Characteristics Of Highly Pathogenic FAdV-4 Strain Infecting Cells And Preparation And Effect Evaluation Of Different Types Of Inactivated Vaccines

Fowl adenoviruses（FAdVs）are important pathogens in poultry.Avian adenovirus type I is divided into 5 subgroups（A～E）and 12 serotypes（FAdV-1～8a and FAdV-8b～11）.Among them,subgroup C serotype 4（FAdV-4）is the main serotype of poultry in China,with the highest incidence and mortality in chickens.At present,the types of FAdV-4infected cells,the changes of blood cells after infection,characteristics of cytokines produced by cells,and the immune effects of different types of vaccines are still unclear.In this study,the following three aspects were studied for the virulent strain FAdV-4 isolated in the laboratory.1.The detoxification time after virus infection was determined,the effect of virus infection on the number of white blood cells in the blood was detected,and the cell type of virus infection in vitro was determined.Firstly,15-day-old SPF chickens were inoculated with viruses at a dose of 5×10^5.5TCID₅₀,and the virus excretion was detected by anal swabs at 24,36,54,72 and 96 h to determine the infection time of the body.Secondly,when the virus was infected in vivo for 72 h,the blood of chickens was collected aseptically.The peripheral blood red blood cells and white blood cells were separated by chicken peripheral blood lymphocyte isolation kit,and the DNA of the two cells was extracted respectively.The virus infection was detected by PCR.Then,the number and proportion of white blood cells in 10⁶blood cells were counted by flow cytometry.Finally.In vitro,the virus was infected with leghorn male hepatocellular cells（LMH）,chicken fibroblasts cells（DF-1）,chicken macrophage-like cells（HD11）,chicken B lymphocyte cells（DT40）and chicken embryonic kidney cells（CEK）.Cells DNA were extracted after incubation for 3～5 days,and PCR was used to detect virus infection.The results showed that virus shedding could be detected more than 54 h after inoculation,and most chickens died at 96～120 h（4～5 d）.The virus could not infect DF-1,HD11 and chicken red blood cells,but could infect DT40 cells,LMH,CEK and chicken white blood cells.The results of flow cytometry showed that the number of white blood cells in the viral infection group was 8.6 times higher than that in the blank control group,and the difference was extremely significant（P<0.01）.2.To explore the types and characteristics of cytokines produced by virus infection in different cells.Firstly,the m RNA of white blood cells of 72 h chicks inoculated with virus was extracted and reversely transcribed into c DNA.RT-PCR was used to detect the expressions of granzyme A（Gzyms-A）,perforin（PFP）,IL-1βand Ii genes.Secondly,RT-PCR was used to detect the expression levels of IFN-αand IFN-γat 12,24 and 36 h after viral infection of LMH.Finally,the expression of IFN-αand IFN-γin CEK infected with 0.2 and 0.4 MOI for 72 h was detected by RT-PCR.The results showed that the expression of activated T cell cytokines（Gzyms-A and PFP）,cytokines（IL-1β）and antigen transport-related Ii genes decreased after 72 h of virus infection,which were 1/3,1/100,1/8 and 1/20 of the blank control group,respectively,and the difference was extremely significant（P<0.01）.After FAdV-4 was infected with LMH cells for 36 h,the expression of IFN-αwas increased by two times,while the expression of IFN-γwas increased by 25,42 and 46 times after 12,24 and 36 h,respectively,which was significantly different from that of the control group（P<0.01）.After 72 h of infection with CEK cells,the expression levels of IFN-αand IFN-γof 0.2 MOI FAdV-4 were increased by 78 and 3200 times,respectively.After 72 h of infection with CEK cells,the expression levels of IFN-αand IFN-γof 0.4 MOI FAdV-4 were increased by 85 and 3700 times,respectively,which were significantly different from those of the control group（P<0.01）.3.The inactivated vaccines of allantoic fluid and CEK cells were prepared and the immune effects were compared.Firstly,the virus solution was prepared,and the virus was inoculated into chicken embryos to prepare allantoic fluid.At the same time,CEK cells were infected with the virus,and the cell supernatant was collected after 72 h to obtain the cytotoxin.Secondly,TCID₅₀of allantoic fluid and cell fluid was determined,and the oil emulsion inactivated vaccine was prepared after inactivation with formaldehyde at the final concentration of 1.5‰.The appearance,viscosity,sterility and safety of the vaccine were tested.Finally,chickens were inoculated with the prepared vaccine,and each chicken was inoculated with 0.05 m L virus after secondary immunization.The protection rate of the vaccine was calculated according to the number of dead chickens.The results showed that the toxicities of allantoic fluid and cytotoxin were 10^6.56TCID₅₀/0.1 m L and 10^6.42TCID₅₀/0.1 m L,respectively.The prepared two types of vaccines were tested to be qualified.The protection rates of inactivated urinary bladder fluid and cell fluid were62.5%and 37.5%,respectively.In summary,this study found that the highly pathogenic FAdV-4 strain was selective to cells,which could infect LMH,CEK,DT40 and chicken white blood cells,and could not infect DF-1,HD11 and chicken red blood cells.After virus infection,white blood cells increased.FAdV-4 infection could not up-regulate the proportion and number of activated T cells,and could not induce effective T cell response.The virus could induce the expression of IFN-αand IFN-γin LMH and CEK cells,especially the expression of cytokines in CEK,which indicated that the kidney was the most important immune organ.oil emulsion inactivated vaccine of FAdV-4 allantoic fluid and CEK cell were prepared,with low protection rates of 62.5%and 37.5%,respectively.