Circulation of Immunosuppressive Viruses and Avian Encephalomyelitis Virus in Backyard Chicken Flocks

The objective of this study was to evaluate the circulation of Chicken Anemia Virus (CAV), Infectious Bursal Disease Virus (IBDV), Avian Reovirus (ARV) and Avian Encephalomyelitis virus (AEV) in properties of backyard chickens and carry out an epidemiological analysis. We evaluated 200 samples of chickens from 19 backyard chicken property. Only one property (P10) did not present serological titers for the diseases evaluated. This property is close to industrial farms as well as the other properties, however, P10 remained a few years without the breeding of chicks and these were the first poultry to be housed on site. This reinforces the importance of the fallow period for poultry production. The prevalence of virus-seroreactive birds was 78% (156/200), 64.5% (129/200), 78% (156/200), 78% (156/200) for CAV, IBDV, ARV and, EA, respectively. All the free-range farms studied are within a radius of 500 meters to 6 Km away from some establishments of industrial poultry. There was a correlation between serological titers for CAV and the frequency of disease in poultry (r = 0.6178). In places where birds are frequently sick, 30.76% reported that the disease occurs in chicks, 30.76% in broilers, 23.07% in broiler chickens and 7.69% in birds of all ages. Birds get sick more often in the summer period. The owners reported that the most common signs of disease were respiratory signs (snoring and nasal discharge) (46.15%), diarrhea (30.76%), and paralysis of wings and/or paws (38.46%). There was a correlation between the presence of untreated water in the property and serological titers for ARV (r = 0.5576). This report draws attention not only to high serological prevalence for the viruses studied but also important epidemiological aspects of backyard chicken diseases that may indirectly influence the industrial production.

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.

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.

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.

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.

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.

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.

Strengthened Monitoring of H5 Avian Influenza Viruses in External Environment in Hubei, 2018

The contamination status of H5 avian influenza viruses and distribution of subtypes of H5N1 and H5N6 in poultry-related environment of Hubei areas were investigated.Urban and rural live poultry markets,poultry farms,intensive livestock farms and other monitoring types of 103 counties in 17 cities were selected in Hubei.Wiping samples from cage surface,wiping samples from chopping board,fecal specimens and other environmental samples were collected and tested by real-time RT-PCR using primers and probes of influenza A,avian influenza of H5,N1 and N6 from December 2017 to March 2018.The avian influenza virus positive rate was compared among different monitoring sites,samples,time and regions.Totally,7132 environmental samples were collected in 1634 monitoring points with a positive rate of 2.24%.The positive rate of H5 avian influenza virus was the highest in urban and rural live poultry markets(3.44%,x^2=61.329,P<0.05)in 6 monitoring sites and wiping samples from chopping board(5.46%,x^2=67.072,P<0.05)in 6 sample types.H5N6 avian influenza viruses were detected more in eastern than western Hubei,and H5N6 avian influenza viruses were detected only in Xiangyang city of western Hubei.There were important high-risk places of human infection with H5 avian influenza virus in urban and rural live poultry markets and the poultry slaughtering plants.H5N6 has been the predominant subtype of H5 avian influenza viruses in the eastern and western Hubei and H5N6 avian influenza viruses were still present in a few areas of Hubei.Outbreaks of human H5N1 and H5N6 avian influenza remain at risk in Hubei province.

Development and Assessment of Two Highly Pathogenic Avian Influenza(HPAI) H5N6 Candidate Vaccine Viruses for Pandemic Preparedness

Objective In China, 24 cases of human infection with highly pathogenic avian influenza(HPAI) H5 N6 virus have been confirmed since the first confirmed case in 2014. Therefore, we developed and assessed two H5 N6 candidate vaccine viruses(CVVs).Methods In accordance with the World Health Organization(WHO) recommendations, we constructed two reassortant viruses using reverse genetics(RG) technology to match the two different epidemic H5 N6 viruses. We performed complete genome sequencing to determine the genetic stability. We assessed the growth ability of the studied viruses in MDCK cells and conducted a hemagglutination inhibition assay to analyze their antigenicity. Pathogenicity attenuation was also evaluated in vitro and in vivo.Results The results showed that no mutations occurred in hemagglutinin or neuraminidase, and both CVVs retained their original antigenicity. The replication capacity of the two CVVs reached a level similar to that of A/Puerto Rico/8/34 in MDCK cells. The two CVVs showed low pathogenicity in vitro and in vivo, which are in line with the WHO requirements for CVVs.Conclusion We obtained two genetically stable CVVs of HPAI H5N6 with high growth characteristics,which may aid in our preparedness for a potential H5N6 pandemic.

