Construction Of Replication-defective Flavivirus And Study On The Production Mechanism Of Subgenomic Flavivirus RNA

The genus Flavivirus includes many important pathogens such as west Nile virus,yellow fever virus,dengue virus,zika virus,Japanese encephalitis virus,and tick-borne encephalitis virus.The genome of flavivirus is an approximately 11 kb single-strand positive RNA.Flavivirus is widely distributed and has great influence on human health.As a group of arbovirus,most flaviviruses are transmitted by the bites of mosquitos or ticks,and the clinical approved vaccines or antiviral drugs are not available,which increases the difficulty to control virus transmission and infection.Therefore,it is important to establish an effective platform for antiviral screening and study the pathogenic mechanism of flavivirus.Omsk hemorrhagic fever virus?OHFV?is a tick-borne flavivirus classified as a biosafety level-4?BSL4?pathogen.The clinical symptoms of OHF include fever,headache,cough,myalgia,hemorrhage,and sometimes meningeal signs with neurological system damage,which seriously threats human health.Studies of OHFV are restricted within BSL4 laboratories.Currently,no commercial vaccines or antiviral drugs are available against OHFV infection.In this study,using full-length infectious clone as the backbone,we constructed a replication-defective OHFV with an NS1 deletion?OHFV-?NS1?and a defective reporter virus with the Gaussia luciferase?Gluc??OHFV-?NS1-Gluc?.Both the defective viruses could replicate efficiently in the BHK-21 cell line expressing NS1(BHK-21_NS1)but not in na?ve BHK-21 cells.The expression of Gluc was used to surrogate the replication of OHFV-?NS1-Gluc on BHK-21_NS1.Using OHFV-?NS1-Gluc reporter virus,we established a high-throughput drug screening method based on 96-well plate format with a calculated Z'value above 0.5.The defective OHFV-?NS1-Gluc reporter virus is a powerful tool for antiviral screening as well as viral replication mechanism and pathogenesis studies in BSL2 laboratories.Subgenomic flavivirus RNA?sfRNA?is a newly discovered important virulence factor of flavivirus.SfRNA is the highly structured noncoding RNA derived from3'UTR produced by incomplete cleavage of viral genomic RNA by host 5'-3'exonuclease XRN1.SfRNA participates in intracellular type I interferon response and RNAi,affecting the virus pathogenicity.Previous literatures showed that the XRN1cleavage of viral genome halted at the conserved stem loop structures?SLII/SLIV?and dumbbell structures?DB1/DB2?within 3'UTR and led to the production of sfRNA.In this study,we uncovered that a new structure,conserved sequences?CS?control the production of specific sfRNA,as well as mouse pathogenicity.We found that two West Nile virus strains?WNV and WNVa?in our laboratory showed different pathogenicity and sfRNA production,implying the correlation between them.By a series of experiments,including sequence alignment of full-length genome,verification of mutation based on the infectious clone,mouse virulence test and the detection of sfRNA,we proved that 10577 nucleotide in 3'UTR is a key site affecting the generation of sfRNA1 and the pathogenicity.We found that 10577 site is located in the RCS3,and sfRNA detection of recombinant mutant virus demonstrated that deletion of RCS3 affects the production of sfRNA1.By a series of sfRNA detection of mutants with site mutations in the stem and loop sequences of RCS3,we further identified that the stem pairing of RCS3 is necessary for the generation of sfRNA1,while the loop sequence is not the necessity.Through the analysis of the 3'UTR sequence of other flaviviruses,we found that the conserved sequence?CS?was very conservative in different flaviviruses.Then we further verified that WNV deletion of CS3,RCS2 and CS2 affected the production of sfRNA2/3/4,respectively.Using multi-site mutants,we also confirmed that the stem paring of CS3 is necessary for the generation of sfRNA2.Similarly,we confirmed that the RCS3 and CS3 also affected the generation of sfRNA in ZIKV and DENV2 viruses,reaching the consistent conclusion with in WNV.Overall,our results systematically demonstrate for the first time that these conserved sequences?CS?are critical determinants of sfRNA production and viral virulence,providing potential novel targets for vaccine design or therapy development.