveillance in capturing viral evolution. We identify distinct ecological patterns among major clades, with 2.3.4.4b showing the widest distribution and acting as the main lineage mediating intercontinental spread. Since 2020, most 2.3.4.4b viruses detected in wild birds in China have clustered with lineages originating outside China, consistent with repeated reintroduction rather than sustained local circulation. This shift underscores the growing role of migratory connectivity in shaping global viral exchange and the need for coordinated international active surveillance.

Influenza A viruses continue to pose a major threat to global public health. In addition to H1N1 and H3N2 subtypes causing seasonal epidemics that result in an estimated 3-5 million severe cases and 290,000-650,000 deaths annually, other subtypes, including avian H5, H7, and H9, have shown cross-species transmission potential, leading to thousands of human infections in multiple countries. The development of broad-spectrum antiviral drugs capable of inhibiting different influenza virus subtypes is key for alleviating the severity of diseases caused by influenza viruses and reducing mortality rates. Here, we constructed five nanobody-based proteolysis-targeting chimeras (Nb-PROTACs) by fusing NP-specific nanobodies to the α-domain of the Von Hippel‒Lindau (VHL) E3 ubiquitin ligase. We found that two of these chimeras (VHL-Nb135 and VHL-Nb170) efficiently induced NP degradation across all 16 recognized influenza A subtypes (H1-H16). VHL-Nb135 and VHL-Nb170 efficiently inhibited the replication of human (H1N1, H3N2) and avian (H5N1, H7N9, H9N2) influenza viruses in vitro. In animal studies, when VHL-Nb170 was administered intratracheally to mice via adeno-associated virus serotype LungM3 (AAV-LungM3), virus replication was significantly inhibited in the respiratory tract, and 90% and 80% of the mice survived infection with lethal H1N1 and H5N1 viruses, respectively. Our study indicates that Nb-PROTACs offer a robust platform for the development of broad-spectrum therapies against influenza viruses and hold potential for clinical translation as innovative antiviral candidate drugs.

Carbapenem-resistant Klebsiella pneumoniae produces the carbapenemase KPC-2, which rapidly confers resistance and poses a significant threat to public health worldwide. Natural compounds have broad application potential, novel structures, and are not easily recognized by existing bacterial resistance mechanisms. Therefore, through virtual screening based on the KPC-2 protein structure and in vitro antimicrobial assessment, the natural anthraquinone compound emodin was identified as a KPC-2 inhibitor (IC50 = 24.4 μM) and a synergist of meropenem. Molecular dynamics simulation and interaction analyses indicated that emodin competitively occupied the active site of KPC-2 at Trp105 and Thr237, resulting in reduced α-helices and increased β-sheets, thereby inhibiting KPC-2 activity. The combination of emodin and MEM exhibited a synergistic effect. Furthermore, emodin modulates succinate dehydrogenase (SDH) activity and its transcriptional expression, resulting in metabolic perturbation that renders bacteria susceptible to meropenem, thereby potentiating the synergistic antibacterial activity of the combination. This disrupted the metabolic homeostasis of the bacteria, thereby compromising the integrity of bacterial cells and biofilms. No resistance development was observed over 30 generations of passage. In a mouse model of pneumonia, the combination therapy significantly reduced MEM usage, decreased the pulmonary bacterial load by 1.43 log CFU/g of model group, attenuated lung inflammation, and restored normal lung histology. These findings suggest that emodin acts as a promising candidate of KPC-2 inhibitor, which is capable of reinstating carbapenem efficacy against carbapenem-resistant Gram-negative pathogens in vitro and in vivo.

Klebsiella pneumoniae has evolved from an opportunistic pathogen into a formidable global threat, with hypervirulent strains now causing severe infections in healthy individuals and carbapenem-resistant variants achieving mortality rates exceeding 42%. This transformation can be driven by mobile genetic elements including plasmids, integrative conjugative elements (ICE), insertion sequences (ISs), transposons, and integrons. Recent discoveries reveal that these elements employ sophisticated mechanisms: conjugative virulence plasmids dissemination across bacterial populations; ICEs-mediated virulence traits transfer; and hybrid genetic elements simultaneously confer virulence and antimicrobial resistance. Understanding these molecular mechanisms is critical for developing targeted diagnostics and therapeutics that disrupt mobile element mobility, offering promising strategies to combat the convergence of hypervirulence and resistance in this WHO priority pathogen.

