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. 2014 Feb 13:180:12-22.
doi: 10.1016/j.virusres.2013.12.001. Epub 2013 Dec 14.

Development of a novel DNA-launched dengue virus type 2 infectious clone assembled in a bacterial artificial chromosome

Jose A Usme-Ciro  1 , Jaime A Lopera  2 , Luis Enjuanes  3 , Fernando Almazán  3 , Juan C Gallego-Gomez  4
Affiliations

Development of a novel DNA-launched dengue virus type 2 infectious clone assembled in a bacterial artificial chromosome

Jose A Usme-Ciro et al. Virus Res. .

Abstract

Major progress in Dengue virus (DENV) biology has resulted from the use of infectious clones obtained through reverse genetics. The construction of these clones is commonly based on high- or low-copy number plasmids, yeast artificial chromosomes, yeast-Escherichia coli shuttle vectors, and bacterial artificial chromosomes (BACs). Prokaryotic promoters have consistently been used for the transcription of these clones. The goal of this study was to develop a novel DENV infectious clone in a BAC under the control of the cytomegalovirus immediate-early promoter and to generate a virus with the fusion envelope-green fluorescent protein in an attempt to track virus infection. The transfection of Vero cells with a plasmid encoding the DENV infectious clone facilitated the recovery of infectious particles that increased in titer after serial passages in C6/36 cells. The plaque size and syncytia phenotypes of the recombinant virus were similar to those of the parental virus. Despite the observation of autonomous replication and the detection of low levels of viral genome after two passages, the insertion of green fluorescent protein and Renilla luciferase reporter genes negatively impacted virus rescue. To the best of our knowledge, this is the first study using a DENV infectious clone under the control of the cytomegalovirus promoter to facilitate the recovery of recombinant viruses without the need for in vitro transcription. This novel molecular clone will be useful for establishing the molecular basis of replication, assembly, and pathogenesis, evaluating potential antiviral drugs, and the development of vaccine candidates for attenuated recombinant viruses.

Keywords: Bacterial artificial chromosome; Dengue virus; Eukaryotic promoter; Flavivirus; Infectious cDNA; Reverse genetics.

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Figures

Fig. 1
Fig. 1
Strategy to assemble a DENV infectious cDNA clone as a BAC: A four-step cloning strategy, in which the overlapping cDNA fragments, termed overlapping PCR 1, PCR 2, PCR 3 and PCR 4, were sequentially cloned into the plasmid pBeloBAC11 and used to generate the infectious clone pBAC-DENV-FL. This plasmid includes the CMV promoter precisely joined to the DENV genome, followed by the hepatitis delta virus ribozyme (RZ) and the bovine growth hormone termination and polyadenylation sequences (BGH). The genomic positions of the relevant restriction sites used for the assembly of the full-length cDNA clone are illustrated at the top.
Fig. 2
Fig. 2
Phenotypic characterization of the rDENV-FL virus: (A) cytopathic effect (CPE) in C6/36 cells. C6/36 cells were mock infected, infected with the parental virus or infected during three passages with the rDENV-FL and subsequently analyzed for the induction of CPE through light microscopy. (B) CPE in Vero cells. Monolayers of Vero cells were mock infected, infected with the parental virus or the rDENV-FL and subsequently analyzed for the induction of CPE through light microscopy. (C) and (D) Plaque size phenotype. The plaque sizes of the rDENV-FL and the parental virus were analyzed as described in Section 2. Circles were drawn to delimitate the plaques and estimate the diameter, which were expressed in millimeters as the mean ± standard deviation. V1C2 corresponds to the history of the virus rescue (in this case, one passage in Vero cells and two passages in C6/36 cells).
Fig. 3
Fig. 3
Specific detection of rDENV-FL virus: (A) Envelope protein detection. C6/36 cells were mock infected or infected with rDENV-FL passage 2 and subsequently the envelope protein expression was analyzed through indirect immunofluorescence using the polyclonal antibody 2133 and a secondary antibody conjugated to Alexa Fluor® 488. Nuclei were stained with Hoechst. (B) Viral genome detection. Supernatants of the serial passages of rDENV-FL in C6/36 cells were used for viral RNA extraction and RT-qPCR quantification. The error bars represent the standard error of the mean (SEM). **p < 0.01.
Fig. 4
Fig. 4
Strategy for the construction of EGFP- and hRLuc-bearing DENV infectious clones: A three-step cloning strategy, in which three overlapping cDNA fragments (PCR1 to PCR3) were sequentially cloned into the plasmid pBeloBAC11 and used to generate the plasmid pBAC-ENV-GFP/hRLuc-NS1-3. After verifying the nucleotide sequence, the Sph I-Nsi I fragment was cloned into pBAC-DENV-FL to generate pBAC-DENV-FL-GFP and pBAC-DENV-FL-LUC. The genetic structure of the DENV infectious clones expressing EGFP or hRLuc, the position of relevant restriction sites and the cleavage site for host cell signalase are illustrated.
Fig. 5
Fig. 5
Characterization of the rDENV-FL-EGFP and rDENV-FL-LUC: (A) Viral genome quantification. The supernatants from passages 1 and 2 of the rDENV-FL-EGFP in C6/36 cells were used for viral RNA extraction and RT-qPCR quantification. (B) EGFP expression. Vero cells were mock transfected or transfected with the plasmid pBAC-DENV-FL-EGFP, fixed at 24 hpt, stained with Hoechst and visualized through confocal microscopy. (C) and (E) Viral genome replication analysis. Cellular lysates from Vero cells transfected with (C) pBAC-DENV-FL-EGFP or (E) pBAC-DENV-FL-LUC were collected at 24 and 48 hpt and used for total RNA extraction and RT-qPCR quantification of negative strand of the viral RNA. (D) Luciferase activity. Cellular lysates from Vero cells transfected with the plasmid pBAC-DENV-FL-LUC were collected at 20 and 44 hpt, and after the first passage in Vero cells, the luciferase activity was quantified. The values are given in Relative Luciferase Units (RLU). The error bars represent the SEM. *p < 0.05; **p < 0.01; ***p < 0.001.
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