Structural and functional characterization of avian influenza H9N2 virus neuraminidase with a combination of five novel mutations.

The influenza virus neuraminidase (NA) protein is responsible for actively cleaving the sialic acid (SA) bound to the viral hemagglutinin. In the present study, we identified a combination of five novel amino acid substitutions in the NA, conferring increased substrate binding and altered surface characteristics to a low pathogenic avian influenza (LPAI) H9N2 virus strain. The H9N2 strain reported from India, A/Environmental/India/1726265/2017 (H9N2-1726265) showed the combination of amino acid substitutions T149I, R249W, G346A, W403R and G435R, which were in the vicinity of the enzyme active site cavity. The strain A/chicken/India/99321/2009 (H9N2-99321) did not show these substitutions and was used for comparison. Virus elution was studied using turkey red blood cells (tRBCs). NA enzyme kinetics assays were carried out using the MUNANA substrate, which is an SA analogue. Homology modelling and molecular docking were performed to determine alterations in the surface characteristics and substrate binding. H9N2-1726265 showed enhanced elution from tRBCs. Enzyme kinetics revealed a lower KM of H9N2-1726265 (111.5 µM) as compared to H9N2-99321 (135.2 µM), indicating higher substrate binding affinity of H9N2-1726265, due to which the NA enzyme cleaved the SA more efficiently, leading to faster elution. Molecular docking revealed a greater number of binding interactions of H9N2-1726265 to SA as compared to H9N2-99321 corroborating the greater substrate binding affinity. Changes in the surface charge, hydrophobicity, and contour, were observed in H9N2-1726265 NA due to the five substitutions. Thus, the novel combination of five amino acids near the sialic acid binding site of NA, resulted in altered surface characteristics, higher substrate binding affinity, and virus elution.

The adaptability of H9N2 avian influenza A virus to humans: A comparative docking simulation study.

Influenza A virus, the H9N2 subtype, is an avian influenza virus that has long been circulating in the worldwide poultry industry and is occasionally found to be transmissible to humans. Evidence from genomic analysis suggests that H9N2 provides the genes for the H5N1 and H7N9 subtypes, which have been found to infect mammals and pose a threat to human health. However, due to the lack of a structural model of the interaction between H9N2 and host cells, the mechanism of the extensive adaptability and strong transformation capacity of H9N2 is not fully understood. In this paper, we collected 40 representative H9N2 virus samples reported recently, mainly in China and neighboring countries, and investigated the interactions between H9N2 hemagglutinin and the mammalian receptor, the polysaccharide α-2,6-linked lactoseries tetrasaccharide c, at the atomic level using docking simulation tools. We categorized the mutations of studied H9N2 hemagglutinin according to their effects on ligand-binding interactions and the phylogenetic analysis. The calculations indicated that all the studied H9N2 viruses can establish a tight binding with LSTc although the mutations caused a variety of perturbations to the local conformation of the binding pocket. Our calculations suggested that a marginal equilibrium is established between the conservative ligand-receptor interaction and the conformational dynamics of the binding pocket, and it might be this equilibrium that allows the virus to accommodate mutations to adapt to a variety of environments. Our results provided a way to understand the adaptive mechanisms of H9N2 viruses, which may help predict its propensity to spread in mammals.

Mineralized State of the Avian Influenza Virus in the Environment.

Although the circulation of avian influenza viruses in humans is limited, they can be transmitted from Aves (birds) to humans, representing a great challenge. Herein, we suggest that influenza viruses from Aves might exist in a mineralized state owing to the high calcium concentrations in the avian intestine. Using two typical influenza viruses as examples, we demonstrate that these viruses can self-mineralize in simulated avian intestinal fluid, resulting in egg-like virus-mineral structured composites. The mineralized viruses are more robust, with enhanced infectivity and thermostability. More importantly, the mineral exterior of mineralized viruses can alter their cell internalization, expanding the possible tropisms. The discovery of a mineralized state of influenza viruses highlights the integration of nanomaterials and viruses in the environment, which provides a new understanding of avian influenza infection and its control.

Evaluation of a smartphone-based rapid fluorescent diagnostic system for H9N2 virus in specific-pathogen-free chickens.

