Abstract
Japanese encephalitis (JE), an acute inflammatory disease of the brain, is caused by infection with Japanese encephalitis virus (JEV). Children are most susceptible to JEV, which is commonly found in Asia. JEV is an enveloped RNA virus that is genetically similar to West Nile, dengue, and yellow fever viruses. No specific antiviral drugs are currently available to treat JE disease, but four types of vaccine are licensed locally to prevent JEV infection. However, the disease continues to be an important global health priority. Of the four vaccines, the most widely used in JE-endemic countries is the live-attenuated vaccine SA14-14-2. In our previous work, we cloned a genomelength cDNA of SA14-14-2 and rescued recombinant viruses entirely from the cloned cDNA (pBac/SA14-14-2). In the present study, we engineered this functional SA14-14-2 cDNA to construct a self-replicating subgenomic replicon (pBac/SA14-14-2Rep) by deleting the coding region for its two viral envelope glycoproteins, prM and E. After transfection of BHK-21 cells with the replicon RNA transcribed in vitro from pBac/SA14-14-2Rep, a combination of confocal microscopic imaging and immunoblotting showed the transient replication of the replicon RNA in the cytoplasm of RNA-transfected cells. Moreover, transfection of the SA14-14-2 replicon RNA into a BHK-21 cell line stably expressing all three JEV structural proteins (C, prM and E) led to the production of singleround infectious particles that packaged the replicon RNA, with a titter of ~105 infectious units per ml. Our SA14-14-2-based replicon represents a valuable tool for studying JEV RNA replication in low-level bio containment facilities and provides a useful platform for developing new vaccine vectors against an array of human and animal pathogens.
Highlights
Japanese encephalitis virus (JEV) belongs to the genus Flavivirus in the family Flaviviridae [1]
We introduced an internal in-frame deletion of 2,001 nucleotides into pBac/SA14-14-2 to remove all of the coding sequence for the two viral surface glycoproteins prM and E, thereby creating pBac/SA14-14-2Rep (Figure 1a)
After transfection into permissive BHK-21 cells, we demonstrated the ability of the SA14-14-2Rep RNA to establish transient replication and gene expression, with no concomitant production of infectious particles
Summary
Japanese encephalitis virus (JEV) belongs to the genus Flavivirus in the family Flaviviridae [1]. JEV is an enveloped RNA virus that has an inner core composed of a plus-strand RNA genome and multiple copies of the capsid (C) protein; this core is surrounded by an outer lipid bilayer with 180 copies of the viral membrane (M) and envelope (E) proteins embedded in it [3,4]. The linear genomic RNA is approximately 11,000 nucleotides long [5] and contains a single long open reading frame (ORF) flanked by two short non-coding regions (NCRs) at the 5’ and 3’ ends that possess cis-acting RNA elements required for viral replication [6,7]. SA14-14-2 cDNA to construct a self-replicating subgenomic replicon (pBac/SA14-14-2Rep) by deleting the coding region for its two viral envelope glycoproteins, prM and E. Transfection oexf ptrheessSiAng14-a1ll4-t2hrreeeplJiEcoVnsRtrNuActuinrtaol a BHK-21 cell line stably proteins (C, prM and E)
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