Rapid generation of stable and sequence specific attenuated dengue viruses as vaccine candidates
Production
of full-length infectious clones of positive-sense RNA (+RNA) viruses has
greatly facilitated studies on the effects of specific mutations on viral replication
and pathogenesis. This has important future implications for the development of
attenuated viruses for vaccine use, with next-generation sequencing allowing an
in depth analysis of the potential for virus adaptation and evolution.
Recently, Aubry et al. (2014) described a simple method for the generation of
+RNA virus infectious clones; termed infectious-subgenomic-amplicons (ISA) [1]. This
approach uses overlapping double-stranded DNA fragments, usually derived by PCR,
which cover the whole viral genome. The cytomegalovirus promoter (pCMV)
is incorporated at the 5' end of the first fragment to drive transcription by cellular
DNA-dependant RNA polymerase II. Transfection of the dsDNA fragments into
mammalian cells resulted in recombination of the fragments to generate
full-length, infectious RNA. Here, this approach has been used to generate infectious
Dengue Virus (DENV), a +RNA virus of the flaviviridae family. DENV contains a
single open-reading-frame which is translated to yield a single polyprotein
comprising all 10 viral proteins, 3-structural and 7 non-structural. Viral
replication and infectivity is dependent on co- and post-translational
processing of the polyprotein by the host proteases furin and signalase,
in addition to a viral typsin-like serine protease encoded on non-structural
protein 3 (NS3). In this research, site-directed alanine mutagenesis was
performed on residues of NS3 surrounding the active-site of the enzyme and
subsequently incorporation into the DENV infectious clone. These residues
showed varied levels of activity compared to the wild-type enzyme when assayed
using in vitro enzyme kinetic assays. As polyprotein processing is vital for
DENV replication and infection, this research is aimed at determining specific
residues of NS3 which attenuate the virus. Deep sequencing of virus passaged
several times has then been used to examine the potential for the emergence of
escape mutants. Our goal in these studies is to develop a streamlined pathway
to the generation of stable sequence/motif specific attenuation of DENV as
vaccine candidates.