Designing Primers for H5 and H7 Subtypes of Avian Influenza Virus and Multiplex RT-PCR Amplification

[Objective] The research aimed to design primers that are suitable for detecting H5 and H7 subtypes of avian influenza virus （AIV） ; [Method] DNAStar was used to analyze the homology of the sequences of H5 and H7 subtypes of AIV accessed in GenBank, and design primers（ by Primer Premier 5.0） on high homologous region of these sequences, and then amplified by RT-PCR. [Result] The multiplex RT-PCR amplification, agarose gel electrophoresis and sequencing results showed that the self-designed primers are successful for detecting AIV. [Conclusion] It is feasible to rapidly diagnose AIV through this method.

Role of feline ANP32 proteins in regulating polymerase activity of influenza A virus

Recently,increasing natural infection cases and experimental animal challenge studies demonstrated domestic cats are susceptible to multiple subtypes influenza A virus(IAV)infections.Notably,some subtype IAV strains could circulate in domestic cats after cross-species transmission and even infected humans,posing a threat to public health.Host factors related to viral polymerase activity could determine host range of IAV and acidic nuclear phosphoprotein 32(ANP32)is the most important one among them.However,role of cat-derived ANP32 on viral polymerase activity and host range of IAV is still unknown.In the present study,a total of 10 feline ANP32(feANP32)splice variants(including 5 feANP32A,3 feANP32B,and 2 feANP32E)were obtained from domestic cats by RT-PCR.Sequence alignment results demonstrated amino acid deletions and/or insertions occurred among feANP32 variants,but all feANP32 proteins were primarily localized to cell nucleus.Minigenome replication systems for several representative IAV strains were established and the support ability of feANP32 on IAV polymerase activity was estimated.The results indicated that most feANP32A and feANP32B splice variants were able to support all the tested IAV strains,though the support activity of a single feANP32 protein on polymerase activity varied among different IAV strains.In addition,the role of feANP32 in supporting H3N2 canine influenza virus was determined by investigating viral replication in vitro.Collectively,our study systematically investigated the support activity of feANP32 on IAV,providing a clue for further exploring the mechanism of susceptibility of cats to IAV.

Genetic and biological properties of H9N2 avian influenza viruses isolated in central China from2020 to 2022

The H9N2 subtype of avian influenza virus(AIV)is widely prevalent in poultry and wild birds globally,and has become the predominant subtype circulating in poultry in China.The H9N2 AIV can directly or indirectly(by serving as a"donor virus")infect humans,posing a significant threat to public health.Currently,there is a lack of in-depth research on the prevalence of H9N2 viruses in Shanxi Province,central China.In this study,we isolated 14 H9N2 AIVs from October 2020 to April 2022 in Shanxi Province,and genetic analysis revealed that these viruses belonged to 7 different genotypes.Our study on animals revealed that the H9N2 strains we identified displayed high transmission efficiency among chicken populations,and exhibited diverse replication abilities within these birds.These viruses could replicate efficiently in the lungs of mice,with one strain also demonstrating the capacity to reproduce in organs like the brain and kidneys.At the cellular level,the replication ability of different H9N2 strains was evaluated using plaque formation assays and multi-step growth curve assays,revealing significant differences in the replication and proliferation efficiency of the various H9N2 viruses at the cellular level.The antigenicity analysis suggested that these isolates could be classified into 2 separate antigenic clusters.Our research provides crucial data to help understand the prevalence and biological characteristics of H9N2 AIVs in central China.It also highlights the necessity of enhancing the surveillance of H9N2 AIVs.

Antibodies elicited by Newcastle disease virus-vectored H7N9 avian influenza vaccine are functional in activating the complement system

