Epidemiological Investigation Of Duck Tembusu Disease In Shandong Province And The Comparison Of Innate Immune Responses Induced By Virulent And Attenuated Strains In Duck Embryo Fibroblast

Duck tembusu disease is an infectious disease caused by the tembusu virus(DTMUV).Which mainly infects ducks.The egg production rate of the laying duck will decrease drastic ally after infection,and the duckling infection will manifest as neurological symptoms and hig h mortality.Duck tembusu disease exists in most duck-raising areas in Shandong Province,w hich threatens the healthy development of the duck industry seriously.There is no reliable pr eventive measure for duck tembusu disease,it is use for preventing and controlling the duck t embusu disease in time to conduct epidemiological investigations for grasping the epidemic d ynamics of DTMUV and the variation of pathogens.According to the pathogenicity of DTM UV to ducks,DTMUV is divided into virulent strains(such as FX2010)and attenuated strains(such as MM1775).At present,comparative studies on host range and pathogenicity of two st rains are common.There are few studies on the innate immune response induced by both strai ns in host or cell,and innate immunity may be related to the pathogenicity and spread of the st rain.Comparing the innate immune responses of the host caused by strong and attenuated stra ins can provide a theoretical basis for revealing the pathogenic mechanism of DTMUV and sc reening the controlling drugs.This study is aimed at the following two aspects.This study investigated the prevalence of tembusu disease in ducks of different breeds an d ages in Shandong Province.4437 serum of unimmunized ducks and 276 suspected cases we re collected from 9 areas including Tai'an,Heze and Liaocheng and so on.The DTMUV anti body positive rates in duck serum were detected by enzyme linked immunosorbent assay.Wit hing RT-PCR method to detect DTMUV nucleic acid in organs such as liver,spleen,brain,lu ng,kidney and heart.After virus isolation and amplification of virulence gene(E gene)sequen ces in positive cases.The sequence is used as a reference to construct an E gene phylogenetic tree and perform sequence homology alignment analysis based on the popular DTMUV E gen e published on Genbank.The prevalent strain FX2010 and the attenuated strain MM1775 of mosquitoes were infected with duck embryo fibroblasts at the same dose.Cell samples were c ollected at different time points after inoculation,and the samples of 24 and 48 h were detecte d the expression levels of mRNA with pattern recognition receptors(TLR2,TLR3,TLR4,T LR7,RIG-I,and MDA5),inflammatory cytokines(IL-1?,IL-6,IL-8,and TNF-?),interferons(IFN-?,IFN-?,and IFN-?)and interferon-stimulated response genes(MX,OAS,PKR)by q PCR.The results of the epidemiological survey of the duck tembusu disease have shown that d ifferent levels of DTMUV antibody positive rates have been detected in different ducks in Sha ndong Province.The highest of which was the egg duck group(23.9%),followed by the meat duck group(13.9%),the lowest is the reserve duck group(12.4%);From regional perspective,the highest positive rate of DTMUV was Taian area(21.0%),the lowest positive rate of DT MUV was Dezhou(11.7%);From breeds perspective,the seroprevalence rate of Compell duc ks(25.3%)was higher than that of Jinding ducks(23.6%),Shaoxing ducks(22.9%)was high er than cherry valley ducks(13.6%)and Fengye ducks(12.9%);The amino acid homology of the isolates and the prevalent strains in shandong in 2017 was 98-99.2%,and that of the isolat es in 2010,2011 and 2012 was 96.8-98.6%.The isolated strain has no key amino acid mutatio n compared with the virulent strain FX2010.The infection of DEFs by DTMUV showed that FX2010 and MM1775 could up-regulate the expression of TLR3,RIG-I and MDA5 mRNA i n DEFs at the early stage of infection,but the amplitude of up-regulation was different,and th e expression of MM1775 was significantly higher than that of FX2010;Both FX2010 and M M1775 could up-regulate the mRNA expression levels of inflammatory cytokines IL-6 and T NF-?,but the expression of FX2010 is significantly higher than that of MM1775.Both FX2010 and MM1775 could cause interferon stimulation of DEFs.The expression of ISGs gene m RNA was up-regulated,but the MX expression caused by MM1775 was significantly higher t han that of FX2010.In 2018,there was still a prevalence of duck tembusu disease in the non-immunized duck populations in Shandong Province.The prevalence of different duck populations varied,and the infection rates in different regions were different.The infection rates of ducks in different varieties were different.The prevalent strains are all virulent strains,and the virulence of the isolated epidemic strains has not changed greatly,and there is no regional difference between the isolates.Both FX2010 and MM1775 could up-regulate the expression of PRRs and their downstream genes in DEFs.The significant differences are TLR3,RIG-I,MDA5,IL-6,TNF-?,IFN-? and MX.