During 2022-2024, a highly pathogenic avian influenza virus (HPAIV) H5N1 strain, designated A/Seagull/Hebei/qhd6/2024 (H5N1), was isolated from migratory birds in Beidaihe National Wetland Park, North China. Phylogenetic analyses revealed that its hemagglutinin (HA) gene belongs to the 2.3.4.4b clade, while the neuraminidase (NA) gene and internal genes clustered with strains originating from multiple continents, consistent with a transcontinental reassortment event. The virus also exhibited 90.1-98.1% nucleotide homology with human-derived H5N1 isolates. Molecular characterization identified key virulence-associated mutations, including the classic HPAIV HA cleavage site, HA-T160A (associated with enhanced human receptor-binding capacity), and NA-I117T (potentially linked to drug resistance). BALB/c mouse infection experiments confirmed systemic replication and high pathogenicity of strain qhd6, with a 50% lethal dose (LD50) of 0.95 log10EID50/mL. Antigenic analysis revealed good cross-reactivity with the widely used H5-Re14 vaccine strain. This study reports the identification, in Beidaihe National Wetland Park, of an HPAIV H5N1 strain whose genetic characteristics suggest intercontinental reassortment and indicate cross-species transmission risk. It clarifies the genetic characteristics and pathogenicity of this strain, providing an important theoretical and practical basis for precise surveillance, risk early warning, and comprehensive prevention and control of AIV at migratory bird stopover sites in North China.

Given that ducks serve as critical reservoirs for avian influenza viruses, achieving high immune coverage in duck flocks is essential for preventing the transmission of avian influenza viruses from wild birds to domestic poultry. Duck enteritis is the most important infectious disease that must be prevented with a live-attenuated vaccine in duck breeding. Therefore, we previously constructed a recombinant duck enteritis virus (DEV), rDEV-dH5/H7, which carries the hemagglutinin (HA) genes of two H5 viruses and an H7 influenza virus. It could induce rapid and complete protection against both lethal DEV and H5 and H7 viruses as early as 7 days post-prime vaccination, although almost no antibodies were detected in ducks at this time. In the present study, we demonstrated that rDEV-dH5/H7 immediately initiated innate immune responses and T-cell proliferation in ducks. The expression levels of IFN-γ and granzyme A were markedly upregulated at 5-7 days post-prime vaccination. The percentages of CD3+ and CD3+CD8+ T cells in peripheral blood mononuclear cells significantly increased from 7 days post-prime vaccination. Moreover, HA-specific CD8+ and CD4+ T cells, as well as those induced by DEV virion, were significantly higher than those in control animals from 7 days post-prime vaccination. Together, the quick response of specific T cells and the innate immune response induced by rDEV-dH5/H7 may confer rapid immune protection against lethal influenza virus and DEV.IMPORTANCEPreviously, we found that recombinant duck enteritis virus, rDEV-dH5/H7, could induce rapid and robust dual protection against lethal DEV and highly pathogenic avian influenza viruses as early as day 7 post-prime vaccination, although almost no antibodies could be detected at this time. In the present study, we reveal that cell-mediated immunity plays a critical role through early upregulation of IFN-γ/granzyme A pathways and robust hemagglutinin (HA)-specific T-cell responses and drives protection even in the absence of detectable antibodies. This is the first mechanistic evidence showing DEV-vectored vaccines activate the robust proliferation of T lymphocytes and HA-specific T-cell responses. Our findings fundamentally advance the understanding of DEV-vectored vaccines, offering new insight for recombinant DEV vaccine design.

