Reusable surface amplified nanobiosensor for the sub PFU/mL level detection of airborne virus.

We developed a reusable surface-amplified nanobiosensor for monitoring airborne viruses with a sub-PFU/mL level detection limit. Here, sandwich structures consisted of magnetic particles functionalized with antibodies, target viruses, and alkaline phosphatases (ALPs) were formed, and they were magnetically concentrated on Ni patterns near an electrochemical sensor transducer. Then, the electrical signals from electrochemical markers generated by ALPs were measured with the sensor transducer, enabling highly-sensitive virus detection. The sandwich structures in the used sensor chip could be removed by applying an external magnetic field, and we could reuse the sensor transducer chip. As a proof of concepts, the repeated detection of airborne influenza virus using a single sensor chip was demonstrated with a detection limit down to a sub-PFU/mL level. Using a single reusable sensor transducer chip, the hemagglutinin (HA) of influenza A (H1N1) virus with different concentrations were measured down to 10 aM level. Importantly, our sensor chip exhibited reliable sensing signals even after more than 18 times of the repeated HA sensing measurements. Furthermore, airborne influenza viruses collected from the air could be measured down to 0.01 PFU/mL level. Interestingly, the detailed quantitative analysis of the measurement results revealed the degradation of HA proteins on the viruses after the air exposure. Considering the ultrasensitivity and reusability of our sensors, it can provide a powerful tool to help preventing epidemics by airborne pathogens in the future.

Comprehensive N-glycosylation analysis of the influenza A virus proteins HA and NA from adherent and suspension MDCK cells.

Glycosylation is considered as a critical quality attribute for the production of recombinant biopharmaceuticals such as hormones, blood clotting factors, or monoclonal antibodies. In contrast, glycan patterns of immunogenic viral proteins, which differ significantly between the various expression systems, are hardly analyzed yet. The influenza A virus (IAV) proteins hemagglutinin (HA) and neuraminidase (NA) have multiple N-glycosylation sites, and alteration of N-glycan micro- and macroheterogeneity can have strong effects on virulence and immunogenicity. Here, we present a versatile and powerful glycoanalytical workflow that enables a comprehensive N-glycosylation analysis of IAV glycoproteins. We challenged our workflow with IAV (A/PR/8/34 H1N1) propagated in two closely related Madin-Darby canine kidney (MDCK) cell lines, namely an adherent MDCK cell line and its corresponding suspension cell line. As expected, N-glycan patterns of HA and NA from virus particles produced in both MDCK cell lines were similar. Detailed analysis of the HA N-glycan microheterogeneity showed an increasing variability and a higher complexity for N-glycosylation sites located closer to the head region of the molecule. In contrast, NA was found to be exclusively N-glycosylated at site N73. Almost all N-glycan structures were fucosylated. Furthermore, HA and NA N-glycan structures were exclusively hybrid- and complex-type structures, to some extent terminated with alpha-linked galactose(s) but also with blood group H type 2 and blood group A epitopes. In contrast to the similarity of the overall glycan pattern, differences in the relative abundance of individual structures were identified. This concerned, in particular, oligomannose-type, alpha-linked galactose, and multiantennary complex-type N-glycans.

Development and evaluation of a monoclonal antibody-based competitive ELISA for the detection of antibodies against H7 avian influenza virus.

BACKGROUND: H7 subtype avian influenza has caused great concern in the global poultry industry and public health. The conventional serological subtype-specific diagnostics is implemented by hemagglutination inhibition (HI) assay despite lengthy operation time. In this study, an efficient, rapid and high-throughput competitive enzyme-linked immunosorbent assay (cELISA) was developed for detection of antibodies against H7 avian influenza virus (AIV) based on a novel monoclonal antibody specific to the hemagglutinin (HA) protein of H7 AIV. RESULTS: The reaction parameters including antigen coating concentration, monoclonal antibody concentration and serum dilution ratio were optimized for H7 antibody detection. The specificity of the cELISA was tested using antisera against H1 ~ H9, H11 ~ H14 AIVs and other avian viruses. The selected cut-off values of inhibition rates for chicken, duck and peacock sera were 30.11, 26.85 and 45.66% by receiver-operating characteristic (ROC) curve analysis, respectively. With HI test as the reference method, the minimum detection limits for chicken, duck and peacock positive serum reached 20, 21 and 2- 1 HI titer, respectively. Compared to HI test, the diagnostic accuracy reached 100, 98.6, and 99.3% for chicken, duck and peacock by testing a total of 400 clinical serum samples, respectively. CONCLUSIONS: In summary, the cELISA assay developed in this study provided a reliable, specific, sensitive and species-independent serological technique for rapid detection of H7 antibody, which was applicable for large-scale serological surveillance and vaccination efficacy evaluation programs.

