Genetic variations in dengue virus type-2 and their effects in viral replication and infectivity
Dengue is an emerging disease mainly spread throughout tropical and subtropical regions of the world. An estimate of three hundred and eighty million infections occurs annually . Dengue virus (DENV) is transmitted to humans primarily by the Aedes aegypti mosquito and causes a variety of clinical illnesses classified as dengue fever (DF), dengue hemorrhagic fever (DHF), and dengue shock syndrome (DHF/DSS). Secondary infection and viral strain are associated with DHF/DSS. Like many RNA viruses, DENV exhibits substantial genetic diversity. Such genetic subtlety may have significant implications for the emergence of new genotype(s) and lead to viruses with altered antigenicity and pathogenesis, ultimately influencing disease transmission patterns. This thesis aims to study the genetic variation of the nonstructural proteins within DENV-2 strains from different genotypes and their influence in RNA replication and infectivity. The protein sequences of highly pathogenic (DHF) versus less pathogenic (DF) patient isolated strains from diverse geographical locations were compared. Single amino acid substitutions of conserved surface exposed residues in the NS3 and NS5 proteins from within strains derived from DHF patients were identified and targeted for mutagenesis using DENV-2 16681 as the parental virus. Rescued viruses displayed varied phenotypes and were characterized by RNA synthesis and infectivity. Substitutions in NS3 protein I160T, S171N and K213N had no effect on RNA synthesis or infectivity. Substitution T399A affected RNA synthesis by 3 logs and produced second site reversions responsible for the wild-type growth kinetics. Substitution of residue I135T, E558K and N730S in the NS5 protein had a 1 log reduction in RNA synthesis whereas I135V, V723N and K800T produced a 3 log reduction. Furthermore, amino acid substitution Q819L in the NS5 protein showed 1 log reduction in RNA synthesis when compared to the wild type, however it completely prevented the production of plaques. Localization of NS5 protein to the nucleus was not affected in the NS5 substitutions. The Q819L substitution is defective in its ability to lyse and spread in BHK cells (i.e CPE) by fifty percent. Q819L amino acid substitution produced 2 to 2.5 logs less numbers of RNA molecules when compared to the wild type, suggesting that there is a defect in the number of RNA containing particles. Single particle tracking experiments showed that wild type as well as Q819L virus particles were able to enter BHK and C636 cells and fuse with cellular membranes, suggesting that the defect might be in another step of the virus life cycle. Thin sections on DENV particles showed that the Q819L mutant produced wild type particles (50 nm in diameter) as well as particles of approximately 30-35 nm in diameter, which were localized in the cytoplasm as well as within cellular compartments, suggesting that a subset of particles produced by the mutant are non-infectious subviral particles, in part explaining the lack of plaque production by plaque assay. Thin section together with qRT-PCR studies lead to the suggestion that Q819L is involved in the assembly process by reducing the number of release virus particles (mature and/or immature) and by increasing the number of subviral particles produced by this mutant. Coupling between genome replication and virus assembly has been reported before in YFV and KUNV NS3 and NS2A proteins. This study is the first to report the role of DENV-2 NS5 in virus assembly. Due to the role of NS5 in replication, as well as virus assembly and production as suggested here, NS5 is a good target for antiviral development.
Kuhn, Purdue University.
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