H7N9 subtype avian influenza virus poses a great challenge for poultry industry.Newcastle disease virus(NDV)-vectored H7N9 avian influenza vaccines(NDV_(vec)H7N9)are effective in disease control because they are protective and allow mass administration.Of note,these vaccines elicit undetectable H7N9-specific hemagglutination-inhibition(HI)but high IgG antibodies in chickens.However,the molecular basis and protective mechanism underlying this particular antibody immunity remain unclear.Herein,immunization with an NDV_(vec)H7N9 induced low anti-H7N9 HI and virus neutralization titers but high levels of hemagglutinin(HA)-binding IgG antibodies in chickens.Three residues(S150,G151 and S152)in HA of H7N9 virus were identified as the dominant epitopes recognized by the NDV_(vec)H7N9 immune serum.Passively transferred NDV_(vec)H7N9 immune serum conferred complete protection against H7N9 virus infection in chickens.The NDV_(vec)H7N9 immune serum can mediate a potent lysis of HA-expressing and H7N9 virus-infected cells and significantly suppress H7N9 virus infectivity.These activities of the serum were significantly impaired after heat-inactivation or treatment with complement inhibitor,suggesting the engagement of the complement system.Moreover,mutations in the 150-SGS-152 sites in HA resulted in significant reductions in cell lysis and virus neutralization mediated by the NDV_(vec)H7N9 immune serum,indicating the requirement of antibody-antigen binding for complement activity.Therefore,antibodies induced by the NDV_(vec)H7N9 can activate antibody-dependent complement-mediated lysis of H7N9 virus-infected cells and complement-mediated neutralization of H7N9 virus.Our findings unveiled a novel role of the complement in protection conferred by the NDV_(vec)H7N9,highlighting a potential benefit of engaging the complement system in H7N9 vaccine design.

High-Value-Added Utilization of Turpentine:Screening of Anti-Influenza Virus Agents fromβ-Pinene Derivatives

Turpentine is a renewable and resourceful forest product.The deep processing and utilization of turpentine,particularly its primary componentβ-pinene,has garnered widespread attention.This study aimed to synthesize 40 derivatives ofβ-pinene,including nopinone,3-cyanopyridines of nopinone,myrtanyl acid,myrtanyl acylthioureas,and myrtanyl amides.We assessed the antiviral activities of theseβ-pinene derivatives against influenza virus A/Puerto Rico/8/34(H1N1)using the 3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method.Theβ-pinene derivatives were used before and after cellular infection with the influenza virus to evaluate their preventive and therapeutic effects against the H1N1 virus.The results showed that only compound 10o exhibited a preventive effect against the H1N1 virus with a half-maximal inhibitory concentration(IC50)value of 47.6μmol/L.Among the compounds,4e,4i,and 4l demonstrated therapeutic effects against cellular infection,with compound 4e displaying the most potent therapeutic effect(IC50=17.5μmol/L),comparable to the positive control ribavirin.These findings indicated that certainβ-pinene derivatives exhibited in vitro antiviral activity against the H1N1 influenza A virus,warranting further investigation as potential anti-influenza agents.

Pudilan Xiaoyan oral liquid regulates tissue inflammation and apoptosis in mice with influenza virus pneumonia

Background:The influenza A virus is the primary cause of respiratory infections and poses a global health risk.Pudilan Xiaoyan oral liquid(PDL)exhibits anti-inflammatory and immunomodulatory properties.PDL is commonly employed in clinical practice to manage upper respiratory tract infections.However,there is still much to uncover regarding its potential therapeutic mechanism.Methods:Institute of cancer research mice were infected with influenza A virus via nasal drip.The general state of the mice,lung index,and lung index inhibition rate were used to evaluate the efficacy of PDL.Enzyme-linked immunosorbent assay,western blotting,and immunohistochemistry were used to observe the presence of proteins and cytokines in the lung tissue.Apoptosis was evaluated using the TUNEL assay.Results:PDL improved the mental state of influenza A virus-infected mice,reduced the lung index,and inhibited viral replication.The expression of interleukin-1βand tumor necrosis factor-αwere decreased,whereas the expression of interleukin-10 in the lung tissue was increased due to PDL treatment.In addition,PDL treatment modulated Toll-like receptor 4 and MyD88 expressions in the lung tissues.PDL significantly reduced apoptosis and decreased cleaved caspase-3 and PARP levels,whereas increased B-cell lymphoma-2 expression in the lung tissue.Notably,the moderate-dose group of PDL exhibited a more pronounced effect.These findings indicate that PDL exerts a protective effect against pneumonia injury in influenza A virus-infected mice.Conclusion:PDL inhibited the inflammatory response and regulated apoptosis by regulating Toll-like receptor 4 and MyD88 protein expressions,thereby protecting the lung tissue from viral infection-induced lung tissue injury.