High-complexity extracellular barcoding using a viral hemagglutinin.

While single-cell sequencing technologies have revealed tissue heterogeneity, resolving mixed cellular libraries into cellular clones is essential for many pooled screens and clonal lineage tracing. Fluorescent proteins are limited in number, while DNA barcodes can only be read after cell lysis. To overcome these limitations, we used influenza virus hemagglutinins to engineer a genetically encoded cell-surface protein barcoding system. Using antibodies paired to hemagglutinins carrying combinations of escape mutations, we developed an exponential protein barcoding system which can label 128 clones using seven antibodies. This study provides a proof of principle for a strategy to create protein-level cell barcodes that can be used in vivo in mice to track clonal populations.

Development and fit-for-purpose verification of an LC-MS method for quantitation of hemagglutinin and neuraminidase proteins in influenza virus-like particle vaccine candidates.

Recombinant influenza Virus-Like Particle (VLP) vaccines are promising vaccine candidates to prevent influenza, contain two major viral antigenic glycoproteins, Hemagglutinin (HA) and Neuraminidase (NA), on the surface of recombinant VLPs. Accurate quantitation of the mass of these antigenic proteins is important to ensure the product quality and proper dosing. Currently, Single Radial Immunodiffusion (SRID) is a recognized assay for determination of the HA immuno-reactive concentration (potency) in vaccine products, based on immuno-reactivity of HA with strain-specific antisera. The SRID assay, however, requires availability of strain-specific and properly calibrated reagents, which can be time-consuming to generate and calibrate. In addition, the assay is not suitable for quantitation of low abundant proteins, such as NA. In order to accelerate the overall production cycle, we have developed and optimized a high-resolution (HR) LC-MS method for absolute quantitation of both HA and NA protein concentrations in influenza VLP vaccine candidates. In this work, we present the method development, optimization and verification of its suitability for the intended purpose, as a prerequisite for its potential application in Quality Control, by assessing specificity, precision and accuracy, detection characteristics, and dynamic linear range. The method can be also used for other HA/NA containing preparations including in-process samples, purified proteins, whole virus preparations, nano-particle and egg-based vaccine preparations, or for calibration of SRID reference antigens.

Subtyping of Swine Influenza Using a High-Throughput Real-Time PCR Platform and a Single Microfluidics Device.

Reverse transcription real-time PCR (RT-qPCR) is one of several techniques used to determine the presence and level of infectious veterinary pathogens in diagnostic laboratories. Here we describe how automation of PCR reactions using integrated fluidic circuits (IFCs), an IFC controller MX and a Biomark HD instrument allows for the testing of 48 field samples with swine influenza for up to 48 different subtypes simultaneously in nanoliter volumes.

Hemagglutinin Quantitative ELISA-based Potency Assay for Trivalent Seasonal Influenza Vaccine Using Group-Specific Universal Monoclonal Antibodies.

The assurance of vaccine potency is important for the timely release and distribution of influenza vaccines. As an alternative to Single Radial Immunodiffusion (SRID), we report a new quantitative enzyme-linked immunosorbent assay (ELISA) for seasonal trivalent influenza vaccine (TIV). The consensus hemagglutinin (cHA) stalks for group 1 influenza A virus (IAV), group 2 IAV, and influenza B virus (IBV) were designed and produced in bacterial recombinant host in a soluble form, and monoclonal antibodies (mAbs) were generated. The group-specific 'universal' mAbs (uAbs) bound to various subtypes of HAs in the same group from recombinant hosts, embryonated eggs, and commercial vaccine lots. The calibration curves were generated to assess the sensitivity, specificity, accuracy, and linear dynamic range. The quantitative ELISA was validated for the potency assay of individual components of TIV- H1, H3, and IBV- with good correlation with the SRID method. This new assay could be extended to pandemic or pre-pandemic mock-up vaccines of H5 of group 1 and H7 virus of group 2, and novel HA stalk-based universal vaccines.

Isolation and Characterization of Avian Influenza H9N2 Viruses from Different Avian Species in Pakistan 2016-17.

