Molecular Analysis Of The Entire Genes Of An Avian Influenza Virus Isolate A/Mallard/ZhaLong/88/04(H4N6)

A H4N6 subtype avian A influenza virus was isolated from mallard in ZhaLong natural reserve of HeiLongjiang province. The virus was named A/Mallard/ZhaLong/88/04(H4N6) (MZL88/04).In this report, the isolate A/Mallard/ZhaLong/88/04(H4N6) was propagated in specific pathogen free (SPF) chicken embryo. Twelve pair of primers were designed and synthesized according to the relevant nucleotide sequences in GenBank, which were used for amplifying the eight full-length genes of isolate MZL88/04. Thereinto, HA, PA, PBl and PB2 genes were all divided into two fragments to amplified. The viral RNAs and MRNAs were extracted and used Reverse Transcription-Polymerase Chain Reaction (RT-PCR) to amplify the eight full-length CDNAs of the virus genes. The purified CDNAs were inserted into PMD18- T vector, and the recombinant plasmid was transformed into competent DH5-a E. Coli, then sequenced when the PCR result was positive for amplifying the inserted gene in recombinant plasmid. The nucleotide sequences and their deduced amino acid sequences were compared and analyzed for the homology and phylogenetic relationships between the isolate MZL88/04 and some other isolates of H4N6 subtype using professional software.The results showed that each of eight gene CDNA fragments contained the whole open reading frame, and some of the genes contained the non-coding region. Analysis of the results of homology comparison and phylogenetic relationships indicated that isolate MZL88/04 can be classified into Eurasian lineage, and all of the eight genes of isolate MZL88/04 were closely related with those of some isolates from Nanchang, implying relevant genes evolving in homothetic route. Considering that non of H4N6 subtype isolates has been found to be highly pathogenic strain and the amino acid component of HA cleavage site of isolate MZL88/04, it can be concluded that isolate MZL88/04 was not highly pathogenic strain.Because isolate MZL88/04 was the first strain of H4N6 subtype AIV isolated from wild birds in motherland of China, the results of molecular clone and analysis of the entire genes of the isolate MZL88/04 had great value for the further research of avian influenza, especially for the research of perpetuation status of AIV in wild waterfowl and the research of molecular epidemiology of avian influenza.