Accidental acquisition of a rescued Japanese encephalitis virus with unspliced introns in the viral genome when using an intron-based stabilization approach

The intron-based stabilization approach is a very useful strategy for construction of stable flavivirus infectious clones. SA(14)-14-2 is a highly attenuated Japanese encephalitis (JE) live vaccine strain that has been widely used in China since 1989. To develop safe and effective recombinant vaccines with SA(14)-14-2 as a backbone vector, we constructed the DNA-based infectious clone pCMW-JEV of SA(14)-14-2 using the intron-based stabilization approach and acquired the rescued virus rDJEV, which retained the biological properties of the parental virus. Unexpectedly, a rescued virus strain with altered virulence, designated rHV-DJEV, was accidentally acquired in one of the transfection experiments. rHV-DJEV showed up to 10(5)-fold increased neurovirulence compared with the SA(14)-14-2 parental strain. Genome sequencing showed that the inserted introns were still present in the genome of rHV-DJEV. Therefore, we think that the intron-based stabilization approach should be used with caution in vaccine development and direct iDNA immunization.