Avian influenza (AI) virus (AIV) subtype H9N2 continues to cause significant outbreaks among commercial and backyard poultry in Pakistan. Despite this, the characterization of H9N2 viruses in avian hosts other than chickens in Pakistan has not been thoroughly investigated. In this study, 12 low pathogenicity avian influenza viruses subtype H9N2 were isolated from peacocks (n = 4), ducks (n = 4), pheasants (n = 2), geese (n = 1), and black swans (n = 1) in Pakistan during 2016 and were characterized on the basis of the hemagglutinin (HA) and neuraminidase genes. All of the viruses possessed an amino acid substitution Q226L in the receptor-binding site of the HA protein, which is known to contribute to increased viral replication and virulence in mammals. In addition, phylogenetic studies showed that these H9N2 AIVs belonged to the Middle East B genetic group of sublineage G1 and were very similar to viruses isolated from an outbreak in chickens in Pakistan in 2017. This demonstrates an epidemiologic link between poultry and other avian species, which is a fact to consider in future H9N2 disease management programs.

Nota de investigación ­ Aplicación in ovo y al primer día de edad de una vacuna viva contra el virus de la enfermedad infecciosa de la bolsa en pollos de engorde comerciales. La enfermedad infecciosa de la bolsa (IBD) es una enfermedad económicamente importante de pollos jóvenes causada por un Avibirnavirus, el virus de la enfermedad infecciosa de la bolsa (IBDV). El virus causal es altamente resistente a los ambientes avícolas y la vacunación es la medida más efectiva para el control de esta enfermedad. Sin embargo, la presunta neutralización de las cepas altamente atenuadas por los anticuerpos maternos y la supuesta virulencia de las cepas parcialmente atenuadas ha limitado la implementación de cepas de vacunas vivas convencionales contra la enfermedad de Gumboro en pollitos antes o después de la eclosión. Sin embargo, los datos preliminares han generado dudas sobre la validez de este dogma prevaleciente. Para analizar la posible aplicación de una cepa de vacuna viva intermedia plus del virus de Gumboro, cepa MB-1, en embriones de pollo con inmunidad materna y a pollos de un día de edad, se realizaron cuatro ensayos de campo a gran escala en distintos lugares del mundo. Los cuatro ensayos midieron la seguridad relativa, los parámetros de inmunización para Gumboro y los rendimientos de producción de MB-1 frente a las vacunas de Gumboro con complejos inmunes establecidas en una variedad de sistemas comerciales de engorde. El desempeño general de la salud y la producción en los cuatro ensayos ha sido similar o mejor en los grupos con la cepa MB-1. Los resultados desafían la noción prevaleciente de que las cepas vivas de del virus de la enfermedad infecciosa de la bolsa pueden neutralizarse o sobrepasar la inmunidad materna e inducir daño permanente a la respuesta inmune de los pollos de engorde jóvenes. Se ha observado un fenómeno de replicación retardada después de la administración parenteral de la cepa de vacuna viva contra el virus de la enfermedad de Gumboro, sin embargo, este mecanismo de replicación retardada aún no se ha dilucidado. Los resultados de este estudio justifican una mayor investigación de las cepas de vacunas vivas convencionales contra la enfermedad de Gumboro como una alternativa para la inmunización activa de pollos de engorde antes y después de la eclosión.

Characterization of influenza virus PR8 strain cultured in embryonated eggs by cryo-electron tomography.

Influenza A viruses, as causative agents of seasonal epidemics and periodic worldwide pandemics, cause enormous mortality loss globally. The PR8 strain cultured in chicken eggs is widely used for scientific research and the production of influenza vaccines. Here, based on Cryo-electron Tomography (CET), we analyzed the morphological and structural characteristics of the influenza virus PR8 strain at different pHs. We found that a large number of defective virions were propagated in embryonated eggs. By comparing virions with/without the matrix layer, it was revealed that the matrix layer played an essential role in the structural integrity of virions and RNPs encapsulation during the influenza virus life cycle. We also utilized hemagglutinin receptor-containing liposomes to mimic the membrane fusion process. Several potential intermediates of HA during membrane fusion were observed at acidic pH. Our observations afford insight into the architecture and function of influenza virus.

Influenza A virus surface proteins are organized to help penetrate host mucus.