H11 subtype avian influenza viruses (AIVs) have been identified in both wild and domestic birds. H11N9 viruses from wild birds provided the NA gene to human H7N9 virus in 2013 in China, which caused five waves of human infections. During active surveillance in wild birds in China, 17 H11 viruses were isolated between December 2022 and January 2024, including six H11N1, one H11N2, one H11N3, and nine H11N9. The epidemiology of H11 subtype viruses in public databases revealed that they distributed across seven continents, and more than 54.9% of H11 viruses originated from wild Anseriformes. Phylogenetic analysis of the HA genes indicated that H11 viruses were classified into Eurasian and North American lineages, and our isolates belonged to the Eurasian lineage. Bayesian phylogeographic analysis suggested that Bangladesh served as a crucial geographical transmission center for H11 viruses in Eurasian lineage. Reassortment indicated that the H11 isolates in the study underwent complex genomic recombination with various subtype AIVs circulating in wild and domestic birds, including the clade 2.3.4.4b H5N1 highly pathogenic viruses, and formed seven genotypes. Notably, 17 H11 isolates acquired several mutations associated with enhanced human-type receptor binding in HA (S137A) and increased mammalian virulence in PB1 (D3V, D622G), PB1-F2 (N66S), M1 (N30D, I43M, T215A), and NS1 (P42S, I106M). Seven representative viruses exhibited dual receptor binding specificity and could infect mice directly without prior adaptation. These findings highlight the potential public health risks posed by H11 viruses from wild birds and emphasize the necessity of enhancing routine surveillance.

Rationale: Phenotyping is used to create clinically useful groups of patients that can be identified by a common set of characteristics; the concept of ‘treatable traits’ is similar in purpose. There have been multiple attempts to phenotype chronic obstructive pulmonary disease (COPD) that have usually included patients with a wide range of severity, assuming that phenotypic characteristics are constant, irrespective of the degree of airflow limitation. The included patients typically had moderate to very severe disease, and the most comprehensive analysis of its kind contained no patients with Global Initiative for Chronic Obstructive Lung Disease (GOLD) I (Rennard et al. Ann Am Thorac Soc. 2015;12:303-312). That analysis was designed to gain fundamental insights into the nature of COPD, but phenotyping for routine practice means restricting the included variables to those that are routinely available. This analysis concerns the identification of key phenotypic factors present in patients with GOLD I and II disease using measures that can be assessed in routine clinical practice. Methods: Data came from COMPASS, a prospective 2.5-year study of COPD, performed in 41 centers in China (Liang Z, et al. ERJ Open Res 2021;7:00201). Exploratory factor analysis (EFA) with Varimax rotation was used to identify key factors: those with eigenvalues >1.0. To reduce the risk of distortion in the factor structure that can occur by the inclusion of multiple highly correlated variables (e.g. spirometric values), an initial analysis was carried out to inform decisions about which to include in the final EFA. Similarly, the Charlson Comorbidity Index was used to aggregate multiple extra-pulmonary comorbidities into a single score. Results: Data from 1299 patients with mild to moderate COPD were available. After initial variable selection, 17 were subject to EFA. Seven factors with eigenvalues >1.0 accounted for 61.7% of the variance (Table). They represented the following domains: 1. lung function (post-bronchodilator spirometry); 2. symptom burden (COPD Assessment Test, Modified Medical Research Council dyspnea and chronic bronchitis); 3. systemic inflammation (blood eosinophils, basophils, and lymphocytes); 4. aging and comorbidities; 5. body mass index; 6. exacerbation risk (moderate exacerbations in the previous 12 months); 7. history of asthma and/or gastroesophageal reflux. Conclusions: Even in patients with mild-moderate airflow limitation, there appear to be seven distinct phenotypic factors that can be identified using routine assessments. Further analysis of these factors is needed to form clinically useful groups.

