Sequence Analysis Of The Whole Genome And Pathogenicity Analysis Of Fowl Adenovirus Serotype 4 From Guangxi

Avian adenovirus serotype 4(FAdV-4),which is a double-stranded DNA virus with the complete genome sequence about 43 kb,is mainly infected with broilers aged 3 to 6 weeks and cause Hydropericardium Hepatitis Syndrome(HHS).It has strong contagious and pathogenicity after infected the broilers.This disease outbroken in China in 2014.After that,it also outbroken in many chicken farms in Guangxi in 2016 and leaded to serious economic losses in poultry industry.However,there are few reports on the prevalence and pathogenicity of FAdV-4 in Guangxi.In this study,a FAdV-4 Guangxi isolate FAdV-4-GX2017-005 was isolated and identified from a chicken liver suspected of being infected with FAdV-4 in a chicken farm in Guangxi.Animal regression tests and whole gene sequencing analysis were conducted to investigate its infection.The pathogenicity infected the chicken embryo liver cell(CEL)and SPF Chicks were tested and analyzed.All results will provide data as reference for epidemic situation about FAdV-4 and lay a foundation for prevention and control FAdV-4.The specific research contents are as follows:In this study,chicken liver tissues suspected of being infected in Guangxi were collected,isolated from chicken embryo liver cells,plaque purification,and pathogenic PCR identification and gene sequencing,determination of half the amount of tissue infection,serum neutralization test,and SPF chickens infected animals regression test.PCR results showed that the strain FAdV-4 was positive,and other common pathogens PCR results were negative;gene sequencing results showed that the strain is FAdV-4,named: FAdV-4-GX2017-005;FAdV-4 positive serum It can neutralize the infection of this strain,which further proves that the strain is FAdV-4;animal regression tests have shown that FAdV-4-GX2017-005 can cause SPF chickens to have anorexia,closed eyes,drowsiness,and yellowgreen thin stool.Symptoms,necropsy revealed pericardial effusion,yellow liver swelling,kidney swelling and congestion.In addition,pathological tissue sections of dead chickens showed pathological changes in the liver,lungs,kidneys,spleen,thymus,and bursa.In short,the isolate showed strong pathogenic ability,which provided reference materials for the pathogenicity and prevention research of FAdV-4 in Guangxi.In this study,the whole gene sequence of Guangxi isolate FAdV-4-GX2017-005 was successfully obtained through whole gene sequencing and bioinformatics analysis.The results showed that the full-length genome of FAdV-4-GX2017-005 isolate strain was 43,722 bp,encoding 43 open reading frames.The nucleotide sequence comparison analysis revealed that the similarities with domestic isolates were 99.8%?100%,and that with foreign isolates were 94.8%?95.7%.The nucleotide sequences of the important structural genes hexon,penton and fiber of this strain were analyzed.The similarities comparison with domestic and foreign reference strains were 96.0%? 100%.After further in-depth analysis of the amino acid sequence encoded by this strain,it was found that some amino acid sites in the amino acid sequence were mutated.Multiple potential glycosylation sites and phosphorylation sites were found in the three important structural proteins,and some amino acids were inserted and missing in fiber proteinIn order to further analyze the pathogenicity,CEL and specific pathogen free(SPF)chickens were used for testing in this study.First,the replication of virus in CEL and its effect on cytokines expression were analyzed after infection with FAdV-4-GX2017-005 isolate strain.The results revealed that FAdV-4-GX2017-005 had a good tropism in CEL.The obvious cell lesions began to appear in 48 hours after infection.The virus began to proliferate rapidly at 24 hours,at 48 hours,the cells showed obvious lesions,and reached its highest level at 60 hours after infection by detecting the expression level of the hexon gene.The quantitative expression of cytokines at the m RNA level detected by fluorescence quantitative PCR.The results found that the expression levels of IL-1?,IL-6,IL-8,IL-12 and IL-15 had no change in 1?72 hours after infection.However,they were significantly up-regulated in 84?120 hrs.The expression of IL-18 had no obvious changes throughout the infection period.To establish an animal infection model and further study the pathogenicity of FAdV-4.4-weeks SPF chickens were then infected with FAdV-4-GX2017-005 isolate strain by nasal drip,the results showed that SPF chickens began to appear depressed,loose feathers and other symptoms after infected 4 days.some chickens died on infected 5 days,and the other chickens began to recover on 7 days.DNA extraction from anal swabs of chickens after infected were detected by PCR.All of detecting samples were positive for the hexon gene.It indicated that SPF chickens had been infected with FAdV-4-GX2017-005.Post-mortem observation revealed that the dying chicken had typical pericardial effusion and hepatomegaly hemorrhage.The expression of IL-1?,IL-6,IL-8,IL-12,IL-15 and IL-18 in heart,liver,kidney,lung,spleen,thymus and bursa of SPF chickens in the infection group were up-regulated in varying degrees by Fluorescence quantitative PCR assay,and the highest expression was found on 5 or 6 days after infection(P < 0.05).One FAdV-4 Guangxi isolate FAdV-4-GX2017-005 was successfully isolated and identified.Animal regression tests showed that the strain could cause severe pathological damage to SPF chicken tissues and organs.The complete gene sequence of FAdV-4-GX2017-005 was obtained,the analysis of whole gene sequencing showed that there were some amino acid site variations,which indicated that there were differences among FAdV-4 epidemic strains in different district in Guangxi.FAdV-4-GX2017-005 strain can proliferate in CEL cells and lead to cytopathy,84-120 h after infection can significantly increase the expression of cytokines IL-1?,IL-6,IL-8,IL-12 and IL-15.After 5 or 6 days of infection with SPF chickens,it leaded to appear the typical hydropericardium hepatitis syndrome.also lead to the cytokines IL-1?,IL-6,IL-8,IL-12,IL-15 and IL-18 in the heart,liver,kidney,lung,spleen,thymus and bursa appear the highest level of expression.It indicated that FAdV-4-GX2017-005 strain had strong pathogenicity.This study results provides a data as reference for the prevention and control of FAdV-4 in Guangxi.It also will lay a foundation for further research on the molecular pathogenic mechanism of FAdV-4 in the future.