Nested RT-PCR method for the detection of European avian-like H1 swine influenza A virus

Swine influenza A virus（swine IAV） circulates worldwide in pigs and poses a serious public health threat, as evidenced by the 2009 H1N1 influenza pandemic. Among multiple subtypes/lineages of swine influenza A viruses, European avian-like（EA） H1N1 swine IAV has been dominant since 2005 in China and caused infections in humans in 2010. Highly sensitive and specific methods of detection are required to differentiate EA H1N1 swine IAVs from viruses belonging to other lineages and subtypes. In this study, a nested reverse transcription（RT）-PCR assay was developed to detect EA H1 swine IAVs. Two primer sets（outer and inner） were designed specifically to target the viral hemagglutinin genes. Specific PCR products were obtained from all tested EA H1N1 swine IAV isolates, but not from other lineages of H1 swine IAVs, other subtypes of swine IAVs, or other infectious swine viruses. The sensitivity of the nested RT-PCR was improved to 1 plaque forming unit（PFU） m L^（-1） which was over 10~4 PFU m L^（-1） for a previously established multiplex RT-PCR method. The nested RT-PCR results obtained from screening 365 clinical samples were consistent with those obtained using conventional virus isolation methods combined with sequencing. Thus, the nested RT-PCR assay reported herein is more sensitive and suitable for the diagnosis of clinical infections and surveillance of EA H1 swine IAVs in pigs and humans.

The Helper Activities of Different Avian Viruses for Propagation of Recombinant Avian Adeno-Associated Virus

To compare the helper activities of different avian viruses for propagation of recombinant avian adeno-associated virus （rAAAV）, AAV-293 cells were cotransfected with the AAAV vector pAITR-GFP containing green fluorescent protein （GFP） gene, the AAAV helper vector pcDNA-ARC expressing the rep and cap genes, and the adenovirus helper vector pHelper expressing Ad5 E2A, E4, and VA-RNA genes. Chicken embryonic fibroblast （CEF） or chicken embryonic liver （CEL） cells were cotransfected with the AAAV vector and the AAAV helper vector, followed by infection with Marek＇s disease virus （MDV）, avian adenovirus, chicken embryo lethal orphan （CELO） virus or infectious bursal disease virus （IBDV）. Infectious rAAAV particles generated by the two strategies were harvested and titrated on CEF and CEL cells. A significantly higher viral titer was obtained with the helper activity provided by the pHelper vector than by MDV or CELO virus. Further experiments showed that rAAAV-mediated green fluorescent protein （gfp） expression was overtly enhanced by MDV or CELO virus super infection or treatment with sodium butyric acid, but not by IBDV super infection. These data demonstrated that MDV and CELO viruses could provide weak helper activity for propagation of rAAAV, and rAAAV- mediated transgene expression could be enhanced by super infection with the helper viruses.

Genetic Analysis of the Entire Genome of a A/duck/Shanghai/Y20/2006 (H4N6) Avian Influenza Virus

[ Objective] This paper aimed to investigate the origin, characteristics and molecular evolution of duck derived H4N6 subtype avian influ- enza virus （DK/SH/Y20/06） and enrich the epidemiologic data of the waterfowl origin AIV. [Method] The entire genome of DK/SH/Y20/06 was amplified and subjected to genome sequencing. The molecular software was used for sequence analysis and phylogenetic tree construction of DK/ SH/Y20/06 with some other reference sequences in GenBank. [Result] The results indicated that the amino acid sequence adjacent to HA cleav- age site was PEKASR ↓ GLF, which was the typical characteristics of the LPAIV. The phylogenetic analysis indicated that the HA gene of the isolate was derived from the Eurasian lineage in the eastern hemisphere. The NA gene was at the same branch with A/rnallard/Yan chen/2005（ H4N6）, sharing 98.3% sequence identity. The PB2, PB1, NP and PA gene of this isolate had genetically close relationships with H6 subtype AIV which is epidemic in China at present. The M gene fell into the same branch with A/environment/Korea/CSM05/2004（ H3N1 ）. The NS segment had the highest similarity with A/wild duck/Korea/YS44/2004（H1N2）. The eight genes were not at the same branch and shared a low similarity with other H4N6 subtype avian influenza viruses isolated in North America. [Condusion] These data showed that DK/SH/Y20/06（H4N6） was possibly a re- combinant virus derived from H4N6 subtype, H6N2, H6N5, H3N1 and H1 N2 subtype AIV by complex gene recombination in duck.

