Supplementary Material for: Generation of a DNA-Launched Reporter Replicon Based on Dengue Virus Type 2 as a Multipurpose Platform

Dengue viruses (DENV) have become the most important arthropod-borne
viruses, causing dengue and severe dengue fever in at least 50-100
million cases each year, mainly in tropical and subtropical countries.
During recent years, important advances in the molecular biology
concerning the life cycle of these viruses have allowed the manipulation
and generation of recombinant viruses and replicons with multiple
applications, mainly in viral biology and the screening of antiviral
compounds. In the present study, we describe the construction of an
enhanced green fluorescent protein-bearing DENV replicon under the
control of the cytomegalovirus immediate early promoter. Following a
rational in silico design and cloning by standard molecular biology
techniques, a reporter DENV-2 replicon and a replication-deficient
mutant were constructed, and characterized by confocal microscopy and
real-time RT-PCR. The results showed successful transcription,
translation, and autonomous viral RNA replication of the DENV replicon
from its DNA clone. This novel DENV replicon will allow the study of
viral replication and testing of antiviral candidates without the need
for in vitro transcription.