The Isolation Identification Of Fowl Adenovirus Serotype 4 And The Phylogenetic Analysis Of Hexon Gene In Liaoning Area

The fowl Ankara disease was an infectious disease caused by ? group of fowl adenovirus serotype 4(FAdV-4).This disease was reported in Islamic republic of Pakistan,Ankara,so that it was named Ankara Disease.The main pathological features of this disease was the heart full of liquid and the liver was multifocal necrosis,so it was also called Hydropericardium-Hepatitis Syndrome,referred to as HHS.At present,the disease is prevalent all over the world,which makes a serious impact on the development of poultry industry in China.In order to understand the prevalence state of fowl adenovirus in Liaoning Province,our laboratory colleted 11 batches of livers which were suspected of fowl adenovirus serotype 4 from Liaoning province,Shenyang,Fushun,Anshan area to do a positive test and do the phylogenetic analysis of Hexon complete gene.The experimental contents and results are as follows:1.According to the Hexon protein gene of FAdV-4 to design a specific primers,to make the PCR amplification for the liver tissue DNA of poultry and wild birds,and the purpose gene are both appeared in 954 bp.By comparing with the reference strains recorded in GenBank,they are all be infected by fowl adenovirus serotype 4.2.To deal with the Liaoning Province Anshan,Fushun,Shenyang area's poultry liver and forest wildlife park acryllium vulturinum with their liver samples on the conventional process,and inoculate the 9 days' SPF chickens,the results showed that they are both curled up and slow growth after 7 days'.Collecting the allantoic fluid of chicken embryo and only the Anshan strain's allantoic fluid of chicken embryo can agglutination the rat's red blood cells.3.According to the Hexon protein gene of FAdV-4 to design a full gene of specific amplification primers.To amplification the Hexon complete gene for Liaoning Province Anshan,Fushun,Shenyang area's poultry liver and forest wildlife park acryllium vulturinum with their liver samples,the results showed that they are both appeared in 2889 bp.This research has amplified the Hexon complete gene of Open Reading Frame is 2814 bp,which approximately coding 937 amino acids.To make a phylogenetic analysis of Hexon complete gene,the difference between five isolated strains each other and the reference strains from GenBank are none,the nucleotide homology with domestic countries are between 99.8%-100%,and the nucleotide homology with foreign countries are between 96.9%-99.8%;The amino acids homology with domestic countries are between 99.6%-100%,and the amino acidshomology with foreign countries are between 96.4%-99.5%,they are showed that they are having a strong conservation.According to the phylogenetic tree structure can be seen that the five strains isolated strains are in the same branch with domestic strains,and they are also in the same branch with Indian isolates,it means that they are closest to Indian strain,and are furthest to USA strain.4.Using DNASTAR software to predict the analysis of epitope,the area between55-66,162-168,266-275,400-407,560-570,765-774,788-804,874-885 and 913-919 of Hexon complete gene maybe the potential antigenic epitopes.However,the amino acid mutation sites such as the acryllium vulturinum's strain(FAdV-4_Shenyang_Acr.*)406,the Anshan's strain(FAdV-4_Anshan_gallus*)773 and 880 are included in these potential advantage epitope antigen area.To do the phylogenetic analysis of Hexon complete gene,and understand the variation trend of the strain are very important,it also lays a theoretical foundation to research the vaccine.It also has an important significance to search for a dominant epitope to diagnosis the ?group of fowl adenovirus.