Virulence Difference Of Five Type ? Dengue Viruses And The Intrinsic Molecular Mechanism

Currently,dengue virus(DENV)is the most popular mosquito-borne virus and widely spread in tropical and subtropical regions.A series of symptoms caused by dengue virus,such as dengue/dengue shock syndrome(DSS/DHF),seriously threaten human health.Dengue virus belongs to a single-stranded positive sense RNA virus and lacks of accurate replication correcting system.Virus nucleotide changes will eventually lead to stronger or weaker virus virulence during the long-term process of virus spread.Secondly,host response is induced upon virus infection and the interactions between host and virus also influence the viral virulence.These two aspects corporately influence the virus pathogenicity and severity of the disease.Therefore,it is of great significance to further find the sites within the virus that is associated with virulence and to investigate the interactions between virus and host.In the past 25 years,eases of dengue infection have been reported in China's Guangdong province every year.The peak of infection happened in 2014,and DENV1 appeared as the major sera type at that year.We acquired five DENV1 isolations with different genotypes from Guangdong province among the outbreak of dengue fever.Firstly,human embryonic kidney cells(293T)and mosquito cells(C6/36)were infected with these five DENV1 subtypes and the viral replication efficiency were investigated.The virulence in vivo and biological characteristics of five variants were analyzed during virus challenge of newborn mice.Data in vitro and vivo suggested that DENV IB showed the strongest replication efficiency in mammalian and mosquito cells and the highest motality to newborn mice than the other variants,while DENV IE was the weakest among the five viruses.To further understand the molecular mechanism underlying the virulence difference of five variants,we studied the ability of variants escaping from host innate immune and the structure and function of the variants proteins.(1)The ability of five variants escaping from host innate immune:when the host was infected by DENV,the host innate immune system,as the first line of defense against virus invasion,was activated immediately.We focused on the mechanism of innate immune response mediated by interferon,and mainly studied the difference of non-structural proteins of five variants in inhibiting the production of interferon.We constructed eukaryotic expression plasmids of five variants non-structural proteins for detection of dual-luciferase reporter(DLR)assays and the results showed that non-structural proteins(NS2A,NS2B,NS4A)of DENV1B more strongly inhibited RIG-I-mediated production of IFN-? compared with DEN VIE.At the same time,the replication capacity of viral replicon became significantly weaker because the 66th NS2A amino acid of DGL2 was mutated.However,we surprisingly found that viral load of DENV1B was still higher than that of DENV1E after IFNAR--(type ? interferon receptor-deficient)mice and their primary cells were infected with viruses.Therefore,we hypothesized that the difference between two variants escaping from the host antiviral immune response was not the only factor that causes stronger virulence of DENV1B than DEN VIE.Amino acids mutations at certain sites of variants led to changes in the structure and functions of viral proteins,which might also result in virulence difference of distinct variants.(2)The structural differences of five variants:? The genome sequences of five variants were obtained from high-throughput sequencing.Phylogenetic analysis suggested that DENV1A/2014,DENV1B/2014 and DENV1C/2014 belonged to DENV-1 genotype ?.DENV1D/2014 and DENV1E/2014 belonged to DENV?1 genotype ?.Phylogenetic relationship between DENV1B and DENV1C was the closest,and far away from DENV1D and DENV1E.? The comparison of 3' UTR sequences and predicated secondary structure suggested that there is a deletion in 3' UTR region of DENV1D&E.The secondary structure of 3' UTR of DENV1D&E might not be as stable as that of DENV1A&B&C.? The NS3(dengue virus non-structural protein 3)serine protease of dengue virus was an essential component for virus replication and maturation.NS2B played a key role in the activation of NS3 serine protease as a cofactor activator.We obtained DENV1B-NS2B3 and DENV1E-NS2B3 recombinant protease with prokaryotic expression and purification methods.We found that DENV1B-NS2B3 showed higher substrate-cleaving efficiency in enzyme activity experiments.The sequence alignment showed that the 55th amino acid of the coenzyme domain NS2B(H)of two variants was different.We mutated lysine(K)at position 55 in DENV1B-NS2B3 protein into arginine(Arginine,R),and found that the substrate cutting efficiency of K55RNS2B(H)-NS3 protease significantly decreased.We further mutated the 55th amino acid of NS2B in dengue virus replicon to verify that this point mutation significantly attenuated the replication of DENV.In summary,we compared the difference of replication efficiency and virulence of five DENV1 variants.We found that DENV1B had the strongest replication capacity and virulence,and DENV IE was the weakest.We further suggested that the 66th amino acid of NS2A and 55th amino acid of NS2B were potential virulence determining sites,which provided a theoretical basis for better understanding the molecular mechanisms of DENV virulence.It also provides new ideas for investigation of DENV protein function,pathogenic mechanism and novel attenuated vaccine.