%0 Journal Article %A Varma, Satya Pavan Kumar %A Kumar, Amit %A Kapur, Neeraj %A Durgapal, Hemlata %A Acharya, Subrat Kumar %A Panda, Subrat Kumar %T Hepatitis E virus replication involves alternating negative- and positive-sense RNA synthesis %D 2011 %J Journal of General Virology, %V 92 %N 3 %P 572-581 %@ 1465-2099 %R https://doi.org/10.1099/vir.0.027714-0 %I Microbiology Society, %X Hepatitis E virus (HEV) is the major cause of epidemic hepatitis and many outbreaks of sporadic hepatitis. The virus responsible has a single-stranded, positive-sense RNA. Its replication and the regulatory process involved therein are poorly understood. Much of the HEV biology studied has been done by using full-length capped RNA transcripts (replicons) and transient transfections in cell cultures. We investigated replicon replication using negative-sense strand-specific molecular beacons in live cell imaging, and quantifying intracellular viral RNA using strand-specific real-time PCR every 2 h until 24 h post-transfection. A graph of the copy numbers of both positive- and negative-sense RNA at the different time points was plotted. This showed a temporal separation and alternating cycles of negative- and positive-sense RNA formation. As a control, a dysfunctional replicase mutant (GDD→GAA) was used, which showed no increase in copy number. The live cell imaging corroborated the quantitative data, in that the maximal amount of negative-sense RNA was observed at 8 h post-transfection. The real-time-PCR copy-number analysis of the subgenome showed the presence of a single subgenomic RNA. Using fluorescent protein genes mCherry and EGFP fused in-frame to ORF2 and ORF3 in separate constructs and immunofluorescence, we showed the formation of both proteins pORF2 and pORF3 from a single subgenomic RNA. Our study demonstrated cyclical bursts of virus replication and the role of subgenomic RNA in the HEV life cycle. %U https://www.microbiologyresearch.org/content/journal/jgv/10.1099/vir.0.027714-0