Influenza A virus (IAV) enters cells by binding to sialic acid on the cell surface. To accomplish this while avoiding immobilization by sialic acid in host mucus, viruses rely on a balance between the receptor-binding protein hemagglutinin (HA) and the receptor-cleaving protein neuraminidase (NA). Although genetic aspects of this balance are well-characterized, little is known about how the spatial organization of these proteins in the viral envelope may contribute. Using site-specific fluorescent labeling and super-resolution microscopy, we show that HA and NA are asymmetrically distributed on the surface of filamentous viruses, creating a spatial organization of binding and cleaving activities that causes viruses to step consistently away from their NA-rich pole. This Brownian ratchet-like diffusion produces persistent directional mobility that resolves the virus's conflicting needs to both penetrate mucus and stably attach to the underlying cells, potentially contributing to the prevalence of the filamentous phenotype in clinical isolates of IAV.

An integrated device for the rapid and sensitive detection of the influenza hemagglutinin.

Influenza is a viral respiratory tract infection responsible for up to 5 million cases of severe infection and nearly 600 000 deaths worldwide each year. While treatments for influenza exist, diagnostics for the virus at the point of care are limited in their sensitivity and ability to differentiate between subtypes. We have developed an integrated two-dimensional paper network (2DPN) for the detection of the influenza virus by the surface glycoprotein, hemagglutinin. The hemagglutinin assay was developed using proteins computationally designed to bind with high affinity to the highly-conserved sialic acid binding site. The integrated 2DPN uses a novel geometry that allows automated introduction of an enzymatic amplification reagent directly to the detection zone. This assay was integrated into a prototype device and demonstrated successful detection of clinically relevant virus concentrations spiked into 70 µL of virus-free pediatric nasal swab samples. Using this novel geometry, we found improved assay performance on the device (compared to a manually-operated dipstick method), with a sensitivity of 4.45 × 102 TCID50 per mL on device.

Validation of two multiplex real-time PCR assays based on single nucleotide polymorphisms of the HA1 gene of equine influenza A virus in order to differentiate between clade 1 and clade 2 Florida sublineage isolates.

We validated 2 multiplex real-time PCR (rtPCR) assays based on single nucleotide polymorphisms (SNPs) of the hemagglutinin-1 ( HA1) gene of H3N8 equine influenza A virus (EIV) to determine clade affiliation of prototype and field isolates. Initial validation of the 2 multiplex rtPCR assays (SNP1 and SNP2) was performed using nucleic acid from 14 EIV Florida sublineage clade 1 and 2 prototype strains. We included in our study previously banked EIV rtPCR-positive nasal secretions from 341 horses collected across the United States in 2012-2017 to determine their clade affiliation. All 14 EIV prototype strains were identified correctly as either Florida sublineage clade 1 or clade 2 using the 2 SNP target positions. Of 341 EIV rtPCR-positive samples, 337 (98.8%) and 4 (1.2%) isolates were classified as belonging to clade 1 and 2 Florida sublineage EIV, respectively. All clade 1 Florida sublineage EIV strains were detected in domestic horses, three clade 2 Florida sublineage EIV strains originated from horses recently imported into the United States, and one clade 2 Florida sublineage EIV strain originated from a healthy horse recently vaccinated with a modified-live intranasal EIV vaccine containing the American lineage strain A/eq/Kentucky/1991. EIV Florida sublineage clade differentiation using a fast and reliable multiplex rtPCR platform will help monitor the introduction of clade 2 Florida sublineage EIV strains into North America via international transportation.

A sandwich ELISA for detecting the hemagglutinin of avian influenza A (H10N8) virus.

Novel influenza A virus (H10N8) infected human with fatality in China during 2013-2014. It is important to detect such nonprevalent subtype influenza A virus in clinic and regular surveillance in the early stage for effective control and prevention from the potential pandemic. Unavailability of convenient rapid diagnosis for this subtype virus in resources-limited setting is an obstacle for timely recognizing human case. In the present study, a panel of mouse H10 specific monoclonal antibodies (mAbs) was generated, two of which were used to develop a sandwich enzyme-linked immunosorbent assay (ELISA) for detecting the hemagglutinin of avian influenza A (H10N8) virus. ELISA results showed high sensitivity with the lowest detection limit of 0.5HAU/50 µL for live virus, which laid a foundation for clinic use as a promising diagnostic methodology.