Background: Filamin A (FLNA) is a member of the filamin family and has been found to be critical for the progression of several cancers. However, its biological function in papillary thyroid cancer (PTC) remains largely unexplored. Methods: Data from The Cancer Genome Atlas (TCGA) databases were utilized to analyze the FLNA expression level and its influence on the clinical implications of patients with PTC. Gene Expression Omnibus (GEO) and qRT-PCR was used to verify the expression levels of FLNA in PTC. Kaplan–Meier survival analysis was conducted to evaluate the prognostic value of FLNA in PTC. Transwell assays and wound healing were performed to examine the biological function of FLNA knockdown in PTC cells. Gene set enrichment analysis (GSEA) and Western blotting were conducted to investigate the potential mechanisms underlying the role of FLNA in PTC progression. In addition, the relationship between FLNA expression and the tumor immune microenvironment (TME) in PTC was explored. Results: FLNA was significantly upregulated in PTC tissues. High expression levels of FLNA was correlated with advanced TNM stage, T stage, and N stage, as well as poor disease-free interval (DFI) and progression-free interval (PFI) time in PTC patients. Moreover, we found that FLNA knockdown inhibited the migration and invasion of PTC cells. Mechanistically, FLNA knockdown inhibited epithelial–mesenchymal transition (EMT) in PTC and affected the activation of the FAK/AKT signaling pathway. In addition, FLNA expression was associated with TME in PTC. Conclusion: FLNA may be regarded as a new therapeutic target for PTC patients.

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

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

During the active surveillance, we isolated nine H4N6 subtype influenza A viruses from wild birds in China. To reveal the epidemiology and biology characteristics of H4 subtype influenza A virus from wild birds, we investigated H4 subtype viruses available in the public source, and found that the H4 viruses have been detected in at least 37 countries to date, and more than 73.6% of the viruses were from wild Anseriformes. Bayesian phylogeographic analysis showed that Mongolia worked as the important transmission centre for Eurasian lineage H4 viruses spreading. Phylogenetic analysis of HA genes indicated that global H4 influenza A viruses were divided into Eurasian and North American lineage, our nine H4N6 isolates fell into the Eurasian lineage. Recombination analysis suggested that nine H4N6 isolates underwent complex gene recombination with various subtypes of influenza A viruses and formed two genotypes. Notably, nine H4N6 isolates acquired mammalian virulence-increasing residues. Two representative H4N6 viruses possessed dual receptor binding specificity, they could efficiently replicate in MDCK and 293 T cells in vitro infection, also could cross the species barrier to infect mice directly without prior adaption in vivo experiments. These findings emphasize the public health issues represented by H4 viruses, and highlight the need to strengthen the active surveillance of H4 viruses from wild birds.

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

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

IntroductionMonoclonal antibodies (mAbs) play a pivotal role in disease diagnosis as well as immunotherapy interventions. Traditional monoclonal antibody generation relies on animal immunization procedures predominantly involving mice; however, recent advances in in-vitro expression methodologies have enabled large-scale production suitable for both industrial applications as well as scientific investigations.MethodsIn this study, two mAbs against H7 subtype avian influenza viruses (AIV) were sequenced and analyzed, and the DNA sequences encoding heavy chain (HC) and light chain (LC) were obtained and cloned into pCHO-1.0 expression vector. Then, the HC and LC expression plasmids were transfected into CHO-S cells to establish stable cell lines expressing these mAbs using a two-phase selection scheme with different concentrations of methotrexate and puromycin. Recombinant antibodies were purified from the cell culture medium, and their potential applications were evaluated using hemagglutination inhibition (HI), western blotting (WB), confocal microscopy, and enzyme-linked immunosorbent assay (ELISA).ResultsThe results indicated that the obtained recombinant antibodies exhibited biological activity similar to that of the parent antibodies derived from ascites and could be used as a replacement for animal-derived mAbs. A kinetic analysis of the two antibodies to the AIV HA protein, conducted using surface plasmon resonance (SPR), showed concordance between the recombinant and parental antibodies.DiscussionThe data presented in this study suggest that the described antibody production protocol could avoid the use of experimental animals and better conform to animal welfare regulations, and provides a basis for further research and development of mAbs-based diagnostic products.

