Abstract
Most live-attenuated tetravalent dengue virus vaccines in current clinical trials are produced from Vero cells. In a previous study we demonstrated that an infectious cDNA clone-derived dengue type 4 (DEN-4) virus retains higher genetic stability in MRC-5 cells than in Vero cells. For this study we investigated two DEN-4 viruses: the infectious cDNA clone-derived DEN-4 2A and its derived 3′ NCR 30-nucleotide deletion mutant DEN-4 2AΔ30, a vaccine candidate. Mutations in the C-prM-E, NS2B-NS3, and NS4B-NS5 regions of the DEN genome were sequenced and compared following cell passages in Vero and MRC-5 cells. Our results indicate stronger genetic stability in both viruses following MRC-5 cell passages, leading to significantly lower RNA polymerase error rates when the DEN-4 virus is used for genome replication. Although no significant increases in virus titers were observed following cell passages, DEN-4 2A and DEN-4 2AΔ30 virus titers following Vero cell passages were 17-fold to 25-fold higher than titers following MRC-5 cell passages. Neurovirulence for DEN-4 2A and DEN-4 2AΔ30 viruses increased significantly following passages in Vero cells compared to passages in MRC-5 cells. In addition, more severe DEN-induced hemorrhaging in mice was noted following DEN-4 2A and DEN-4 2AΔ30 passages in Vero cells compared to passages in MRC-5 cells. Target mutagenesis performed on the DEN-4 2A infectious clone indicated that single point mutation of E-Q438H, E-V463L, NS2B-Q78H, and NS2B-A113T imperatively increased mouse hemorrhaging severity. The relationship between amino acid mutations acquired during Vero cell passage and enhanced DEN-induced hemorrhages in mice may be important for understanding DHF pathogenesis, as well as for the development of live-attenuated dengue vaccines. Taken together, the genetic stability, virus yield, and DEN-induced hemorrhaging all require further investigation in the context of live-attenuated DEN vaccine development.
Highlights
The four dengue serotype viruses DEN-1 to dengue type 4 (DEN-4) are single stranded, positive-sense RNA viruses transmitted to humans primarily by Aedes aegypti mosquitoes [1]
Based on 74% of DEN genome sequencing, our results indicate stronger genetic stability for the infectious cDNA clone-derived viruses DEN-4 2A or DEN-4 2AD30 following passages in MRC-5 cells compared to passages in Vero cells
In an earlier study we examined DEN-4 2A virus mutations following Vero and MRC-5 cell passages by sequencing multiple clones of DNA fragments synthesized from DEN-4 2A RNA by RT-PCR [34]
Summary
The four dengue serotype viruses DEN-1 to DEN-4 (genus Flavivirus, family Flaviviridae) are single stranded, positive-sense RNA viruses transmitted to humans primarily by Aedes aegypti mosquitoes [1]. Their shared RNA genome contains coding sequences for three structural protein genes (core C, precursor membrane prM, and envelope E), seven non-structural protein genes (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5), and two flanking non-translating regions (NTRs) [2]. DEN infections in humans result in illnesses ranging from dengue fever (DF) to dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Increases in DEN-related diseases in the past two decades are likely the result of growing human populations, rapid urbanization, the effects of global warming on mosquito vector control, and expanded international travel [9]
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