Abstract
Passage of virus in different cell lines is possible to produce host-cell specific mutations, which may affect cell tropism and virus virulence. We have previously reported that a dengue type 4 virus vaccine candidate passaged in human fetal lung fibroblast (MRC-5) cells rapidly acquired a Glu345-Lys substitution in the envelope protein domain III (Liu et al., PLoS ONE, 2008;e1810). It was also reported that another Glu327Gly substitution of the same vaccine candidate was selected after three passages in fetal rhesus lung (FRhL) cells with reduced infectivity and immunogenicity in rhesus monkeys (Anez et al., J Virol. 2009;83(20): 10384-94). Therefore, single point mutation at Glu327Gly and Glu345Lys, respectively, was constructed in two infectious cDNA clones of the dengue type 4 virus vaccine candidates, DEN-4 2A and its derived 3’ NCR deletion mutant DEN-4 2AΔ30. Using PCR-mediated site-directed mutagenesis method, the infectious cDNA clone-derived Glu345-Lys mutants of DEN-4 2A and DEN-4 2AΔ30 were passaged in MRC-5 cells for three consecutive times. Similarly, the infectious cDNA clone-derived Glu327Gly DEN-4 2A and DEN-4 2AΔ30 were three time-passaged in Vero cells. Single point mutation of Glu345-Lys was found to revert to Glu345 when the virus was passaged in Vero cells. The Glu345-Lys substitution predicted using molecular modeling show the increase of more positive charges on the surface of E protein than the Glu327Gly substitution. The virulence of these recombinant mutant viruses were analyzed in newborn ICR mice. The neurovirulence of these mutated viruses was significantly less than the DEN-2 NGC strain and their parent strain virus. Virulence attenuation inducing by adaptation mutation implicated important information to the development of live-attenuated dengue vaccine.