Repeated interspecies transmission of H9N2 virus from poultry to humans and human infections transmitted via aerosols highlight the need for a highly sensitive, rapid diagnostic system for the detection of this virus. However, no such test exhibiting high performance has been developed. In this study, the performance of a smartphone-based rapid fluorescent diagnostic system (SRFDS) was optimized for the diagnosis of an H9N2-virus-infected animal. To suppress the nonspecific reactivity of the bioconjugate in oropharyngeal (OP) and cloacal (CL) samples derived from chickens, different blocking reagents were tested, and a mixture of casein and sucrose was found to be optimal. To assess the performance of SRFDS, OP and CL samples were obtained from specific-pathogen-free chickens and used for comparison of this method with real-time reverse transcription PCR (rRT-PCR) at time points of three, five, and seven days postinfection (dpi). The limit of detection of SRFDS was found to be 7.5 PFU/mL, which was 138-fold higher than that of a conventional colloidal-gold-based avian influenza rapid diagnostic test. In the animal study, the presence of viral antigen was monitored with SRFDS, and the relative sensitivity (relative to rRT-PCR results) was 94.44 % (17/18) and 95.23 % (20/21) in OP and CL specimens, respectively. The specificity of SRFDS was 100 %. These results imply that the diagnostic performance of SRFDS might be comparable to that of rRT-PCR for diagnosis of H9N2 in chickens and that this test can be used as a highly sensitive rapid diagnostic method in field studies on broiler poultry and wild birds.

[Genetic evolution and substitution frequency of avian influenza virus HA gene in chicken H9N2 subtype in China in the last 20 years].

OBJECTIVE: Low pathogenic avian influenza (LPAI) H9N2 subtype virus has been prevalent in domestic poultry in China over two decades. This study was to determine the genetic evolution trend of H9N2 avian influenza virus (AIV) under immune pressure of vaccine. METHODS: H9 HA sequences of 40 isolates from the present study and 136 pandemic strains and 7 classical strains from China downloaded from GenBank, were genetically analyzed to determine evolution, molecular characteristic, and mutation frequency. RESULTS: Phylogenetic trees analysis suggested that H9N2 subtypes AIV could be clustered into 5 distinct lineages: G1-like, BJ94-like, Y280-like, S2-like and Americans lineages. Most H9N2 isolates in 2005-2014 belonged to S2-like sub-genotype, suggesting that this genotype was the dominate isolates in China. Further more, comparison based on the amino acid sequence showed that different lineages have their distinct characteristics, and significant accumulations of amino acid variation were also found. In addition, in comparison with reference Ck/BJ/1/1994 HA gene, average annual substitution rates of H9N2 pandemic strain nucleotide and amino acid were 5.73 x 10⁻³ and 4.25 x 10⁻³ from 1994 to 2014, respectively. Substitution rate during 2011-2014 were 6.35 x 10⁻³ and 5.32 x 10⁻³, higher than that during the period of 2006-2010 (5.22 x 10⁻³ and 3.70 x 10⁻³) and even much higher than that during the 1999-2005 (0.74 x 10⁻³ and 0.50 x 10⁻³), when the vaccines were initially applied in the field. CONCLUSION: Overall, these data indicate that the mismatch between H9N2 vaccine strains and pandemic strains drives the virus to quickly mutate.

Twenty amino acids at the C-terminus of PA-X are associated with increased influenza A virus replication and pathogenicity.

The PA-X protein, arising from ribosomal frameshift during PA translation, was recently discovered in influenza A virus (IAV). The C-terminal domain 'X' of PA-X proteins in IAVs can be classified as full-length (61 aa) or truncated (41 aa). In the main, avian influenza viruses express full-length PA-X proteins, whilst 2009 pandemic H1N1 (pH1N1) influenza viruses harbour truncated PA proteins. The truncated form lacks aa 232-252 of the full-length PA-X protein. The significance of PA-X length in virus function remains unclear. To address this issue, we constructed a set of contemporary influenza viruses (pH1N1, avian H5N1 and H9N2) with full and truncated PA-X by reverse genetics to compare their replication and host pathogenicity. All full-length PA-X viruses in human A549 cells conferred 10- to 100-fold increase in viral replication and 5-8% increase in apoptosis relative to corresponding truncated PA-X viruses. Full-length PA-X viruses were more virulent and caused more severe inflammatory responses in mice. Furthermore, aa 233-252 at the C terminus of PA-X strongly suppressed co-transfected gene expression by ∼ 50%, suggesting that these terminal 20 aa could play a role in enhancing viral replication and contribute to virulence.