Kinetic analysis of the influenza A virus HA/NA balance reveals contribution of NA to virus-receptor binding and NA-dependent rolling on receptor-containing surfaces.

Interactions of influenza A virus (IAV) with sialic acid (SIA) receptors determine viral fitness and host tropism. Binding to mucus decoy receptors and receptors on epithelial host cells is determined by a receptor-binding hemagglutinin (HA), a receptor-destroying neuraminidase (NA) and a complex in vivo receptor-repertoire. The crucial but poorly understood dynamics of these multivalent virus-receptor interactions cannot be properly analyzed using equilibrium binding models and endpoint binding assays. In this study, the use of biolayer interferometric analysis revealed the virtually irreversible nature of IAV binding to surfaces coated with synthetic sialosides or engineered sialoglycoproteins in the absence of NA activity. In addition to HA, NA was shown to be able to contribute to the initial binding rate while catalytically active. Virus-receptor binding in turn contributed to receptor cleavage by NA. Multiple low-affinity HA-SIA interactions resulted in overall extremely high avidity but also permitted a dynamic binding mode, in which NA activity was driving rolling of virus particles over the receptor-surface. Virus dissociation only took place after receptor density of the complete receptor-surface was sufficiently decreased due to NA activity of rolling IAV particles. The results indicate that in vivo IAV particles, after landing on the mucus layer, reside continuously in a receptor-bound state while rolling through the mucus layer and over epithelial cell surfaces driven by the HA-NA-receptor balance. Quantitative BLI analysis enabled functional examination of this balance which governs this dynamic and motile interaction that is expected to be crucial for penetration of the mucus layer and subsequent infection of cells by IAV but likely also by other enveloped viruses carrying a receptor-destroying enzyme in addition to a receptor-binding protein.

An immuno-assay to quantify influenza virus hemagglutinin with correctly folded stalk domains in vaccine preparations.

The standard method to quantify the hemagglutinin content of influenza virus vaccines is the single radial immunodiffusion assay. This assay primarily relies on polyclonal antibodies against the head domain of the influenza virus hemagglutinin, which is the main target antigen of influenza virus vaccines. Novel influenza virus vaccine candidates that redirect the immune response towards the evolutionary more conserved hemagglutinin stalk, including chimeric hemagglutinin and headless hemagglutinin constructs, are highly dependent on the structural integrity of the protein to present conformational epitopes for neutralizing antibodies. In this study, we describe a novel enzyme-linked immunosorbent assay that allows quantifying the amount of hemagglutinin with correctly folded stalk domains and which could be further developed into a potency assay for stalk-based influenza virus vaccines.

Establishment of the cross-clade antigen detection system for H5 subtype influenza viruses using peptide monoclonal antibodies specific for influenza virus H5 hemagglutinin.

The H5 subtype of highly pathogenic avian influenza (H5 HPAI) viruses is a threat to both animal and human public health and has the potential to cause a serious future pandemic in humans. Thus, specific and rapid detection of H5 HPAI viruses is required for infection control in humans. To develop a simple and rapid diagnostic system to detect H5 HPAI viruses with high specificity and sensitivity, we attempted to prepare monoclonal antibodies (mAbs) that specifically recognize linear epitopes in hemagglutinin (HA) of H5 subtype viruses. Nine mAb clones were obtained from mice immunized with a synthetic partial peptide of H5 HA molecules conserved among various H5 HPAI viruses. The antigen-capture enzyme-linked immunosorbent assay using the most suitable combination of these mAbs, which bound specifically to lysed H5 HA under an optimized detergent condition, was specific for H5 viruses and could broadly detect H5 viruses in multiple different clades. Taken together, these peptide mAbs, which recognize linear epitopes in a highly conserved region of H5 HA, may be useful for specific and highly sensitive detection of H5 HPAI viruses and can help in the rapid diagnosis of human, avian, and animal H5 virus infections.

Electrochemical detection of influenza virus H9N2 based on both immunomagnetic extraction and gold catalysis using an immobilization-free screen printed carbon microelectrode.