In silico evaluation of kuwanon compounds as antiviral agents targeting H9N2 influenza virus

Background:The threat of avian influenza a subtype avian influenza A(H9N2)virus remains a significant concern,necessitating the exploration of novel antiviral agents.This study employs network pharmacology and computational analysis to investigate the potential of kuwanons,a natural compounds against H9N2 influenza virus.Methods:Leveraging comprehensive databases and bioinformatics tools,we elucidate the molecular mechanisms underlying Kuwanons pharmacological effects against H9N2 influenza virus.Network pharmacology identifies H9N2 influenza virus targets and compounds through integrated protein-protein interaction and Kyoto Encyclopedia of Genes and Genomes analyses.Molecular docking studies were performed to assess the binding affinities and structural interactions of Kuwanon analogues with key targets,shedding light on their potential inhibitory effects on viral replication and entry.Results:Compound-target network analysis revealed complex interactions(120 nodes,163 edges),with significant interactions and an average node degree of 2.72.Kyoto Encyclopedia of Genes and Genomes analysis revealed pathways such as Influenza A,Cytokine-cytokine receptor interaction pathway in H9N2 influenza virus.Molecular docking studies revealed that the binding free energy for the docked ligands ranged between-5.2 and-9.4 kcal/mol for the human interferon-beta crystal structure(IFNB1,Protein Data Bank:1AU1)and-5.4 and-9.6 kcal/mol for Interleukin-6(IL-6,PDB:4CNI).Conclusion:Our findings suggest that kuwanon exhibits promising antiviral activity against H9N2 influenza virus by targeting specific viral proteins,highlighting its potential as a natural therapeutic agent in combating avian influenza infections.

Development of a reverse-transcription loop-mediated isothermal amplification assay to detect avian influenza viruses in clinical specimens

In recent years,the avian influenza has brought not only serious economic loss to the poultry industry in China but also a serious threat to human health because of the avian influenza virus(AIV) gene recombination and reassortment.Until now,traditional RT-PCR,fluorescence RT-PCR and virus isolation identification have been developed and utilized to detect AIV,but these methods require high-level instruments and experimental conditions,not suitable for the rapid detection in field and farms.In order to develop a rapid,sensitive and practical method to detect and identify AIV subtypes,4 specific primers to the conserved region of AIV M gene were designed and a loop-mediated isothermal amplification(RT-LAMP) method was established.Using this method,the M gene of H1–H16 subtypes of AIV were amplified in 30 min with a water bath and all 16 H subtypes of AIV were able to be visually identified in presence of fluorescein,without cross reaction with other susceptible avian viruses.In addition,the detection limit of the common H1,H5,H7,and H9 AIV subtypes with the RT-LAMP method was 0.1 PFU(plaque-forming unit),which was 10 times more sensitive than that using the routine RT-PCR.Further comparative tests found that the positivity rate of RT-LAMP on detecting clinical samples was 4.18%(14/335) comparing with 3.58%(12/335) from real-time RT-PCR.All these results suggested that the RT-LAMP method can specifically detect and identify AIV with high sensitivity and can be considered as a fast,convenient and practical method for the clinic test and epidemiological investigation of AIV.

Effect of Curcumin on Influenza Virus HIN1 and H3N2 in Vitro

Objective：To investigate the inhibitory effect of curcumin on influenza virus HIN1 and H3N2 in vitro, Methods：The directly killing role of cureumin extract in vitro to influenza virus type A subtype H1N1 and H3N2 was evaluated by the canine kidney cells （MDCK）, Results：The largest non toxic concentration of curcumin extract was 12, 5g/L and the effective inhibitory concentration to H1N1 and H3N2 was 6, 25G/1 AND 1,56g/L respectively, Conclusion： Curcumin extract have directly killing effect on H1N1 and H3N2 infections.

Development and Characterization of Monoclonal Antibody Specific to Nuclear Protein of Avian Influenza Virus Type A

Five monoclonal antibodies（Mabs） to nuclear protein of avain influenza virus（AIV） were developed by syncretizing SP 2/0 and the spleen cells from BALB of mice immuized with H9 subtype AIV. Specificity of these Mabs were identified by immunofluorescent assay（IFA） and enzyme linked immunosorbent assay （ELISA）. These five Mabs which were named as AIV-NP-2C3, AIV-NP-6A5, AIV-NP-3 H9, AIV-NP-7B4, AIV-NP-2H4 could react with all viruses of AIV-H9 strains in tests. The result of Western blotting showed that only the 60 ku protein antigen of AIV-H9 could be recognized by the Mabs but never recognized by New castle disease virus, REV and infectious bursa disease virus. The result of preliminary application showed that avian influenza viruses could be deetected bv Mabs in IFA and ELISA. All these Mabs will probably play important roles in preventing and monitoring avian influenza viruses.