A novel activation mechanism of avian influenza virus H9N2 by furin.

Avian influenza virus H9N2 is prevalent in waterfowl and has become endemic in poultry in Asia and the Middle East. H9N2 influenza viruses have served as a reservoir of internal genes for other avian influenza viruses that infect humans, and several cases of human infection by H9N2 influenza viruses have indicated its pandemic potential. Fortunately, an extensive surveillance program enables close monitoring of H9N2 influenza viruses worldwide and has generated a large repository of virus sequences and phylogenetic information. Despite the large quantity of sequences in different databases, very little is known about specific virus isolates and their pathogenesis. Here, we characterize a low-pathogenicity avian influenza virus, A/chicken/Israel/810/2001 (H9N2) (Israel810), which is representative of influenza virus strains that have caused severe morbidity and mortality in poultry farms. We show that under certain circumstances the Israel810 hemagglutinin (HA) can be activated by furin, a hallmark of highly pathogenic avian influenza virus. We demonstrate that Israel810 HA can be cleaved in cells with high levels of furin expression and that a mutation that eliminates a glycosylation site in HA(1) allows the Israel810 HA to gain universal cleavage in cell culture. Pseudoparticles generated from Israel810 HA, or the glycosylation mutant, transduce cells efficiently. In contrast, introduction of a polybasic cleavage site into Israel810 HA leads to pseudoviruses that are compromised for transduction. Our data indicate a mechanism for an H9N2 evolutionary pathway that may allow it to gain virulence in a distinct manner from H5 and H7 influenza viruses.

Amino acid 316 of hemagglutinin and the neuraminidase stalk length influence virulence of H9N2 influenza virus in chickens and mice.

H9N2 influenza viruses with an A316S substitution in hemagglutinin (HA) and a shorter neuraminidase (NA) stalk have become predominant in China. The A316S was shown to increase HA cleavage efficiency when combined with short stalk NA, and the short stalk NA improved NA enzyme activity and release of virus from erythrocytes. Single mutations or combinations of these mutations strengthened the virulence of H9N2 virus in chickens and mice.

Sequence and phylogenetic analysis of surface protein genes of emerging H9N2 influenza viruses isolated from poultry in two geographical regions of China.

Subtype H9N2 avian influenza viruses (AIVs) circulating in China have aroused increasing concerns for their impact on poultry and risk to public health. The present study was an attempt to elucidate the phylogenetic relationship of H9N2 AIVs in two geographically distinct regions of China where vaccination is routinely practiced. A total of 18 emerging H9N2 isolates were identified and genetically characterized. Phylogenetic analysis of hemagglutinin (HA) and neuraminidase (NA) genes confirmed that the isolates belonged to the Y280 lineage. Based on the HA genes, the isolates were subdivided into two subgroups. The viruses from Zhejiang Province were clustered together in Group I, while the isolates from Guangdong Province were clustered together in Group II. Antigenic characterization showed that the tested viruses were antigenically different when compared to the current used vaccine strain. It was notable that 14 out of total 18 isolates had an amino acid exchange (Q→L) at position 216 (226 by H3 Numbering) in the receptor-binding site, which indicated that the virus had potential affinity of binding to human like receptor. These results suggest that the emerging viruses have potential risk to public health than previously thought. Therefore, continuous surveillance studies of H9N2 influenza virus are very important to the prognosis and control of future influenza pandemics.

Age- and sex-associated differences in the glycopatterns of human salivary glycoproteins and their roles against influenza A virus.

Recent studies have elucidated that expression of certain glycoproteins in human saliva is increased or decreased according to age; meanwhile, human saliva may inhibit viral infection and prevent viral transmission. However, little is known about the age- and sex-associated differences in the glycopatterns of human salivary glycoproteins and their significant roles against influenza A virus (IVA). Here, we investigate the glycopatterns of human salivary glycoproteins with 180 healthy saliva samples divided into six age/sex groups using lectin microarrays and fabricate saliva microarrays to validate the terminal carbohydrate moieties of glycoproteins in individual saliva samples. Furthermore, we assess the inhibiting and neutralizing activity of saliva against two strains of influenza A (H9N2) virus. We find that seven lectins (e.g., MAL-II and SNA) show significant age differences in both females and males, and seven lectins (e.g., WFA and STL) show significant sex differences in children, adults and elderly people. Interestingly, we observe that elderly individuals have strongest resistance to IVA partly by presenting more terminal α2-3/6-linked sialic acid residues in their saliva, which bind with the influenza viral hemagglutinations. We conclude that age- and sex-associated differences in the glycopatterns of human salivary glycoproteins may provide pivotal information to help understand some age related diseases and physiological phenomena.

