A ‘minimal’ approach in design of flavivirus infectious DNA

Abstract

The 'infectious DNA' approach, which is based on in vivo transcription of (+)RNA virus genome cDNA cassettes from eukaryotic promoters in transfected cells, became a popular alternative to the classical scheme in the infectious clone methodology. Its use, however, is often limited by the instability of plasmids due to a transcriptional activity of eukaryotic promoters in Escherichia coli resulting in synthesis of products toxic for the bacterial host. Using a highly unstable representative infectious clone of Japanese encephalitis (JE) flavivirus, we tested a new approach in design of such problematic 'infectious DNA' constructs, which is based on minimizing unwanted transcription in the bacterial host. A plasmid containing full genome size JE cDNA under control of the minimal cytomegalovirus (CMV) promoter can be propagated in E. coli with growth and stability characteristics similar to that of constructs controlled by the T7 promoter. Transfection of this plasmid into susceptible cells leads to the establishment of a productive infectious cycle. Reinsertion of the CMV enhancer at the 3'-end of the JE cassette substantially increased the specific infectivity without affecting the stability and growth characteristics of the construct. This approach can be useful when stabilization of infectious clones by modification of a viral cDNA cassette is not the feasible or suitable alternative.

Development and Application of a Reverse Genetics System for Japanese Encephalitis Virus

“…Previous attempts to develop such a system (23,38,39,51), including our own, were thwarted by the instability of the cloned JEV cDNA. The system described in this paper will greatly facilitate the study of the molecular mechanisms of replication, virulence, and pathogenesis employed by the virus.…”

“…A variety of vector systems were used previously in attempts to assemble a full-length JEV cDNA (23,38,39,51), including a cosmid vector in E. coli (51). In all cases, the cloned cDNA became genetically unstable during its propagation in a host.…”

“…However, the construction of a full-length infectious cDNA clone for Japanese encephalitis virus (JEV) has been hampered, largely because of the genetic instability of the cloned cDNA. Despite extensive efforts (23,38,39,51), a genetically stable full-length infectious cDNA molecular clone for JEV does not exist.…”

Development of a human live attenuated West Nile infectious DNA vaccine

“…When used directly for inoculation, an iDNA vaccine may lead to early activation of IL-12, TNFα, and IFNα through TLR9, thus bypassing mechanisms of viral counterdefense. We have shown earlier Mishin et al, 2001;Yamshchikov et al, 2004) that viruses are detectable in the media 12 h after transfection with corresponding iDNA. On the other hand, DNA of bacterial origin (for example, a plasmid produced in E. coli) was shown to induce inflammatory cytokine responses within hours after inoculation (Klaschik et al, 2010;Klinman et al, 1996), which peak at 12 h. Thus, the infectious vaccine virus emerging from iDNA encounters the innate response already activated by inflammatory cytokines, which in effect may create an additional safety barrier against runaway infection.…”

Construction of an infectious molecular clone of Japanese encephalitis virus genotype V and its derivative subgenomic replicon capable of expressing a foreign gene

“…To overcome this problem, several approaches have been employed; such as the use of in vitro ligated cDNA, cosmid vector, bacterial artificial chromosome and chemical synthesized cDNA (Li et al, 2014;Mishin et al, 2001;Pu et al, 2011;Sumiyoshi et al, 1995Sumiyoshi et al, , 1992Tajima et al, 2010;Yamshchikov et al, 2001;Yun et al, 2003;Zhang et al, 2001). As to JEV, infectious molecular clones of genotypes I and III have been established so far, and their usefulness in studying the viral replication mechanism has been demonstrated.…”