Pathogenicity Identification And Comparative Analysis Of Major Structural Protein Genes Of Different Generations Of FAdV-4 Strains

FAd V-4 can infect a variety of poultry and cause high infection rate and mortality in poultry.Chickens are the most susceptible animal and can induce HPS in chickens.At present,there are few studies on the changes of virulence,infection modes,mortality of different infection modes and mutations of genes encoding major structural proteins of FAd V-4 strains during passage.Therefore,the following three aspects were studied in this study.Firstly,FAd V-4 strains of 2～15 generations were prepared and TCID₅₀of different generations were determined.The diseased chicken liver infected with FAd V-4 was ground and inoculated SPF chicken embryo through allantoic cavity,and the allantoic fluid was collected to observe the pathological changes of dead chicken embryos.The 2^nd,4^th,7^th,8^thand 15^thgenerations of LMH strains were selected to infect LMH cells,and TCID50 was determined.The results showed that all chicken embryos inoculated with the virus strain died within 6～7 days.Pathological changes such as embryo dysplasia,skin hemorrhage and liver hemorrhage and edema occurred in the dead chicken embryos.TCID₅₀of the 6substrains were 10^6.56/0.1m L,10^6.37/0.1m L,10^6.45/0.1m L,10^6.45/0.1m L,10^6.53/0.1m L and10^6.50/0.1m L,respectively.There was no significant difference in virulence among the substrains（P>0.05）.This indicated that there was no significant difference in virulence between different generations of strains.Secondly,to explore the pathogenicity of different generations of strains and different ways of infection to chickens and determine the pathogenicity of viruses to chickens of different ages.Select the 2^nd,4^th,7^th,8^thand 15^thgeneration strains to inoculate 7-day-old SPF chicks with a dose of 0.05 m L/chicken through subcutaneous administration,mucosal administration（eyedrop application）and oral administration at the neck and back,and set up a blank control group.Observe the dead time,mortality and pathological changes of chickens in different vaccination groups.The results showed that all chickens inoculated subcutaneously died within 3～6 days.The mucosa of group a died partially within 5～7days,and the mortality rates of five generations were 80%,50%,40%,30%and 30%in turn.After autopsy,all the dead chickens showed obvious pericardial effusion.There was no chicken disease in the oral administration group,and the mortality rate was 0%,and there was no chicken disease in the blank group.The second generation strain was inoculated subcutaneously into 10～40-day-old chickens.It was found that the death of 10～25-day-old chickens was earlier,and the death time of 25～40-day-old chickens was relatively delayed,but the mortality of different age groups was 100%.This indicated that different generations of strains had strong pathogenicity to chickens,and the virus could infect chickens through subcutaneous and mucosal infections but could not be infected by oral administration.The death time of chickens was relatively prolonged with the increase of age.Thirdly,the gene sequences encoding major structural proteins of different generations of strains were compared and analyzed.The DNA of different generations of strains were extracted for PCR amplification,and the sequences were compared and analyzed after sequencing.At the same time,the DNA were extracted from the liver of the diseased dead chicken and compared with the gene sequences of the parental strains.The results showed that the major structural proteins Hexon,Fiber1 and Fiber2 genes of the strains with different generations were not mutated,and the structural protein genes of the parental strains were not mutated in chickens.Therefore,it can be seen that the major structural proteins Hexon,Fiber1 and Fiber2 genes encoded by FAd V-4 strain were not mutated during passage and in the body.In conclusion,no significant difference in virulence was found in the virulence of the strains during passage,the mortality of different infection modes and the mutation analysis of genes encoding major structural proteins.The strain can be infected by subcutaneous and mucosal infection,not by oral infection;Genes encoding major structural proteins did not mutate during passage or in the body.