Deletions in the neuraminidase stalk region of H2N2 and H9N2 avian influenza virus subtypes do not affect postinfluenza secondary bacterial pneumonia.

We investigated the synergism between influenza virus and Streptococcus pneumoniae, particularly the role of deletions in the stalk region of the neuraminidase (NA) of H2N2 and H9N2 avian influenza viruses. Deletions in the NA stalk (ΔNA) had no effect on NA activity or on the adherence of S. pneumoniae to virus-infected human alveolar epithelial (A549) and mouse lung adenoma (LA-4) cells, although it delayed virus elution from turkey red blood cells. Sequential S. pneumoniae infection of mice previously inoculated with isogenic recombinant H2N2 and H9N2 influenza viruses displayed severe pneumonia, elevated levels of intrapulmonary proinflammatory responses, and death. No differences between the WT and ΔNA mutant viruses were detected with respect to effects on postinfluenza pneumococcal pneumonia as measured by bacterial growth, lung inflammation, morbidity, mortality, and cytokine/chemokine concentrations. Differences were observed, however, in influenza virus-infected mice that were treated with oseltamivir prior to a challenge with S. pneumoniae. Under these circumstances, mice infected with ΔNA viruses were associated with a better prognosis following a secondary bacterial challenge. These data suggest that the H2N2 and H9N2 subtypes of avian influenza A viruses can contribute to secondary bacterial pneumonia and deletions in the NA stalk may modulate its outcome in the context of antiviral therapy.

A mild and reliable method to label enveloped virus with quantum dots by copper-free click chemistry.

Real-time tracking of the dynamic process of virus invasion is crucial to understanding the infection mechanism. For successful tracking, efficient labeling methods are indispensable. In this paper, we report a mild and reliable method for labeling viruses, especially with regard to easily disabled enveloped viruses. The copper-free click chemistry has been used to label enveloped viruses with quantum dots (QDs) by linking virions modified with azide to the QDs derived with dibenzocyclooctynes (DBCO). Both vaccinia virus (VACV) and avian influenza A virus (H9N2) can be specifically and rapidly labeled under mild conditions, with a labeling efficiency of more than 80%. The labeled virions were of intact infectivity, and their fluorescence was strong enough to realize single-virion tracking. Compared to previously reported methods, our method is less destructive, reliable, and universal, without specific requirements for the type and structure of viruses to be labeled, which has laid the foundation for long-term dynamic visualization of virus infection process.

Robust and highly sensitive fluorescence approach for point-of-care virus detection based on immunomagnetic separation.

In this work, robust approach for a highly sensitive point-of-care virus detection was established based on immunomagnetic nanobeads and fluorescent quantum dots (QDs). Taking advantage of immunomagnetic nanobeads functionalized with the monoclonal antibody (mAb) to the surface protein hemagglutinin (HA) of avian influenza virus (AIV) H9N2 subtype, H9N2 viruses were efficiently captured through antibody affinity binding, without pretreatment of samples. The capture kinetics could be fitted well with a first-order bimolecular reaction with a high capturing rate constant k(f) of 4.25 × 10(9) (mol/L)(-1) s(-1), which suggested that the viruses could be quickly captured by the well-dispersed and comparable-size immunomagnetic nanobeads. In order to improve the sensitivity, high-luminance QDs conjugated with streptavidin (QDs-SA) were introduced to this assay through the high affinity biotin-streptavidin system by using the biotinylated mAb in an immuno sandwich mode. We ensured the selective binding of QDs-SA to the available biotin-sites on biotinylated mAb and optimized the conditions to reduce the nonspecific adsorption of QDs-SA to get a limit of detection low up to 60 copies of viruses in 200 µL. This approach is robust for application at the point-of-care due to its very good specificity, precision, and reproducibility with an intra-assay variability of 1.35% and an interassay variability of 3.0%, as well as its high selectivity also demonstrated by analysis of synthetic biological samples with mashed tissues and feces. Moreover, this method has been validated through a double-blind trial with 30 throat swab samples with a coincidence of 96.7% with the expected results.

