Screening And Mechanism Of Chinese Herbal Medicine Against Fowl Adenovirus Serotype 4(FAdV-4)

Hydropericardium hepatitis syndrome（HHS）is a highly contagious infectious disease caused by group I fowl adenovirus serotype 4（FAdV-4）.Disease mainly affects broilers,and the typical symptoms are sudden death of 3 to 5 weeks old broilers,accompanied by HHS,and the mortality rate can be as high as 90%.Since 2015,the outbreak of FAdV-4 in many chicken farms in China has seriously hindered the development of my country’s poultry industry.At present,the prevention and treatment of fowl adenovirus are mainly based on live attenuated vaccines and antibodies.In clinical application,live vaccines have a certain risk of virulence reversion.Traditional Chinese medicine has the advantages of multi-target mechanism of action and fewer adverse reactions,showing attractive application prospects.Therefore,effective Chinese herbal medicines against fowl adenovirus are screened.It has certain guiding significance for the treatment and prevention of FAdV-4.In this study,FAdV-4 was successfully isolated from dead chickens suffering from HHS in the farm,and the sequences were compared with domestic and foreign FAdV strains.Eight Chinese herbal medicines including Forsythia,Houttuynia cordata and Banlangen were selected for anti-FAdV-4 tests in vitro and in vivo,and the anti-FAdV-4 Chinese herbal medicines were screened out,and the antiviral mechanism of Chinese herbal medicines in vitro was preliminarily studied.The specific research results obtained are as follows:1.FAdV-4 was identified in dead chickens suspected of suffering from HHS was used PCR technology,and the isolated strains were sequenced and compared with classic FAdV strains at home and abroad.The results showed that the results of PCR identification were positive for FAdV-4;after comparing the FAdV-4 isolates with domestic and foreign virus strains,it was found that the FAdV-4 isolates were shorter than foreign strains but longer than domestic strains,and the isolates were similar to foreign classics.Compared with the virus strain,a nucleotide deletion of 1966bp appeared.The homology and genetic evolution analysis showed that the isolate had the highest homology with the domestic SD1601 strain,which was 94.3%.2.Eight kinds of Chinese herbal medicines were extracted by boiling and leaching method,and the maximum safe concentration of eight kinds of Chinese herbal medicines on LMH cells was determined by a combination of CPE observation and CCK-8 reagent detection.The results showed that the maximum safe concentrations of the eight traditional Chinese medicines ranged from 0.3125 mg/m L to 2.5 mg/m L.3.Effective Chinese herbal medicines against FAdV-4 were preliminarily screened by CPE observation and CCK-8 reagent detection.The results showed that the TCID₅₀ of FAdV-4 was 10^-4.5/0.1m L;the effective inhibition rate of 6 kinds of traditional Chinese medicines on FAdV-4 was more than 50%,and the antiviral effect of Houttuynia cordata and Bupleurum chinensis was better than that of other Chinese herbal medicines.The effective inhibition rate of the virus is above 70%.4.Using three different modes of action（direct killing of viruses by Chinese herbal medicines,blocking of adsorption of viruses by Chinese herbal medicines,and blocking of replication of viruses by Chinese herbal medicines）,the antiviral methods of Chinese herbal medicines were explored.In the mode of direct killing,the effective inhibition rates of Bupleurum and Forsythia on the virus were as high as 88.87%and83.97%,respectively,which were 8.62%and 3.73%higher than that of ribavirin,respectively.It shows that Bupleurum and Forsythia can effectively kill viruses in vitro;in the mode of adsorption blocking,Bupleurum,Licorice and Houttuynia cordata are better than the other three Chinese herbal medicines,and the effective inhibition rate is about 70%.The effective inhibition rate of Hu was 20%higher than that of ribavirin.It shows that these three drugs have relatively strong effect on virus adsorption blocking;in the mode of replication blocking,licorice has the strongest effect on virus replication blocking,with an effective inhibition rate of 64.08%,followed by an effective inhibition rate of 60.95%.The effective inhibition rate of ribavirin in this way is46.34%,and the licorice and bupleurum have better blocking effect on virus replication.5.Bupleurum,Glycyrrhiza,Houttuynia cordata,Forsythia,Banlangen and Honeysuckle can up-regulate the transcription levels of cytokines to varying degrees.Honeysuckle has the strongest promoting effect on IL-1;licorice and honeysuckle have higher up-regulation levels of IL-2 and IL-4 than other Chinese herbal medicines;licorice,houttuynia cordata and isatis root can regulate IL-6 better than other Chinese herbal medicines;Chai Hu and licorice had better up-regulation effect on TNF-αthan other Chinese herbs,and Radix isatidis and licorice had the strongest promoting effect on IFN-γ.6.Combined with in vitro screening of effective Chinese herbal medicines against FAdV-4,Chinese herbal prescriptions were formed according to the theory of traditional Chinese medicine,and the research on anti-FAdV-4 in Chinese herbal medicines was carried out.The results showed that the survival rate of the virus infection group was 0%,and the survival rate of the Chinese medicine group was 40%.The blood biochemical indexes of the chicks were detected,and the results showed that compared with the virus infection group,the immune globulin Ig A and Ig G of the chickens in the Chinese medicine group were higher.The content of IL-2 and IFN-γin serum increased significantly.It shows that the traditional Chinese medicine formula can effectively improve the immune function of chicks.In conclusion,we have successfully isolated and cultured a strain of FAdV-4.Eight kinds of Chinese herbal medicines were initially screened and 6 kinds of Chinese herbal medicines,namely Bupleurum,Licorice,Forsythia,Houttuynia cordata,Banlangen and Honeysuckle,had certain inhibitory effects on FAdV-4.The most effective way to detect the antiviral effect of Chinese herbal medicine through three modes of action is the direct killing effect.The expression levels of cytokines were up-regulated by the six Chinese herbal medicines to varying degrees.The survival rate of FAdV-4-infected chicks was increased through the treatment of traditional Chinese medicine prescriptions,and it was detected that the traditional Chinese medicine prescriptions could improve the immune function of the body,thereby enhancing the body’s anti-virus ability.