Typing And Subtyping Avian Influenza Virus Using DNA Microarrays

Avian Influenza Virus (AIV) viruses are subtyped on the basis of antigenic differences of the surface glycoproteins, the hemagglutinin (HA) and the neuraminidase(NA) proteins. So far, sixteen HA (H1 through H16) and nine NA (N1 through N9) subtypes have been identified. Influenza viruses are classified into Highly pathogenic avian influenza (HPAI) and low pathogenic avian influenza (LPAI). Historically, highly pathogenic AIVs have only belonged to the H5 and H7 hemagglutinin (HA) subtypes. Poultry can infected mainly by H5N1 and H9N2 AIV subtypes. There have been regular reported of the natural infections of humans with H5, H7 and H9 AIV. Rapid detection and subtyping of influenza viruses are necessary to control AI outbreaks and maintain routine surveillance, and they are becoming increasingly important in face of concerns over an influenza pandemic.In this paper, we sought to develop an efficient approach for simultaneous typing and subtyping of AIV based on cDNA microarrays. The processes included:(1) Preparation of specific probesPrimers based on conserved sequences of the matrix (M) protein gene of AIV, the haemagglutinin gene of AIV subtypes H5, H7, H9 and the neuraminidase gene of N1, N2 were designed in this study. We also designed primers of the positive control from the GAPDH gene of chicken. All of the primers were derived from the alignments and analyses of the nucleotide sequences retrieved from the enormous GenBank data, and conducted by the MegAlign program. After cDNA cloning and PCR, the concentration of purified PCR products was adjusted to 300 ng/mL for spotting purposes.(2) Fabrication and opitmization of the microarraysAldehyde slide were spotted using a biochip array . Probes were diluted to 0.5μg/μl. The space of spots was 450μm and the diameter of spot was 220μm . We dried the spotted slide at room temperature for at least 24h, hydrated for 10s at 65℃. Quickly dry at 80℃. UV cross-linking by placing the slides, in a crosslinker, exposed the slides to 65mJ of energy. After blocking with 0.1 mol/L NaBH4 and denaturation at 95°C. The microarrays were stored in a dark, humidity-free environment.(3) Evaluation efficiency of the microaraysTo evaluate the specificity of the microarray, Newcastle disease virus, infectious bronchitis virus, infectious laryngotracheitis virus, infectious bursal disease virus, All fifteen HA (H1 through H15) and nine NA (N1 through N9) AIV subtypes were tested using cDNA microarrays; Sensitivity of our microarray-based assay was tested using serial dilutions of H5N1 prototype strains, as well as clinical samples infected with H5N1 AIV; The reproducibility of tests within-run and between-run were evaluated; After preservated for different time(10,20,30,40, 50,60,70 d), the diagnostic microarray was hybridizated to test the storage periods. The results were that the microarray can unambiguously detected and typed samples, and found no cross-reactions among non-related probes. A comparison with RT-PCR revealed that when RNA with 10-4 dilutions, microarray can test with high fluorescent intensity ,but RT-PCR array can not give a clear conclusion, The reproducibility of this AIV diagnostic microarray was well and has fine detection effect after keeping for two month.(4) Application of the microaraysWe tested 30 infacted samples and 21 field samples. For these samples,microarray test have 100% coincidence rate with chicken embryo inoculation and 96% coincidence rate with RT-PCR. The results showed that our method as validates here can identify type A influenza via the conserved matrix gene, differentiate between the H5, H7,and H9 hemagglutinin subtypes, and differentiate between the N1 and N2 subtypes of avian influenza through one test. It is more sensitive and specific than chicken embryo inoculation and RT-PCR. It is a new diagnosis method which is reproducible, fast, precise and highthroughput.