H9N2 influenza virus acquires intravenous pathogenicity on the introduction of a pair of di-basic amino acid residues at the cleavage site of the hemagglutinin and consecutive passages in chickens.

BACKGROUND: Outbreaks of avian influenza (AI) caused by infection with low pathogenic H9N2 viruses have occurred in poultry, resulting in serious economic losses in Asia and the Middle East. It has been difficult to eradicate the H9N2 virus because of its low pathogenicity, frequently causing in apparent infection. It is important for the control of AI to assess whether the H9N2 virus acquires pathogenicity as H5 and H7 viruses. In the present study, we investigated whether a non-pathogenic H9N2 virus, A/chicken/Yokohama/aq-55/2001 (Y55) (H9N2), acquires pathogenicity in chickens when a pair of di-basic amino acid residues is introduced at the cleavage site of its HA molecule. RESULTS: rgY55sub (H9N2), which had four basic amino acid residues at the HA cleavage site, replicated in MDCK cells in the absence of trypsin after six consecutive passages in the air sacs of chicks, and acquired intravenous pathogenicity to chicken after four additional passages. More than 75% of chickens inoculated intravenously with the passaged virus, rgY55sub-P10 (H9N2), died, indicating that it is pathogenic comparable to that of highly pathogenic avian influenza viruses (HPAIVs) defined by World Organization for Animal Health (OIE). The chickens inoculated with the virus via the intranasal route, however, survived without showing any clinical signs. On the other hand, an avirulent H5N1 strain, A/duck/Hokkaido/Vac-1/2004 (Vac1) (H5N1), acquired intranasal pathogenicity after a pair of di-basic amino acid residues was introduced into the cleavage site of the HA, followed by two passages by air sac inoculation in chicks. CONCLUSION: The present results demonstrate that an H9N2 virus has the potential to acquire intravenous pathogenicity in chickens although the morbidity via the nasal route of infection is lower than that of H5N1 HPAIV.

Assessing the effects of a two-amino acid flexibility in the Hemagglutinin 220-loop receptor-binding domain on the fitness of Influenza A(H9N2) viruses.

The enzootic and zoonotic nature of H9N2 avian influenza viruses poses a persistent threat to the global poultry industry and public health. In particular, the emerging sublineage h9.4.2.5 of H9N2 viruses has drawn great attention. In this study, we determined the effects of the flexibility at residues 226 and 227 in the hemagglutinin on the receptor avidity and immune evasion of H9N2 viruses. The solid-phase direct binding assay showed that residue 226 plays a core role in the receptor preference of H9N2 viruses, while residue 227 affects the preference of the virus for a receptor. Consequently, each of these two successive residues can modulate the receptor avidity of H9N2 viruses and influence their potential of zoonotic infection. The antigenic map based on the cross-hemagglutination inhibition (HI) titers revealed that amino acid substitutions at positions 226 or 227 appear to be involved in antigenic drift, potentially resulting in the emergence of H9N2 immune evasion mutants. Further analysis suggested that increased receptor avidity facilitated by residue 226Q or 227M resulted in a reduction in the HI titer. Among the four naturally-occurring amino acid combinations comprising QQ, MQ, LQ, and LM, the number of viruses with LM accounted for 79.64% of the sublineage h9.4.2.5 and the rescued virus with LM exhibited absolute advantages of in vitro and in vivo replication and transmission. Collectively, these data demonstrate that residues 226 and 227 are under selective pressure and their synergistic regulation of receptor avidity and antigenicity is related to the evolution of circulating H9N2 viruses.

Identification of unusual truncated forms of nucleocapsid protein in MDCK cells infected by Avian influenza virus (H9N2).

Unusual truncated forms of nucleocapsid protein (NP) were identified in the lysate of MDCK cells infected by Avian influenza virus (H9N2) using MS-based proteomics approach. Moreover, O-sulfonation that was considered as an unusual modification was identified in one of the tryptic peptides from the truncated NP. The findings might have implications on better understanding on the role of nucleoprotein in Avian influenza virus-host interaction.

Mass spectrometry-based qualitative and quantitative N-glycomics: An update of 2017-2018.

