Analysis Of The Pathogenicity Of Multiple Serotypes Of Avian Adenovirus And Hexon And Fiber Gene Sequence And Its Antibody Cross-neutralization Test

Fowl Adenovirus(Fadv)has spread rapidly around the world since its first outbreak in 1987,causing significant losses to the poultry farming industry.Avian adenoviruses can be divided into three groups according to the length of virus gene structure and sequence: group Ⅰ,group Ⅱ,group Ⅲ.At present,the main group I of poultry adenoviruses that harm our country’s poultry breeding industry.Group,I avian adenoviruses can be divided into 12 according to serotypes.After a long-term epidemiological investigation and research,it has been found that the farming enterprises or scattered households mainly focusing on terrestrial poultry farming are Fadv-4,Fadv-8a,Fadv-8b The four serotypes of Fadv-11 are the most common.This study aims to study the pathogenicity and serum cross-protection of avian adenovirus isolates to provide data support for subsequent vaccine development.This research involves molecular biological identification of Fadv isolates of group I stored in the laboratory from 2014 to 2020 and determination of the serotype of each isolate.The results were: 5 isolates of type 4(GDDL-4,GDHQY-4,GDLHX-4,GDZMY-4,GDZYQ-4);2 isolates of type 8(GDB3-8a,GDT08-8b);2 isolates of 11 There are 9 types of isolates(GDWYT-11,GDZWJ-11).Subsequently,the genetic evolution of Hexon genes and Fiber genes of each serotype isolate was analyzed,and the results were GDDL-4,GDHQY-4,GDLHX-4,GDZMY-4,GDZYQ-4 isolates,and Fadv-4 reference sequence(Gen Bank: MG856954.1)Homology 90%～100%;GDB3-8a,GDT08-8b isolates and Fadv-8 reference sequence(Gen Bank: KT862810.1)homology 97.1%～97.5%;GDWYT-11,GDZWJ-11 isolated The strain has 97.4%-97.9%homology with the Fadv-11 reference sequence(Gen Bank: KT862816.1),and each isolate contains multiple amino acid mutations;according to the genetic evolution analysis,the serotype 4 isolates are involved in this study Hexon and Fiber genes evolved from domestic.Designed different specific fluorescent quantitative primers for the target isolates(GDDL-4,GDB3-8a,GDT08-8b,GDWYT-11),and successfully established a realtime fluorescent quantitative PCR detection method for the three serotypes of group I avian adenovirus.The result is Fadv-4 standard curve: y =-3.5237 x + 44.979,correlation coefficient R2=0.9987,amplification efficiency E=91.89%;Fadv-8standard curve: y =-3.387 x + 44.038,correlation coefficient R2=0.9993,Amplification efficiency E=97.62%;Fadv-11 standard curve: y =-3.673 x + 48.257,correlation coefficient R2=0.9966,amplification efficiency E=86%;the construction and testing showed good specificity,sensitivity and stability Sex.In the next step,a challenging experiment was conducted to explore the pathogenicity of the isolates(GDDL-4,GDB3-8a,GDT08-8b,GDWYT-11)on chicken embryos,duck embryos,and LMH cells.The results showed that the isolates were weakly pathogenic to chicken embryos and duck embryos.No dead embryos were found after inoculation.The allantoic fluid was clarified and the embryos did not change abnormally.The study found that it was more pathogenic to LMH cells and could cause cells.Severe CPE appeared at the 5th generation;the target isolate reached a peak titer48 to 60 h after infection of the cells,and GDWYT-11 proliferated more efficiently on LMH cells.TCID50 shows that the target isolates all have good virus titer,which indicates that the virus strain selected in this study is more suitable for growth in cells.To explore the impact of the pathogenicity of target isolates(GDDL-4,GDB3-8a,GDT08-8b,GDWYT-11)on 10-day-old SPF chicks,this experiment established the GDDL-4 group,GDB3-8a group,and GDT08-In the 8b group,GDWYT-11 group,and negative control group,the leg muscles were injected with 105TCID50/0.2 m L virus solution.Observe the death of SPF chicks and autopsy lesions.The results showed that the four target strains can all cause severe pericardial effusion-inclusion body hepatitis syndrome.The GDDL-4 group has the most severe symptoms,with a case fatality rate of up to 85%,and an overall mortality rate between 0-85%.The results of detoxification analysis showed that the target isolates can cause repeated detoxification of diseased chickens,large titer detoxification(≥10000 copies/μL)in GDDL-4 group and GDB3-8a group,and only detectable in GDT08-8b group and GDWYT-11 group Low titer(≤1000 copies/μL)detoxification.The results of viral load analysis showed that the target isolates had a wide range of tropism,with the highest viral load in the liver and intestines.Subsequent cross-neutralization test results showed that Fadv-4 serum can neutralize Fadv-11 virus,Fadv-8b cannot produce cross-neutralization between Fadv-8a,and Fadv-8b serum can neutralize Fadv-11 virus,which indicates The crossprotection effect between serotypes is complicated and the cross-protection effect is poor.In summary,this study conducted a molecular biological analysis of Fadv isolates,studied its pathogenicity test on chicken embryos,cells,and animals,combined with the results of serum cross-neutralization tests,and reached the following conclusions(1)Isolation of avian adenovirus Strains(GDDL-4,GDB3-8a,GDT08-8b,GDWYT-11)are more suitable for LMH cell isolation and culture;(2)GDDL-4 strain is the most pathogenic to 10-day-old chicks(the mortality rate is as high as 85%);(3)The crossprotection between serotypes is complex and the cross-protection is weak.