Isolation And Identification Of Duck Tembusu Virus JS-2019 Strain And Its Sensitivity To Interferon

Duck Tembusu virus disease first appeared and broke out in 2010 in our country.It causes serious decline in duck egg production,fever,loss of appetite,ataxia and other symptoms,which brings huge economic losses to the duck industry in our country.The pathogen of the disease,Duck Tembusu virus(DTMUV),is a single-stranded positive-stranded RNA virus belonging to the Flaviviridae family,with a full-length genome of approximately 11 kb.DTMUV mainly harms ducks,but poultry such as chickens and geese can also be infected.Mosquitoes are one of the important vectors of the disease,so the disease has a high incidence in the summer and autumn when mosquitoes are active.In addition,DTMUV can be transmitted through direct contact,aerosol transmission and other horizontal or vertical transmission methods.Interferon is a secreted protein produced by cells that has biological functions such as anti-virus,anti-tumor,and participation in immune regulation.It is divided into three types,namely type I,type II and type III.Among them,type I interferon has a strong anti-viral function,and type III interferon also plays an anti-viral auxiliary function.It is reported in the literature that many flaviviruses such as DTMUV are more sensitive to the antiviral effect induced by interferon.This study collected tissues such as liver and spleen of a diseased duck suspected of being infected with DTMUV in a duck factory in Jiangsu in 2019,and a Duck Tembusu virus was successfully isolated and identified.Determine the whole genome sequence and perform genetic evolution analysis,the results showed that the genetic variation of this strain was mostly distributed in the NS4a and NS4b genes.The E.coli system was successfully used to produce duck interferonα,β,andλwith good antiviral activity.By comparing the antiviral activities of the three interferons against the JS-2019 strain,XZ-2012 L strain and XZ-2012 S strain,we found that duck interferonα,β,andλhad weaker activity against DTMUV JS-2019 strain,indicating that it may evolve the function of antagonizing interferon.The specific research content is as follows:1.Isolation,identification and genetic evolution analysis of DTMUV JS-2019 strainIn 2019,a suspected case of Duck Tembusu virus infection occurred in a duck farm in Jiangsu.The sick ducks showed symptoms such as fever,decreased appetite and decreased egg production.In this study,liver,spleen,brain and other tissues of sick ducks were collected,and one strain of DTMUV was successfully isolated and identified through BHK-21 cell inoculation,named as DTMUV JS-2019 strain.The titer of the fourth generation virus proliferated on BHK-21 cells is 9×10~4 PFU/m L.We successfully amplified the entire gene sequence of the virus by RT-PCR,with a total length of 10990 nt.It has only one open reading frame,and the encoded polyprotein consists of 3425 amino acids.According to the homology of the whole genome sequence and phylogenetic tree analysis,it is found that the virus has a high homologies of 96.5%to 98.6%with the 26 strains of TMUV reported in the literature in recent years.Among them,the virus has different host origins from chickens,geese,and mosquitoes.The nucleotide homology is also very high.Comparing the nucleotide and amino acid homology with the DTMUV JS-2010 strain and DTMUV XZ-2012 strain preserved in our laboratory,it is found that the gene variation is more distributed in the NS4a and NS4b genes.2.Preparation of duck interferonα,β,λand determination of their anti-DTMUV activityAccording to the duckα,β,λgene sequence registered on Gene Bank,on the basis of the E.coli codon preference and annexation,the duck interferonα,β,λmature protein coding sequence was replaced with E.coli partial codon.The whole gene was synthesized and cloned into the prokaryotic expression plasmid p ET28a to obtain three recombinant prokaryotic expression plasmids p ET28a-Du IFN-α,p ET28a-Du IFN-βand p ET28a-Du IFN-λ.The recombinant plasmid was transformed into E.coli BL21 and Rosetta(DE3),and the expression was induced by adding IPTG at 37℃for 4 hours.SDS-PAGE electrophoresis showed that the recombinant duckαprokaryotic expression plasmid could be expressed in E.coli BL21 host strain.The recombinant duckβandλprokaryotic expression plasmids can be highly expressed in E.coli Rosetta host strain.The relative molecular masses obtained are respectively 28KD,26.96KD and 20.24KDα,β,λinterferon fusion proteins,they all exist in the form of inclusion bodies.Theα,βandλinterferon fusion proteins with relative molecular masses of 28KD,26.96KD and 20.24KD are obtained,and they all exist in the form of inclusion bodies.After ultrasonic fragmentation,extraction of inclusion bodies,PBS washing,urea dissolution and denaturation and overnight renaturation in 4℃renaturation solution,relatively pure recombinant duck interferonα,βandλwere obtained.The specific anti-VSV activity and anti-DTMUV virus activity of recombinant duck interferonα,βandλwere determined by microcytopathic inhibition method.The results showed that the specific anti-VSV activities of recombinant duck interferonα,βandλwere 1.4×10~6 U/mg,4.16×10~6U/mg and 2.46×10~6U/mg.All the three kinds of interferons had anti-DTMUV activity.Compared with DTMUV XZ-2012strain,the antiviral activity of recombinant duck interferon against DTMUV JS-2109 strain decreased significantly,indicating that DTMUV JS-2109 strain may have evolved some antagonistic function of duck interferon,and the specific mechanism needs to be further explored.