Sequence Analysis of HA Genes from Three H9N2 Subtype Avian Influenza Viruses

[ Objective] The study aimed to understand the genetic characters of H9N2 subtype avian influenza viruses isolated in Belling area. [ Method] HA genes of three H9N2 subtype avian influenza viruses A/Chicken/Beijing/xu/00, A/Chicken/Beijing/bei/00 and A/Chicken/Beijing/ liu/00 were amplified by RT-PCR and then sequenced. [ Result] The results of phylogenetic analysis showed that A/Chicken/Beijing/xu/00, A/ Chicken/Beijing/bei/00 and A/Chicken/Beijing/liu/00 shared the nucleotide homologies of 84.8% （ Dk/HK/Y439/97 ） -98.0% （ Ck/GX17/00 ）, 85.1% （Dk/HK/Y439/97） - 99.1% （ Ck/GXl 7/00）, 90.7% （ Ck/BJ/3/01 ） - 99.1% （Ck/GX17/00） with the isolates from Hongkong and other are- as of Chinese Mainland respectively. At the same time, the analysis of amino acid indicated that the three isolates belonged to low pathogenic H9N2 isolates of avian origin. The 226^th amino acid of them were L （ Leu）, suggesting their high binding affinity to human cells. There were seven glyco- sylation sites in HA protein, five from HA1 and two from HA2. [ Cenclusien] By analysis at molecular level, it could be concluded that A/Chicken/ Beijing/xu/00, A/Chicken/Beijing/bei/00 and A/Chicken/Beijing/liu/00 were low pathogenic H9N2 isolates of avian origin.

A Review on 2009 Influenza A Virus

[Objective] The paper was to introduce the research progress of 2009 influenza A virus. [Method] 2009 influenza A virus was introduced from the aspects of classification and host, virology, molecular characteristics and vaccine. [Result] A novel influenza A/H1N1 virus emerged in early April 2009 quickly spread worldwide through human-to-human transmission. The virus contained a group of novel gene segments, the nearest known precursor was the virus found in swine. The virus appeared to retain the potential to infect swine again and thus continued reassort with swine viruses. All registered 2009 influenza A vaccines were tested for safety and immunogenicity in clinical trials on human volunteers, and all vaccines were found to be safe, single dose of vaccine could cause protective antibody responses. [Conclusion] The paper provided basis for further study on 2009 influenza A virus.

Complete Genome Sequencing and Genetic Variation Analysis of Two H9N2 Subtype Avian Influenza Virus Strains

[Objective] The study aimed to investigate the genetic variation characters of entire sequences between two H9N2 subtype avian influenza virus strains and other reference strains.[Method] The entire sequences of 8 genes were obtained by using RT-PCR,and these sequences were analyzed with that of six H9N2 subtype avian influenza isolates in homology comparison and genetic evolution relation.[Result] The results showed that the nucleotide sequence of entire gene of the strain shared 91.1%-95.4% homology with other seven reference strains,and PG08 shared the highest homology 91.3% with C/BJ/1/94;ZD06 shared the highest homology 92.3% with D/HK/Y280/97.HA cleavage sites of two H9N2 subtype avian influenza virus isolated strains were PARSSR/GLF,typical of mildly pathogenic avian influenza virus.[Conclusion] Phylogenetic tree for entire gene of eight strains showed that the genetic relationship was the closest between ZD06 and C/Pak/2/99 strains,which belonged to the Eurasian lineage;PG08 shared the highest homology 91.3% with ZD06,it may be the product of gene rearrangements of other sub-lines.

Cloning and Phylogenetic Analysis of NS1 Genes from Different Isolates of H9N2 Subtype Duck Influenza Virus