During the routine surveillance, we isolated nine H4N6 subtype avian influenza viruses (AIVs) in Jiangsu Province, China, in March 2023. Phylogenetic analysis revealed that nine H4N6 viruses belonged to the Eurasian lineage and underwent complex genetic recombination among Asian countries during their evolution. It is particularly noteworthy that the PB2 and PB1 genes of our representative virus were descended from clade 2.3.4.4b H5 high-pathogenic AIVs in Japan. Mutations of D3V and D622G in PB1, N66S in PB1-F2, N30D, I43M, and T215A in M1, and P42S and I106M in NS1 were observed in nine isolates, which may increase the pathogenicity of the viruses in mice. The receptor binding analysis showed that the tested H4N6 virus could bind to both avian-type and human-type receptors. Vitro infection kinetics revealed that the representative virus could efficiently replicate in mammalian cells, including MDCK and 293T cells. Pathogenicity tests in mice indicated that the representative virus could replicate in nasal turbinates and lungs without prior adaptation. Our data reveal the potential public health issues represented by H4N6 viruses from wild birds and highlight the need to strengthen routine surveillance of wild birds.

Subclade 2.3.4.4b H5Nx highly pathogenic avian influenza (HPAI) viruses, emerged in 2013 with multiple subtypes of H5N8, H5N1, and H5N6, had unprecedently caused a global epizootic by H5N1 since 2021, which had devasted multiple species of wild birds, poultry, and wild mammals (terrestrial and marine) with a high mortality, causing severe ecological damage. The infected wild mammals may become new "mixers" for influenza viruses, posing the potential transmission to human. Frequent outbreaks of subclade 2.3.4.4b H5Nx viruses among wild birds and poultry had exposed major gaps in our knowledge on their evolution, spatiotemporal diffusion, and species-crossing transmission. Here, we integrated the phylogenetic and epidemiological data of subclade 2.3.4.4b H5Nx viruses in public database and used Bayesian phylodynamic analysis to reveal the pattern of the global large-scale transmission. Phylogenic analysis demonstrated that the HA gene of these viruses diverged into two dominant clusters around 2015 and 2016. The Bayesian phylodynamic analysis illustrated that the viruses presented spatiotemporally complex transmission network with geographical and host relative expansion and recombination with different subtypes of NA segment. Spatially, the Russian Federation (Siberia) was identified as the primary hub for virus transmission, which was further facilitated by the establishment of strong epidemiological linkages between West Europe and broader regions, such as North America. As for hosts, wild Anseriformes were the primary species for the virus spillover, contributing to the spatial expansion and rapid diffusion globally of subclade 2.3.4.4b viruses. We investigated the phylogeny of subclade 2.3.4.4b H5Nx viruses and the spatiotemporal pattern of transmission with initial location and the primary host, which could provide comprehensive insights for subclade 2.3.4.4b H5Nx viruses. Due to the wild birds involved the widespread of subclade 2.3.4.4b H5Nx viruses, the epizootics in poultry are inevitable, so we highly recommend to apply the policy of culling plus with vaccination to protect the poultry industry and potentially protect the public health.

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

Avian influenza virus (AIV) causes huge losses to the global poultry industry and poses a threat to humans and other mammals. Fast, sensitive, and portable diagnostic methods are essential for efficient avian influenza control. Here, a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas13a based platform was developed to detect AIV. This novel method was developed to specifically detect H1–H16 subtypes of AIV with fluorescence and lateral flow-based readouts and exhibited no cross-reactivity with Newcastle disease virus, avian infectious bronchitis virus, or infectious bursal disease virus. The limit of detection was determined to be 69 and 690 copies/μL using fluorescence and lateral flow as readouts, respectively. The developed assay exhibited 100% consistency with quantitative real-time polymerase chain reaction in detecting clinical samples. The heating of unextracted diagnostic samples to obliterate nuclease treatment was introduced to detect viral RNA without nucleic acid extraction. Single-step optimization was used to perform reverse transcription, recombinase polymerase amplification, and CRISPR-Cas13a detection in a tube. These advances resulted in an optimized assay that could specifically detect AIV with simplified procedures and reduced contamination risk, highlighting the potential to be used in point-of-care testing.