Whole-Genome Sequencing Analysis,Detection Method And Pathogenicity Studyof Fowl Adenovirus Serotype 4 Isolated From Guangxi

Fowl adenovirus serotype 4(FAdV-4)can cause hydropericardium hepatitis syndrome(HHS)and other poultry diseases.It was first reported in Pakistan in1987.Since 2015,a emerging FAdV-4(FAdV-4 variant strain)has become an outbreak in chicken flocks in Shandong,Henan,Jilin,Guangdong,and Guangxi,mainly infected 3?6 weeks old broiler chickens,and the mortality rate is high,causing serious economic losses to the poultry industry in China.In this study,the whole genome sequencing and genetic evolution analysis of six FAdV-4 isolates from Guangxi showed that the total length of the genome was 43714?43721 bp,including 43 open reading frames.The nucleotide sequences of six FAdV-4 isolates from Guangxi showed significant variation compared with the previous FAdV-4 strain(FAdV-4 non-variant strain),mainly due to the 1966 bp deletion at the right end of the genome.The amino acid sites of the main structural proteins of Hexon,Penton and Fiber had different degrees of variation,and the mutation sites of Fiber-2 were the most,which were similar to the variation of highly pathogenic FAdV-4 isolates reported in China in recent years.At present,there is no detection method to identify FAdV-4 variant and non-variant simultaneously.In this study,the nucleotide sequences of FAdV-4variant strains and non-variant strains were downloaded from Gen Bank,combined with Primer Explorer V5 and Premier 5.0 primer design software,designed and screened two sets of loop-mediated isothermal amplification(LAMP)primers and two molecular beacon probe.The reaction time,reaction temperature and primer concentration were optimized.A duplex LAMP was established to differentially diagnose FAdV-4 variant and non-variant strains.The specificity of this method is good.Under the dual fluorescence channels of520 nm and 670 nm,the results of amplification of FAdV-4 variant and non-variant strains were respectively green and red,and the amplification results of mixed infection samples of FAdV-4 variant and non-variant strains were yellow,while the results for other serotypes of FAdV,newcastle disease virus and avian reovirus and other virus strains were all negative.The sensitivity is high,the detection limit is 100 copies/?L.The interference was small,and the high concentration template did not affect the amplification of the low concentration template.The established duplex LAMP detection method can distinguish FAdV-4 variant strains,non-variant strains and mixed infections.In order to understand the pathogenicity of FAdV-4 isolated from Guangxi,SPF chickens were infected with different doses and routes.4-week-old SPF chickens were infected by intramuscular injection at different doses,and the incidence and death of each group were recorded.SPF chickens were infected by intramuscular injection and oral administration at a dose of 10^1.5TCID₅₀.The lesions were observed by necropsy,and tissue samples and cotton swabs were collected,the viral load was detected by fluorescence quantitative PCR,and the pathological changes were observed by histological sections.The results showed that doses of 10⁷?10³TCID₅₀caused 100%death of chickens;the dose of 10²TCID₅₀?10 TCID₅₀and 1 TCID₅₀caused 90%,30%and 10%mortality,respectively.Intramuscular injection was more susceptible than oral administration,and dissection revealed significant pericardial effusion and liver swelling and yellowing.Extensive cell degeneration and necrosis and nuclear inclusion bodies appeared in the liver,and extensive lymphocyte degeneration and necrosis occurred in the three immune organs of the spleen,bursa and thymus.FAdV-4 invades the liver,heart and other tissues,and the main target organ is the liver.Chickens infected with FAdV-4 can shed through the throat and cloaca.In this study,the whole genome of six FAdV-4 isolates from Guangxi were sequenced,compared with the domestic and foreign FAdV-4 strains,and the sequence information and variation of FAdV-4 strains isolated from Guangxi were obtained.By designing and screening two sets of LAMP primers and optimizing the reaction conditions,a duplex fluorescent LAMP assay was established for the differential diagnosis of FAdV-4 variant and non-variant strains.To infect SPF chickens in different doses and routes,the results showed that FAdV-4 was highly pathogenic to 4-week-old SPF chickens,and was more susceptible to infection by intramuscular injection,and has a wide range of tissue tropism.The main target organ was liver,which could shed through cloaca and throat.In this study,the whole genome sequencing analysis of FAdV-4,differential diagnosis and pathogenicity of FAdV-4 variant strains were studied,provide theoretical basis and technical support for the prevention and control of my country's emerging FAdV-4.