[ Objective] The study aimed to lay a foundation for the further studies on function mechanism of NS1 protein in the interspecies transmission of waterfowl influenza virus. [Method] Using the serologic assay and the specific RT-PCR method, some strains of H9 subtype waterfowl influenza virus were isolated from the 12 to 20 day-old muscovy duck flocks without any clinical symptoms in different areas of Guangdong Province. Four of these strains, including A/duck/ZQ/303/2007（H9N2） （A3 for short）, A/Duck/FJ/301/2007 （H9N2） （C1 for short）, A/Duck/NH/306/2007（H9N2） （ D6 for short）, A/duck/SS/402/2007（H9N2） （ E2 for short）, and a strain named A/duck/ZC/2007（H9N2） （L1 for short） from a muscovy duck died of avian influenza virus （AIV）, were used for NSl gene cloning and sequencing. Subsequently, the obtained NSl gene sequences were compared with other NS1 sequences registered in GenBank, and the phylogenetic analysis was also conducted. [Result] When compared with the H9N2 AIV NS1 sequences in GenBank, the NSl genes of the four AIV strains A3, C1, 136 and E2 displayed homologies ranging from 99% to 100% at nucleotide level, and 95% to 100% at amino acid level; while the NSl gene of L1 strain displayed homology ranging from 94% to 97% at nucleotide level, and 93% to 98% at amino acid level. The phylogenetic tree demonstrated that A3, C1, D6 and E2 were highly resemblant, and L1 was closest to AY66473 （chicken, 2003）. By comparison with the NS1 gene sequences of L1, AF523514 （duck）, AY664743 （chicken） and EF155262.1 （quail） using DNAstar, A3, C1, D6 and E.2 presented nucleotide variations at site 21 （ R→Q）, 70, 71 （ KE→EG）, 86 （ A→S）, 124 （V→M） and 225 （ S→N）, and amino acid variations at site 21,70, 71 and 86 in dsRNA- dependent protein kinase （PKR） binding domain of NSl gene, which induced the evident variations of antigenic determinant and surface proba- bility plot of NS1 protein. [ Conclusion] This study suggested that the amino acid sequence variation in PKR binding domain of NS1 protein had something to do with the virus pathogenicity.

The role of wild goose(Anser) populations of Russia and theTibet Plateau in the spread of the avian influenza virus

Wild birds of the orders Anseriformes and Charadriiformes represent a natural reservoir of low pathogenic avian influenza(LPAI) viruses(family Orthomyxoviridae).Wild geese(order Anseriformes)relating to waterfowls undertake extensive migration flights reaching thousands of kilometers.Isolation of the avian influenza virus(AIV) from wild geese is quite low or absent.The aims of this study are to monitor the AIV in different wild goose species,nesting on Russian territory and the Tibet Plateau and to analyze the derived data for the purpose of determining the role of these wild bird species in spreading pathogens.In our study 3245 samples from nine wild goose species in nine regions of Russia and on the territory of the Tibet Plateau(the Xizang Autonomous Region) were tested and no AIV were detected.Our study shows the non-essential role of wild geese in the spread of the AIV over long distances and reaches theconclusion that geese are probably not natural reservoirs for the primary viruses.However,further inquiry of AIV in wild goose populations is required.Studies of wild geese and AIV ecology will allow us to obtainmore information about pathogen-host relationships and to make arrangements for the maintenance ofwild goose populations.

Study on Piezoelectric Immunosensor for the Detection of H9-subtype Avian Influenza Virus

[Objective] The aim is to develop the piezoelectric immunosensor to detect H9-subtype avian influenza virus（AIV）.[Method] The immunosensor chip was constructed by self-assembling mercaptopmpionic acid（MPA） to be monolayer on the silver-coated electrode of quartz crystal and coupling the monoclonal antibody to H9 subtype AIV with N-ethy-N＇-（3-dimethyl aminopropyl）carbodiimide hydrochloride（EDC） and N-hydroxysuccinimide（NHS）.The immunosensor to detect H9 subtype AIV was established.[Result] The results showed that the immunosensor displayed better specificity to H9 AIV and had no response to H5AIV and NDV when it was used for detection.The sensitivity test indicated the detection sensitivity for the H9 subtype AIV could reach 20-100 EID50.[Conclusion] The research provided a foundation for further research on the immunosensor for detecting AIV and it could be a new approach to detect other related viruses.

Astragalus polysaccharide enhances immunity and inhibits H9N2 avian influenza virus in vitro and in vivo

This study investigated the humoral immunization of Astragalus polysaccharide （APS） against HgN2 avian influenza virus （H9N2 AIV） infection in chickens. The effects of APS treatment on H9N2 infection was evaluated by an Mqq- [3（4, 5-dimethylthiazol-2-yl）-2, 3-diphenyl tetrazolium bromide] assay and analysis of MFIC and cytokine mRNA expression. The effect on lymphocyte and serum antibody titers in vivo was also investigated. IL-4, IL-6, IL-10, LITAF, IL-12 and antibody titers to H9N2 AIV wet enhanced in the first week after APS treatment. The results indicated that APS treatment reduces H9N2 AIV replication and promotes early humoral immune responses in young chickens.This study investigated the humoral immunization of Astragalus polysaccharide （APS） against HgN2 avian influenza virus （H9N2 AIV） infection in chickens. The effects of APS treatment on HgN2 infection was evaluated by an M]q- [3（4, 5-dimethylthiazol-2-yl）-2, 3-diphenyl tetrazolium bromide] assay and analysis of MHC and cytokine mRNA expression. The effect on lymphocyte and serum antibody titers in vivo was also investigated. IL-4, IL-6, IL-10, LITAF, IL-12 and antibody titers to PIgN2 AIV were enhanced in the first week after APS treatment. The results indicated that APS treatment reduces HgN2 AIV replication and promotes early humoral immune responses in young chickens.

