Isolation And Identification Of Fowl Adenovirus Type 4 And The Effect Of Chicken Viperin Protein On Its Replication

Fowl adenovirus serotype 4(FAdV-4)can cause hydropericardium-hepatitis syndrome (HHS)in chickens.Since 2015,HHS has spread in many places in China,which is difficult to prevent and control,causing heavy losses to the poultry industry.Therefore, strengthening the research of anti-FAdV-4 is of great significance to the development of the poultry industry.The function of the innate immune factor Viperin(Virus inhibitory protein,endoplasmic reticulum-associated,interferon-inducible)is related to the endoplasmic reticulum and has antiviral effects,but it is still unclear whether Viperin has antiviral activity against FAdV-4.Therefore,this study explored the effects of chicken Viperin on FAdV-4 replication to provide a theoretical basis for the host's natural immunity against viral infections.The main research contents and results are as follows:1.Isolation,identification,and pathogenicity analysis of FAdV-4The disease material in this study was a liver tissue sample from a 120-day-old hen suspected of HHS at a poultry farm in Suzhou City,Anhui Province.The homogenate after removing bacteria and impurities with filters was used to inoculate chicken embryos and Chicken Liver Hepatocellular Carcinoma(LMH)cell line,and a FAdV-4 strain was isolated and named AH-F19 strain.The primer design was carried out concerning the hexon sequence of the ON1 strain in Gen Bank.In this study,viral DNA was extracted,PCR amplification and gel electrophoresis verification were performed.At the same time,to identify the strain,the indirect immunofluorescence test(IFA)and observation with the transmission electron microscope(TEM)were performed.To study the pathogenicity of the AH-F19 strain,the TCID₅₀ of the isolated strain was determined in this study,and 10 3-week-old SPF chickens were injected intramuscularly with the virus at a dose of 1×10⁵ TCID₅₀ per head for animal regression experiment.Infected chicken embryos showed developmental delay and diffuse bleeding.After LMH cells were inoculated,the cells became rounded and fell off,the refractive index became stronger,and the grape cluster-like lesions appeared.PCR amplified fragment of the corresponding band size,which was verified by sequencing.Observed by TEM,the virus particles have an icosahedral symmetrical structure without a capsule,with a diameter between 70-90 nm,which is consistent with the structural characteristics of FAdV-4.In the IFA test,the infected cells showed green fluorescence,which further verified that the isolated virus was FAdV-4.The TCID₅₀of the isolated strain was determined to be 1×10^-6.667.In the animal return experiment,the chicks all died within the fourth day of the challenge,and the sick and dead chickens showed typical HHS lesions.2.Genome amplification,sequencing,and genetic evolution analysis of AH-F19 strainIn this study,specific primers were designed concerning the genome of the SD1601 strain in Gen Bank,and the extracted viral DNA was used as a template to amplify each fragment gene,and the PCR products were verified by gel electrophoresis.The fragments whose amplified size matches the expected size were recovered and sequenced.The sequencing results of each fragment were assembled to obtain the genome of the AH-F19strain.The genome was compared with other strains to construct a phylogenetic tree of strain AH-F19.Assembling and sequencing results showed that the length of the AH-F19strain was 43719 bp and the G+C content was 54.88%,which was in line with the characteristics of FAdV.The AH-F19 strain is highly similar to other domestic FAdV-4isolates,and is grouped into a cluster with them in the phylogenetic tree,but is far from foreign strains.However,the genetic evolution analysis of the 100 K gene revealed that the AH-F19 strain is not in the same branch as the domestic FAdV-4 strains,but is in the same branch as the foreign strains.This indicates that AH-F19 may have recombined with foreign strains during the evolution of its related strains.3.Amplification of chicken viperin sequence and exploration of chicken Viperin protein's inhibitory effect on FAdV-4To obtain the sequence of chicken viperin and further analyze it,this study used chicken cDNA as a template to amplify the sequence of chicken viperin.Subsequently,the bioinformatics analysis was carried out,the phylogenetic tree of viperin sequences of different species was constructed,and the subcellular localization and tissue distribution experiment were carried out.The results showed that chicken viperin is closely related to the viperin of turkey,guinea fowl,and Japanese quail,which conforms to the law of species genetic evolution.Through bioinformatics analysis,chicken Viperin protein is a hydrophilic protein with a molecular weight of 40.58 kDa,without transmembrane region and signal peptide,and contains a large amount of?-helix and random coil structure.At the same time,chicken Viperin has an Elp3 domain at 66?275 AA,which belongs to the Mia B family of free radical S-adenosylmethionine(SAM)enzymes.Through subcellular localization,chicken Viperin protein was found to be localized in the cytoplasm.To explore whether chicken Viperin protein has the effect of anti-FAdV-4,an LMH cell line stably overexpressing chicken Viperin was constructed and infected with the AH-F19 strain.Then the FAdV-4 one-step growth curve was drawn,and the virus replication was detected by IFA and Western Blot.The results showed that Viperin protein has a certain inhibitory effect on FAdV-4 on LMH cells.In summary,this study isolated and identified a highly pathogenic FAdV-4 named AH-F19.Subsequently,the genome sequence of the strain was amplified,and genetic evolution analysis was performed,and it was found that its sequence was different from other FAdV-4.At the same time,it was found that Viperin protein has a certain inhibitory effect on FAdV-4 on LMH cells.This study provides a scientific basis for the study of innate immune factors that inhibit FAdV-4.