Biological Characteristics Of A Non-pathogenic Serotype 1 Fowl Adenovirus Strain And Preparation Of Monoclonal Antibodies Against Serotype 1 Fowl Adenovirus

Adenovirus is a common vector for the development of recombinant virus.At present,human and mammalian adenovirus vectors have been extensively studied,while there are few studies on fowl adenovirus(FAdV).The purpose of this study is to isolate and identify serotype 1 fowl adenovirus(FAdV-1).The biological characteristics of FAdV-1 strain were studied to lay a foundation for the development of recombinant virus vaccine with FAdV as vector.In addition,monoclonal antibodies against FAdV-1 were prepared to provide basic materials for the establishment of methods for detecting FAdV-1.1.Isolation,identification and biological characteristics of serotype 1 fowl adenovirusIn this study,cloacal swabs were collected from healthy chickens and detected by PCR with the universal primer Hex-F1/R1 of FAdV.The virus was isolated from the 9-day-old specific-pathogen-free chicken eggs via the chorioallantoic membrane inoculation.The virus proliferated and cultured on chicken hepatocellular carcinoma cell line(LMH)until it produced stable cytopathy.The virus was purified by plaque purification test,identified by PCR and sequenced.The serotype was determined by comparative analysis,and then the biological characteristics of the virus were analyzed.A FAdV-1 strain was isolated from 50 samples,named FAdV-1 JS2017.The virus was inoculated into leghorn male hepatocellular(LMH)can produce typical cytopathic changes of fowl adenovirus,which are characterized by round cells and enhanced refractive index.The viral titer was detected by SYBR Green Ireal-time fluorescence quantitative PCR with 6.34×107 copies.The virus particles were observed by transmission electron microscopy(TEM).It was found that the virus was spherical with no envelope and its diameter was 70-90 nm.The complete genome sequence of the JS2017 was amplified by PCR using 34 pairs of primers,which showed 43,739 bp in length,with 54.29%G+C content.The JS2017 contains 34 ORF coding regions and the inverted terminal repeats(ITR)sequences are 54 nt in length.JS2017 belongs to a large evolutionary branch with reference strains of FAdV-1 CELO,61/11Z,JMI/1 and W-15,and the homologies of nucleotide sequences were 99.3%,99.7%,99.3%and 99.6%,respectively.One-week-old SPF chickens were tested for artificial pathogenicity by intramuscular injection of 106 86 TCID50 JS2017 virus solution.No obvious clinical symptom was found in the test chickens.After 10 days of challege,no visceral lesion was found in the larynx,trachea,heart,liver,stolach,kidney and other visceral organs.Histopathological sections showed no obvious abnormalities in the histological structure of the organs.The results of virus shedding showed that the peak of detoxification was reached in 7 days after infection.The virus can also be detected in liver and kidney.These results indicated that FAdV-1 JS2017 strain had no pathogenicity and strong replication ability.The right end of the genome of FAdV-1 JS2017 strain is 40 000 bp-43 620 bp,which is the non-essential region for replication.Primers are designed at both ends of the non-essential region for amplification of homologous arms.Homologous recombination of pFAdl-EGFP and FAdV-1 JS2017 strains in LMH cells by transfer plasmid with homologous arm sequence and enhanced green fluorescent protein(EGFP)gene.Recombinant virus deletes the non-essential region of replication between homologous arm sequences and replaces it with the complete expression box of EGFP gene.After 2 hours of infection with LMH cells by FAdV-1 JS2017 strain,the transfer vector pFAdl-EGFP was transfected into LMH cells by liposome transfection.The recombinant virus was purified by plaque purification.A recombinant virus FAdl-EGFP stably expressing EGFP was successfully obtained.The results showed that the deletion of the non-essential region of the right-end replication of FAdV-1 JS2017 strain did not affect its stable replication in LMH cells.JS2017 strain lays a foundation for the development of recombinant virus vaccine with FAdV as vector.2.Preparation of monoclonal antibody against FAdV-1FAdV-1 JS2017 was purified and immunized to BALB/c mice after the proliferation of LMH cells.Spleen cells of immunized mice were fused with mouse myeloma cells(SP2/0)after antibody titer reached 1:25600.JS2017 virus solution was purified by ultracentrifugation as antigen.Hybridoma cells were screened by indirect ELISA and subcloned hybridoma cells were detected.Nine hybridoma cell lines targeting FAdV-1 and stably secreting antibodies were obtained.Nine hybridoma cell lines were identified by indirect ELISA using prokaryotic expression of FAdV-1 hexon protein as antigen.Five hybridoma cell lines,named 2D11,3C7,6G8,9D6 and 12H7,were obtained for stable secretion of antibodies against FAdV-1 hexon protein.Further purified FAdV-4(8/?g/mL)and FAdV-8b(16?g/mL)were used as antigens respectively.The five hybridoma cell lines were identified by indirect ELISA,indirect immunofluorescence test and Western blot.The results showed that 6G8 and 9D6 could react specifically with FAdV-4 and FAdV-8b.In conclusion,five monoclonal antibodies against FAdV-1 hexon protein were obtained,of which 6G8 and 9D6 reacted specifically with FAdV-1,FAdV-4 and FAdV-8b.These monoclonal antibodies provide basic materials for establishing methods for detecting FAdV-1.