Characterization of Recombinant Dengue-2 Virus Derived from a Single Nucleotide Substitution in the 5′ Noncoding Region

Abstract

Variants of wild-type dengue serotype 2 (DEN-2) virus containing nucleotide substitutions at positions 14, 15, or 57 in the 5′ NCR were constructed by PCR-mediated site-directed mutagenesis. All three viruses containing a single point substitution demonstrated attenuation phenotype as evidenced by decreases replication and plaque size in cell culture assay. All three variants were less neurovirulent in newborn mice compared to the wild type. The mutants were immunogenic in adult mice immunogenicity and maintained stable replication characteristics following passage in mice. The variant viruses were competent for replication in Aedes aegypi mosquito vector, albeit at lower levels of infection and dissemination in the mosquito than the wild-type Den-2 16681 virus. Although all of the viruses, including the wild type, were found transmissible in mosquito life cycles, they were found subsequentially decreased in efficiency of infection, transmission, and dissemination rates along the mosquito generations and all of them remained genetically stable.

Development of Dengue type-2 virus replicons expressing GFP reporter gene in study of viral RNA replication

“…Using the Δ-DENV/GFP replicons, we were able to dissect the mutation effects of seven 5′NCR mutations that we have previously analyzed using the infectious clone system (Leardkamolkarn et al, 2010;Sirigulpanit et al, 2007). Our results suggested that deletion of nt 57 or 57-58 severely diminished the GFP expression and RNA replication.…”

“…Supernatants of the transfected cells collected on days 1, 4, 6 and 8 pt were determined for infectious viral titer by standard plaque titration assay as previously described (Leardkamolkarn et al, 2010). Briefly, 200 µl of serially diluted supernatant was inoculated onto the confluent monolayer of BHK-21 cells.…”

“…Full-length DENV-2 cDNA plasmids containing various mutations in the 5′NCR (Table 1) were made previously (Sirigulpanit et al, 2007;Leardkamolkarn et al, 2010). A cDNA fragment with A69T substitution isolated from full-length DENV-2.5′-69M plasmid was cloned into the FL-DENV/GFP plasmid to replace the corresponding wt fragment.…”

“…For this purpose, we chose five 5′NCR mutations previously shown to have attenuation effects in mouse neurovirulence and 2 deletion mutants that were lethal for virus (Sirigulpanit et al, 2007;Leardkamolkarn et al, 2010) to be incorporated in the replicons (Table 1), and evaluated whether the mutation effects could be measured using the replicon reporter system in vitro. Positive GFP expressions were observed in cells transfected with all 4 types of Δ-DENV/GFP replicons containing 5′-55M, 5′-60M, 5′-14M, 5′-15M, 5′-57M, but the positive cell numbers were less than those of the Δ-DENV/GFP replicons with wt 16681 5′NCR at 48 h pt (Fig.…”

“…Sequences of 5′NCR together with capsid protein gene and 3′NCR are required to structurally interact during virus replication (Alvarez et al, 2005;You et al, 2001). The functional significance of dengue 5′NCR is demonstrated by the restricted growth of DENV-4 deletion mutants (Cahour et al, 1995), attenuation effect of the DENV-2 PDK-53 vaccine candidate (Butrapet et al, 2000), and reduced virus replication of DENV-2 5′NCR mutants (Leardkamolkarn et al, 2010;Sirigulpanit et al, 2007).…”