Molecu Lar Epide Miology Of Fowl Adenovirus Isolated In China,and Biological Characteristics And Establishment Of An Indirect ELISA For FAdV-4 Serotype Strain

Fowl adenovirus 4 can cause an acute and highly contagious disease that is mainly characterized by hydropericardium syndrome?HPS?and inclusion body hepatitis?IBH?.Fowl adenovirus 4 was rarely reported before 2010 in China.This disease,however,started to break out in northern C hina after 2015 and gradually spread throughout the country,causing huge economic loss in the po uLtry industry.The study on fowl adenovirus 4 is still at an initial stage.Its biological characteristics and genome function are unclear.The detection method for fowl adenovirus 4 is mainly based on its antigen,and there is no commercial ELISA kit.For the prevention and control of fowl adenovirus 4,it is important that research is conducted on its biological characteristics and genome,and antibody detection methods need to be developed.Between 2015 and 2016,more than one hundred thirty samples were collected from diseased chicken farms from 10 provinces in C hina.Eighty-three fowl adenovirus 4 strains were isolated and identified,and 81 strains belonged to fowl adenovirus 4 dominated in Jiangsu,Zhejiang,Guangdong and Yunnan.Two isolates were adenovirus 8b from Jiangsu.Homology analysis of Hexon gene of the 81 FAdV-4 strains showed nucleotide sequence similarities that ranged from 98.7%to 100%;nucleotide sequence homology was over 97%with other strains reported early in C hina,which demonstrated that its variation was small.Whole genomics sequencing analysis on CH/JS/TCZHP2015,CH/GD/XCJFQ2016 and CH/SC/MSLQH2016,which were representatives of East C hina,South China and Southwest China,respectively,indicated that nucleotide sequence homology was nearly identical even when compared with CHSDDZ2015,CHSXCZ2015,CHJSXZ2015,CHAHBZ2015,C HHNJZ2015,HB1501,and HB1510,which were isolated recently in China.Meanwhile,it is different from representative strains in foreign countries?6%⁶.4%difference?,which suggested that FAdV-4 had increased mutations when it spread to China from abroad.In addition,recent research showed that strains from C hina existed the ORF29mutation and ORF19 deletion.However,it is not known whether this mutation wo uLd lead to outbreaks of fowl adenovirus in C hina.To study the biological characteristics of fowl adenovirus 4,we establish a Taq Man real-time PCR assay.The resuLts showed the established assay detected a linear relationship and the correlation coefficient of the standard curve was 0.997,and were good in specificity,sensitivity and stability.FAd V-4 was inocuLated into specific-pathogen-free chicken embryos through the embryo yolk sac,chorioallantoic membrane and chorioallantoic antrum.The viral load was tested in the emb ryo liver,chorioallantoic membrane,chorioallantoic fluid and yolk with a Taq Man real-time PCR assay.The assay revealed that the virus reproduced quickly in chicken embryos through yolk sac inocuLation,and the virus titer was highest in the liver.A cell replication assay revealed that FAdV-4 had a good effect on replication and infection of chicken liver cancer cells.Chicken embryo fibroblasts can reproduce the virus but need to blind pass several generations.Animal artificial infection experiments s howed that CH/JS/TCZHP2015,CH/GD/XCJFQ2016 and CH/SC/MSLQH2016 had a high fatality rate(inocuLated with10^5.0 EID₅₀,the mortality rate couLd reach 90%¹00%).The most strong tissue tropism of FAdV-4 was the liver,followed by the splee n,pancreas and large intestine.Vaccination with an inactivated C H/JS/TC ZHP2015 strain and inoc uLation with CH/JS/TCZHP2015,CH/GD/XCJFQ2016 and CH/SC/MSLQH2016 from different districts protected chickens well.In this study,a pCold-GST expressing vector was used for constructing hexon Domain 1functional protein.After induced at a low temperature,the recombinant hexon protein was purified,and used as a coating antigen to establish an indirect ELISA.Many conditions were optimized,including the coating,blocking buffer and blocking time,primary antibody and secondary antibody dilution ratio and incubation time,and the TMB incubating time.Finally,the optimal ELISA process was determined as follows:the recombinant protein was diluted 100 times and coated overnight at 4?;1%BSA blocking was carried out for 1 hour;serum incubation,diluted 200 times,was carried out for 1 hour;conjugated secondary antibody diluted 2,000 times,was incubated for 1 hour.The reaction was developed for 10minutes at room temperature and its positive threshold was 0.191.This method can detect FAdV-4 antibodies with good specificity,repeatability and sensitivity and has the potential to be applied in the laboratory and clinic for FAdV-4 antibody detection.