| The main targets of serum type 4 Fowl adenovirus（FAdV-4）infection are chicks aged3-5 weeks,and they will die within one week after infection.Autopsy of dead chickens showed typical symptoms of the pericardial effusion-hepatitis syndrome（Hydropericardium-Hepatitis Syndrome,HHS）.A large amount of fluid accumulated in the pericardium,obvious viral inclusion bodies appeared in the liver,and the mortality rate reached 30-80%.The virus is highly contagious.In addition to the horizontal transmission through contact and aerosols,FAdV-4 can also be transmitted vertically through chicken embryos.Since 2015,the highly virulent avian adenovirus type 4（hv FAdV-4）has become the main pathogen of hepatitis-pericardial effusion syndrome（HHS）in China,leading to a continuous rise in chicken mortality and posing a great threat to China’s poultry farming and related industries.In the vaccine research of FAdV-4,although inactivated vaccine and attenuated vaccine have a good protective effect,these vaccines have unavoidable shortcomings in safety and price,so there is an urgent need to develop a safe and efficient new vaccine for effective prevention and control of the disease.In the study of pathogenic mechanism and drug therapy,although FAdV-4 has attracted wide attention,the potential molecular mechanism of viral pathogenesis is still little known,let alone the specific therapeutic drugs for FAdV-4.In this study,a hypervirulent FAdV-4 strain was isolated for the experimental,and the mechanism of hepatocyte steatosis caused by the virus was explored.Finally,the exocrine expression of Fiber-2 protein in yeast was optimized,and the feasibility of the protein as a subunit vaccine was proved.It provides basic data for fermentation production of FAdV-4 subunit vaccine and determination of target sites for specific drug therapy.The specific results are as follows:1.In this study,a hypervirulent FAdV-4 strain（designated GD616）was isolated from25-day-old meat-type chickens with severe HHS in Guangdong Province China in 2017.The whole genome of the GD616 strain shares high homology with those in the recently isolated hypervirulent FAdV-4 strains in China,with natural deletions of ORF19 and ORF27.A comparative analysis of Hexon and Fiber-2 proteins revealed that two unique amino acid residues at positions 378 and 453 of the Fiber-2 protein might be associated with virulence due to its occurrence in all the hypervirulent FAdV-4 isolates only.Next,the study on the rule of virus replication in LMH cells showed that when MOI was infected by 0.1 TCID50,the nucleic acid,protein,and titer of the virus reached the peak after 72 hours.To systemically evaluate the effect of age on the susceptibility of chickens to hypervirulent FAdV-4,we used this hv FAdV-4 strain to intramuscularly inoculate 7-to 180-day-old specific-pathogen-free chickens for the evaluation of pathogenicity.These results showed that the pathogenicity of the hv FAdV-4 strain GD616 to chickens exhibited age-relatedness,with younger than 59-day-old chickens showing 100%morbidity and mortality,while 180-day-old chickens still exhibited a hydropericardium syndrome-like clinicopathology with 60%morbidity and 20%mortality.These findings enrich the currently available knowledge regarding the pathogenicity of the hypervirulent FAdV-4 virus in chickens with a wide range of ages,which assists with the selection of suitable-aged chickens for the evaluation of FAdV-4 vaccines.The study also provides the virus strain for the study of subunit vaccine and pathogenic mechanism of FAdV-4.2.FAdV-4 is a hepatophilic virus,which can cause severe liver damage,but the pathogenesis is still unclear.Here,we found that FAdV-4-mediated hepatic injury is accompanied by accumulation of oil droplets（Triglyceride）in the cytoplasm of hepatocytes,a typical indicator of steatosis,in FAdV-4-infected chickens.Significant upregulation of adipose synthesis-related genes,such as LXR-α,PPAR-γ,SREBP-1C,and significant downregulation of low-density lipoprotein secretion-related genes and lipid oxidation and decomposition-related genes were observed in the infected chickens.FAdV-4 infection in cultured LMH cells showed similar steatosis with differential alterations of various lipogenesis-related genes.It was also found that there was a significant correlation intracellular between TG content and viral load.We further determined the effect of LXR-αactivity on FAdV-4-induced steatosis and found that LXR-αreverse agonist（SR9243）and RNA interference（si-LXR-α）treatment not only reduced the number of oil droplets and the expression of adipogenic factors but also significantly decreased viral DNA level,protein expression,and viral titer.LXR-αagonist（T0901317）showed the opposite phenomenon,with a significant increase in the number of oil droplets,the expression of adipogenic factor,the level of virus DNA,protein expression,and virus titer.However,inhibition of the activity of SREBP-1C,a downstream protein of LXR-α,had no significant effect on virus production.Collectively,FAdV-4-induced steatosis depends on the activation of LXR-αand its regulation of key proteins in downstream fat metabolism,and LXR-αis closely related to the replication of FAdV-4 in cells,but those achieved through a signal pathway independent of SREBP-1C.These results highlight the therapeutic potential of targeting the LXR-αpathway in the treatment of FAdV-4 infection and provide important implications for the development of specific drugs.3.In order to find an efficient expression system to develop a type 4 avian adenovirus genetic engineering vaccine,a methanol nutritional yeast expression system was used to express Fiber-2 protein.We first optimized the Fiber-2 gene of the virus according to the codon preference of yeast and integrated it into the genome of yeast GS115 strain by p PIC9K plasmid.Finally,3 different concentrations of G418 were used for screening and 8recombinant high copy strains were obtained.Two high copy recombinant strains were randomly selected for the expression test,and the results showed that the recombinant strain secreting Fiber-2 protein was successfully obtained.In order to improve the expression of Fiber-2 protein in yeast,the induction conditions were optimized.The results showed that the optimum concentration,times,and temperature of methanol induced in shake flask fermentation were 1.0%（v/v）,72 h,and 30℃,respectively.After optimization,the total protein content in the supernatant of the fermentation broth was about 50 mg/L,and the protein of Fiber-2 reached 8.7 mg/L.Then,a single band of Fiber-2 protein was purified by nickel column and then mixed with adjuvant（ISA-201-VG）at 1:1 to prepare subunit vaccine.Immune protective tests showed that the subunits vaccine prepared by the optimized optimized Pichia pastoris expressing Fiber-2 protein could induce high antibody levels and provide up to 100%attack protection against hypervirulent FAdV-4（GD616）.This study laid a foundation for the research and development of FAdV-4 subunit vaccine using recombinant Fiber-2 protein expressed in Pichia pastoris. |
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