Full-length infectious clone of a low passage dengue virus serotype 2 from Brazil

Abstract

Full-length dengue virus (DENV) cDNA clones are an invaluable tool for many studies, including those on the development of attenuated or chimeric vaccines and on host-virus interactions. Furthermore, the importance of low passage DENV infectious clones should be highlighted, as these may harbour critical and unique strain-specific viral components from field-circulating isolates. The successful construction of a functional Brazilian low passage DENV serotype 2 full-length clone through homologous recombination reported here supports the use of a strategy that has been shown to be highly useful by our group for the development of flavivirus infectious clones and replicons.

Fig. 1:. construction of a low passage full-length dengue virus serotype 2 (DENV2) infectious clone named pSVJS01-DENV2. Five fragments within the DENV2 genome were amplified and inserted into the shuttle vector pSVJS01 through homologous recombination in yeast. E: envelop; F: fragment; NS: nonstructural; UTR: untranslated region.

Fig. 2:. in vitro characterisation of pSVJS01-DENV2 virus. A: detection of dengue virus serotype 2 (DENV2) in baby hamster kidney (BHK)-21 cells transfected with RNA derived from pSVJS01-DENV2 clone 10 by indirect immunofluorescence assay. Cells were collected at five days post-transfection and labelled with anti-flavivirus group B as the primary antibody and anti-mouse IgG-fluorescein isothiocyanate as the secondary antibody; B, C: plaque phenotypes of the wild type DENV2 (BR-3808) and the virus derived from pSVJS01-DENV2. BHK-21 cells were infected with the different viruses for five days at 37ºC. Plaque formation was revealed by immunoperoxidase assay; B: DENV2 BR-3808; C: pSVJS01-DENV2 clone 10.

Fig. 3:. growth curve analysis of parental (BR-3808) and infectious clone-derived dengue virus serotype 2 (DENV2) (pSVJS01) assessed by quantitative real-time polymerase chain reaction. Viral RNA was quantified in baby hamster kidney-21 cells infected with either viruses at 0 h, 24 h, 48 h and 72 h post-infection.