Influenza is a viral infectious disease considered as a source of many health problems and enormous socioeconomic disruptions. Conventional methods are inadequate for in-field detection of the virus and generally suffer from being laborious and time-consuming. Thus, studies aiming to develop effective alternatives to conventional methods are urgently needed. In this work, we developed an approach for the isolation and detection of influenza A virus subtype H9N2. For this aim, two specific influenza receptors were used. The first, anti-matrix protein 2 (M2) antibody, was attached to iron magnetic nanoparticles (MNPs) and used for the isolation of the virus from allantoic fluid. The second biomolecule, Fetuin A, was attached to an electrochemical detectable label, gold nanoparticles (AuNPs), and used to detect the virus tacking advantage from fetuin-hemagglutinin interaction. The MNP-Influenza virus-AuNP formed complex was isolated and treated by an acid solution then the collected gold nanoparticles were deposited onto a screen printed carbon electrode. AuNPs catalyzes the hydrogen ions reduction in acidic medium while applying an appropriate potential, and the generated current signal was proportional to the virus titer. This approach allows the rapid detection of influenza virus A/H9N2 at a less than 16 HAU titer.

A latex agglutination assay to quantify the amount of hemagglutinin protein in adjuvanted low-dose influenza monovalent vaccines.

To formulate inactivated influenza vaccines, the concentration of hemagglutinin (HA) must be accurately determined. The standard test currently used to measure HA in influenza vaccines is the Single Radial Immunodiffusion (SRID) assay. We developed a very rapid, simple and sensitive alternative quantitative HA assay, namely the Latex Agglutination Assay (LAA). The LAA uses the Spherotest® technology, which is based on the agglutination of HA-specific immunoglobulin-coated latex beads. The amount of HA in a sample is calculated from the level of bead agglutination by a simple absorbance measurement at 405nm against a standard curve generated using a monovalent vaccine standard. In less than 2hours, tens of samples could be quantified using the LAA as opposed to 2days for the SRID assay. Ten steps are required to complete an SRID assay as compared to 6 steps for the LAA, from sample preparation through spectrophotometric analysis. Furthermore, the limit of detection of the LAA was found to be approximately 15ng HA/mL, similar to an ELISA, with the quantification of less than 1.8µg HA/mL. The quantification limit of the SRID is usually considered to be approximately 5µg HA/mL. The development of the assay and a comparison of the titers obtained by SRID and LAA for several monovalent vaccines corresponding to various strains were performed. For A/H5N1 and A/H1N1 monovalent vaccines, the LAA was found to be linear and accurate as compared to the SRID. The precision of the LAA was close to that of the standard test, and good reproducibility from one laboratory to another was observed. Moreover, the LAA enabled HA quantification in AlOOH-adjuvanted and in emulsion-adjuvanted low-dose vaccines as well as unadjuvanted vaccines. In conclusion, LAA may be useful to rapidly and accurately measure influenza HA protein in monovalent vaccines, especially in those containing less than 5µg/mL of HA in the presence of an adjuvant.

Rapid and broad detection of H5 hemagglutinin by an immunochromatographic kit using novel monoclonal antibody against highly pathogenic avian influenza virus belonging to the genetic clade 2.3.4.4.

Highly pathogenic avian influenza viruses (HPAIVs) of H5 subtype have persistently caused outbreaks in domestic poultry and wild birds worldwide and sporadically infected humans. Rapid and accurate diagnosis is one of the key strategies for the control of H5 HPAIVs. However, the sensitivity of the diagnosis of H5 HPAIVs has gradually reduced due to extensive antigenic variation during their evolution. Particularly, the previously developed immunochromatographic diagnosis kit for H5 viruses, Linjudge Flu A/H5, exhibits reduced detection of H5 HPAIVs isolated in recent years. In the present study, we established a new advanced H5 rapid immunochromatographic detection kit (New Linjudge Flu A/H5) by a combination of two anti-H5 hemagglutinin monoclonal antibodies, A64/1 previously applied in the Linjudge Flu A/H5 and A32/2, a novel monoclonal antibody generated from a clade 2.3.4.4 H5 HPAIV. The new kit broadly detected all classical and recent H5 influenza viruses and showed a higher specificity and sensitivity than the original Linjudge Flu A/H5 with recently circulating H5 HPAIVs. Furthermore, the applicability of the New Linjudge Flu A/H5 was demonstrated by detecting antigens from the swabs and tissue homogenates of naturally infected birds and experimentally infected chickens with H5N6 HPAIVs belonging to the genetic clade 2.3.4.4. Our study, therefore, can provide an effective point-of-care rapid antigen detection kit for the surveillance of H5 avian influenza viruses and as a prompt countermeasure against the current widespread of the clade 2.3.4.4 H5 HPAIVs in domestic and wild birds.