Multiple RT-PCR Detection of H5,H7,and H9 Subtype Avian Influenza Viruses and Newcastle Disease Virus

Objective:This paper focuses on the multiple detection RT-PCR technology of H5,H7,AND H9 subtype avian influenza viruses and Newcastle disease virus,and points out the specific detection methods and detection procedures of avian influenza and Newcastle disease virus.Methods:The genes of Newcastle disease virus carrying out the HA gene sequence of H5,H7 and H9 subtype AIV in GenBank were used to establish a strategy for simultaneous detection of three subtypes of avian influenza virus and Newcastle disease virus.Results:The results showed that the program can detect and distinguish H5,H7 and H9 subtype avian influenza viruses and Newcastle disease virus at one time.Conclusion:Multiple RT-PCR detection method has high detection sensitivity and can detect and determine different subtypes of avian influenza virus and Newcastle disease virus quickly and accurately,therefore,it has a crucial role in the detection and control of avian influenza H5,H7 and H9 subtypes and Newcastle disease.

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.

Variation and evolution of NP genes of human avian H_5N_1 virus strains

In order to reveal variation and revolution of NP genes of human avian H5 N1 influenza virus strains, the NP gene of a human avian H5 N1 influenza virus strain in Guangdong was sequenced and the global NP genes of strains were retrieved. The sequences were analyzed by DNAStar 5.0, and the evolutionary speed was studied with reference to the epidemiological data. It was found that NP genes of 45 strains during 1997-2006 were homologically classified into three groups： strains in 1997-1998, strains in 2004-2005 and strains from 2003 to 2006. There were 35 substitutions in NPs in all strains accounting for a ratio of 7.03% （35/498）. An additional glycoprotein domain （NGT430-432） was found in NP genes in the strains of 2003-2006, the mutation of N370S in GD-01-06 resulted in occurrence of one more glycoprotein domain （NES368-370）. In the synonymous variation, Ks values in NP were 2.03 × 10^-5-2.55 × 10^-5 Nt/d and K. values in NP were 1.58 × 10^-6-3.10 × 10^-6 Nt/d. There didn＇t exist obviously selective pressure. An additional glycoprotein domain in every strain of 2003-2006 and one more in strain GD-01-06 might change the antigenicity of human avian H5 N1 influenza virus. The variation on human avian H5 N1 influenza strains occurred frequently in the natural world, which would result in high probability of human-human transmission along with the natural evolution of the virus.

Isolation and Identification of a Subgroup A Avian Leukosis Virus from Imported Meat-type Grand-parent Chickens

An exogenous avian leukosis virus (ALV) strain SDAU09C1 was isolated in DF-1 cells from one of 240 imported 1-day-old white meat-type grand parent breeder chicks. Inoculation of SDAU09C1 in ALV-free chickens induced antibody reactions specific to subgroup A or B. But gp85 amino acid sequence comparisons indicated that SDAU09C1 fell into subgroup A; it had homology of 88.8%-90.3% to 6 reference strains of subgroup A, much higher compared to other subgroups including subgroup B. This is the first report for ALV of subgroup A isolated from imported breeders.

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.

Subtype Identification of Avian Influenza Virus on DNA Microarray

We have developed a rapid microarray-based assay for the reliable detection of H5, H7 and H9 subtypes of avian influenza virus （AIV）. The strains used in the experiment were A/Goose/Guangdong/1/96 （H5N1）, A/African starling/983/79 （H7N1） and A/Turkey/Wiscosin/1/66 （H9N2）. The capture DNAs clones which encoding approximate 500-bp avian influenza virus gene fragments obtained by RT-PCR, were spotted on a slide-bound microarray. Cy5-labeled fluorescent cDNAs, which generated from virus RNA during reverse transcription were hybridized to these capture DNAs. These capture DNAs contained multiple fragments of the hemagglutinin and matrix protein genes of AIV respectively, for subtyping and typing AIV. The arrays were scanned to determine the probe binding sites. The hybridization pattern agreed approximately with the known grid location of each target. The results show that DNA microarray technology provides a useful diagnostic method for AIV.