N-glycosylation is one of the most frequently occurring protein post-translational modifications (PTMs) with broad cellular, physiological and pathological relevance. Mass spectrometry-based N-glycomics has become the state-of-the-art instrumental analytical pipeline for sensitive, high-throughput and comprehensive characterization of N-glycans and N-glycomes. Improvement and new development of methods in N-glycan release, enrichment, derivatization, isotopic labeling, separation, ionization, MS, tandem MS and informatics accompany side-by-side wider and deeper application. This review provides a comprehensive update of mass spectrometry-based qualitative and quantitative N-glycomics in the years of 2017-2018.

Reconstruction of a chicken BF2 protein complex and identification of binding nonamer peptides derived from avian influenza virus hemagglutinin.

In order to reconstruct the system for identification of short antigenic peptides, the chicken BF2 gene of Chinese Sanhuang (SH) chicken line was linked to the beta(2)m gene via (G4S)3, a linker encoding a 15-amino acid glycine-rich peptide, by splicing overlap extension PCR (SOE-PCR). The MBP-BF2-(G4S)3-beta(2)m fusion protein was expressed and purified in a pMAL-p2X/E. coli TB1 system. The purified MBP-BF2-(G4S)3-beta(2)m protein was cleaved by Factor Xa protease, and further purified by DEAE-Sepharose chromatography. The conformation of the BF2-(G4S)3-beta(2)m protein was determined by circular dichroism (CD). In addition, the refolded BF2-(G4S)3-beta2m protein was used to bind three predicted nonameric peptides derived from the hemagglutinins of the avian influenza virus (AIV) H5N1 and H9N2 subtypes. The BF2-(G4S)3-beta2m-associated peptides were detected by mass spectrometry. The molecular weights and amino acid sequences of the peptides were confirmed by primary and tandem mass spectrometry analysis, respectively. The results indicate that the secondary structures and predicted three-dimensional crystal structure of BF2-(G4S)3-beta(2)m are similar to those of the monomers of chicken BF2 and beta(2)m. The BF2-(G4S)3-beta(2)m protein could bind two of the three predicted nonamer peptides derived from AIV hemagglutinin. The experimental system demonstrated that the reconstructed BF2-(G4S)3-beta(2)m protein complex can be used to identify nonamer peptides, including T-cell epitopes in chicken.

Pandemic Avian Influenza and Intra/Interhaemagglutinin Subtype Electrostatic Variation among Viruses Isolated from Avian, Mammalian, and Human Hosts.

Host jump can result in deadly pandemic events when avian influenza A viruses broaden their host specificity and become able to infect mammals, including humans. Haemagglutinin-the major capsid protein in influenza A viruses-is subjected to high rate mutations, of which several occur at its "head": the receptor-binding domain that mediates specific binding to host cell receptors. Such surface-changing mutations may lead to antigenically novel influenza A viruses hence in pandemics by host jump and in vaccine escape by antigenic drift. Changes in haemagglutinin surface electrostatics have been recently associated with antigenic drift and with clades evolution and spreading in H5N1 and H9N2 viruses. We performed a comparative analysis of haemagglutinin surface electrostatics to investigate clustering and eventual fingerprints among representative pandemic (H5 and H7) and nonpandemic (H4 and H6) avian influenza viral subtypes. We observed preferential sorting of viruses isolated from mammalian/human hosts among these electrostatic clusters of a subtype; however, sorting was not "100% specific" to the different clusters. Therefore, electrostatic fingerprints can help in understanding, but they cannot explain alone the host jumping mechanism.

Lectin affinity based elemental labeling with hybridization chain reaction for the sensitive determination of avian influenza A (H9N2) virions.

Avian influenza virus (AIV) as a type of highly pathogenic influenza A virus, can not only cause serious illness and death in poultry but also threat human health and lead to public panic. Rapid, sensitive detection of AIV is urgent and significant for prevention and timely control of influenza epidemics. Herein, we reported an inductively coupled plasma mass spectrometry (ICP-MS) based method for the analysis of AIV virions on the basis of the selective recognition between lectin Con A and glycoproteins on AIV surface and signal amplification of hybridization chain reaction (HCR). With H9N2 as the model AIV, the limit of detection was down to 0.12 ng mL-1 due to the dual amplification effect of AuNPs and HCR, and the linear range was 0.4-50 ng mL-1 with the relative standard deviation of 7.9% for seven replicate detections of 2 ng mL-1 H9N2 virions. Furthermore, the applicability of the method for the analysis of real biological samples was demonstrated by the spiking tests. The proposed approach is highly specific and sensitive for the detection of AIV with good application potential in early diagnosis, which is helpful for the prevention of influenza outbreak.