Use of a Multiplex RT-PCR Assay for Simultaneous Detection of the North American Genotype Porcine Reproductive and Respiratory Syndrome Virus,Swine Influenza Virus and Japanese Encephalitis Virus

A multiplex reverse transcriptase-polymerase chain reaction(multiplex RT-PCR) assay was developed and subsequently evaluated for its efficacy in the detection of multiple viral infections simultaneously,in swine.Specific primers for each of the 3 RNA viruses,North American genotype porcine reproductive and respiratory syndrome virus,Japanese encephalitis virus,and swine influenza virus,were used in the testing procedure.The assay was shown to be highly sensitive because it could detect as little as 10-5 ng of each of the respective amplicons in a single sample containing a composite of all 3 viruses.The assay was also effective in detecting one or more of the same viruses in various combinations in specimens,including lymph nodes,lungs,spleens,and tonsils,collected from clinically ill pigs and in spleen specimens collected from aborted pig fetuses.The results from the multiplex RT-PCR were confirmed by virus isolation.The relative efficiency(compared to the efficiency of separate assays for each virus) and apparent sensitivity of the multiplex RT-PCR method show that this method has potential for application in routine molecular diagnostic procedures.

Antiviral activity of Basidiomycete mycelia against influenza type A(serotype H1N1) and herpes simplex virus type 2 in cell culture

In this study, we investigated the in vitro antiviral activity of the mycelia of higher mushrooms against influenza virus type A(serotype H1N1) and herpes simplex virus type 2(HSV-2), strain BH. All 10 investigated mushroom species inhibited the reproduction of influenza virus strain A/FM/1/47(H1N1) in MDCK cells reducing the infectious titer by 2.0–6.0 lg ID50. Four species, Pleurotus ostreatus, Fomes fomentarius, Auriporia aurea, and Trametes versicolor, were also determined to be effective against HSV-2 strain BH in RK-13 cells, with similar levels of inhibition as for influenza. For some of the investigated mushroom species—Pleurotus eryngii, Lyophyllum shimeji, and Flammulina velutipes—this is the first report of an anti-influenza effect. This study also reports the first data on the medicinal properties of A. aurea, including anti-influenza and antiherpetic activities. T. versicolor 353 mycelium was found to have a high therapeutic index(324.67), and may be a promising material for the pharmaceutical industry as an anti-influenza and antiherpetic agent with low toxicity. Mycelia with antiviral activity were obtained in our investigation by bioconversion of agricultural wastes(amaranth flour after CO2 extraction), which would reduce the cost of the final product and solve some ecological problems.

Sequence and phylogenetic analysis of hemagglutinin genes of H9N2 influenza viruses isolated from chicken in China from 2013 to 2015

H9N2 avian influenza virus（AIV） infection is a major problem in poultry industry worldwide. In this study, molecular characterizations and phylogenetic relationships of hemagglutinin（HA） gene sequences of H9N2 AIV of 5 Chinese isolates in 2014 recently available in Gen Bank, 3 widely used vaccine strains, and 52 novel isolates in China from 2013 to 2015 were analyzed. The homology analysis showed that the nucleotide sequences of HA gene of these recent Chinese H9N2 AIV isolates shared homologies from 94.1 to 99.9%. Phylogenetic analysis showed that all isolates belonged to AIV lineage h9.4.2.5. Fifty-six out of the 57 recent Chinese H9N2 AIV isolates had the motifs PSRSSR↓GLF at the cleavage sites within the HA protein, while one isolate PWH01 harbored LSRSSR↓GLF. Remarkably, all of the recent Chinese H9N2 AIV strains had the Q216 L substitution in the receptor binding site, which indicated that they had potential to infect humans. Most of recent Chinese H9N2 AIV isolates lost the potential N-linked glycosylation site at residues 200–202 compared with vaccine strains. This present study demonstrated that AIV lineage h9.4.2.5 was more predominant in China than other lineages as it harbored all the H9N2 AIV isolated between 2013 and 2015. Also we showed the importance of continuous surveillance of emerging H9N2 AIV in China and update of vaccine formulation accordingly in order to prevent and control H9N2 AIV.