| The fowl adenovirus serotype 4(FAd V-4)is a viral infection of poultry with a major feature of pericardial effusion and hepatitis,and is therefore commonly referred to as pericardial effusion-hepatitis syndrome.The early report of the disease was in the Ankara area of Pakistan in 1987,so it is often referred to as avian Ankara disease.After 1989,the disease was prevalent in the world,and in 2015,it broke out in large areas in China.The disease has a rapid onset,rapid spread,and is susceptible to 3-5 weeks old broilers,with a mortality rate between 20% and 80%.In order to understand the current molecular prevalence of avian Ankara disease and explore the trend of FAd V-4 mutation in Liaoning Province,the laboratory isolated and identified the suspected infectious Ankara disease and the Penton,Fiber1,Fiber2 gene cloning and genetic evolution analysis.as follows:1.Design specific primers for the FAd V-4 hexon gene fragment,and PCR-amplify the liver DNA of the diseased chicken.The results showed that the 954 bp fragment showed a bright target band.The results were compared with the isolates in the public gene pool and found to be 99.9% homologous to the avian Ankara virus.So it can be confirmed as a FAd V-4 virus infection.2.Inoculated 9-day-old SPF chicken embryo,the allantoic fluid did not agglutinate 1% cock red blood cells;using the allantoic fluid DNA as template,the target band was identified by PCR,indicating that the virus proliferated successfully;the transmission electron microscopy showed that the virus was Spherical particles with a diameter of 70-80 nm without capsules are icosahedral typical avian adenovirus structures,indicating successful isolation of avian adenovirus.3.The Penton,Fiber1 and Fiber2 genes were amplified by PCR using the isolated virus DNA as template.The two strains showed target bands at 1578 bp,1296 bp and 1440 bp fragments.After cloning and sequencing,the results of comparison with domestic and foreign reference strains showed that the nucleotide sequences of Penton,Fiber1 and Fiber2 genes of the two strains were 99.9%-100%,99.8%-100% and 100%,and the homology with the reference strains of foreign countries are 98.8%-99.2%,96%-97.1% and 95.8%-97.2%,respectively.Among them,the nucleotide sequence of Penton gene of Anshan strain changed from T to C to the missense mutation at position 1078,and the amino acid sequence of position 360 changed from tryptophan to arginine.The nucleotide sequence of the Fiber1 gene of Anshan strain changed from A to T at position 540,which is a synonymous mutation.Through the phylogenetic tree,it can be seen that the three genes of the two strains are all in the same branch as the domestic isolate.It is not in the same branch as the foreign isolate.4.Epitope analysis predicts that fragments 13-24,66-79,91-100,160-171,217-228,286-291,359-375,402-409,and 498-504 may be potential for Penton proteins.Epitope epitopes;fragments 15-50,100-104,306-310,315-319,328-335,400-404,and 423-427 may be potential epitopes of the Fiber1 protein;4-19,27-33,241-247 and 258-262 fragments may be potential epitopes of the Fiber2 protein,and the amino acid site mutation occurs in the 360 th position of the Anshan strain,which is in the potential antigen dominant epitope region.Whether or not the immunogenicity and pathogenicity of the isolate are affected remains to be further studied.In summary,the genetic evolution analysis of Penton,Fiber1 and Fiber2 genes in two isolates in Liaoning Province provides basic data for the current molecular prevalence of poultry Ankara disease in our province,and also to further study the pathogenicity of isolates.The virulence and the development of a group of avian adenovirus vaccines laid the foundation. |
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