Isolation And Identification Of Group Ⅰ Fowl Adenovirus And Establishment Of PCR-HRM Typing Method

Inclusion Body Hepatitis（IBH）is a poultry-infected disease with liver damage caused by fowl adenovirus I（FAV-1）.The typical symptoms of the disease are hydronephrosis and necrotizing hepatitis.FAV-1 mainly infects with broilers aged 3⁶ weeks,leading to the mortality rate as high as 80%.Once having occurred,the disease can spread to the surrounding area quickly.In recent years,it is reported that the disease has occurred in many provinces and cities in China,which has caused huge economic losses to the national aquaculture industry.Strengthening the research of FAV is of great significance to promoting the healthy development of the poultry industry.In this study,the avian adenovirus was detected by PCR from the suspected samples of"hepatitis containing body"send from poultry farms in Shandong Province,Jiangsu Province,Shanxi and Hebei Province.The isolated virus and sequenced hexon gene are used to understand the infection and distribution status of FAV.At the same time,using the FAV PCR-HRM typing method established to achieve accurate and rapid genotyping of the isolated adenovirus.The method can be used as a model to develop other pathogen genotyping techniques.In this study,SPF chicken embryos and chicken embryo liver cell（CEL）were used to separate the FAV from suspected avian tissues infected with adenovirus clinically.The virus isolates were identified by PCR detection,Hexon gene sequencing and Hexon gene sequence analysis.Inoculation of SPF chicken embryos with diseased tissue homogenate supernatant can cause embryo body dysplasia,body surface flushing and hemorrhage,liver necrosis and swelling.The peak of death is 6⁷ days after inoculation.After inoculation of chicken embryo liver cell（CEL）,some cells begin to round and shrink after 48h.After 72h,70%⁸0%of cells begin to fall off causing grape-like aggregation.The lessioms of embryoid bodies and cells are consistent with the pathological process of group I avian adenoviruses.The TCID₅₀of 15 isolates are between 10^-4.754.75 and 10^-7.57.5 respectively.The result of PCR identification shows that the amplified product is consistent with the expected size.A total of 15 group I avian adenoviruses are isolated and named as SD151225,SD150808,SD150930,SD150909,JS151208,SD160309,SD160402,SD160402,SD160318,SD160827,SD160720,SX160723,SD160826,SD160829,SD160930.The hemagglutination test shows that the isolated strains are not hemagglutinating.The hexon gene of the isolated strains are amplified by PCR and compared with the Hexon gene sequence of other serotypes of the group I avian adenovirus published in GenBank.The results show that 10 out of 15 isolates have higher homology with serum type 1 and 5 strains have higher homology with serum type 4.The results of genetic evolution analysis show that SD160827,SD151225,SD150808,SD150930,SD150909,JS151208,SD160309,SD160402,SD160401 and SD160318 isolates are on the same branch as the serotype 1 avian adenovirus and the remaining isolates are serotype 4 avian adenovirus.On the same branch,15 strains of FAV-I isolates can be divided into two serotypes including10 strains of serum type 1 and 5 strains of serum type 4.A specific primer was designed based on Hexon gene sequence of each serotype of the I group of avian adenoviruses published in GenBank for real-time PCR and subsequent high-resolution melting（HRM）curve analysis to distinguish 12 FAdV serotypes.The results are compared with the previous Hexon gene PCR results,which shows that PCR-HRM analysis proves to be a sensitive and specific method for distinguishing between the 12 serotypes.All melting curves are highly reproducible and the replication of each serotype is correctly genotyped by using a normalized HRM curve.The PCR-HRM typing method for group I avian adenovirus established in this study has high sensitivity,specificity and reproducibility and it can be used for clinical detection of FAV-1.