Increased virulence of Marek's disease virus type 1 vaccine strain CVI988 after adaptation to QT35 cells

CVI988/Rispens strain of Marek's disease virus type 1 (MDV-1) is widely use d as efficient vaccine to control Marek's disease (MD) in chicken flocks. S imilarly to other live MD vaccine viruses it is propagated in freshly prepa red chicken embryo fibroblasts (CEF). In this study, MDV-1 CVI988/Rispens s train was adapted to QT35 cells. The adapted virus, designated QT-CVI, exhi bited similar cytopathic effect (CPE) in vitro to that of parental virus pr opagated in CEF. In contrast, QT-CVI induced MD symptoms typical for mild M DV-1 strains after injection to birds. For identification of differentially expressed transcripts that might be involved in increased virulence of QT- CVI, we performed subtractive suppression hybridization (SSH). Subtracted P CR products mapped within MDV-1 BamHI-A and -H fragments and differential g ene expression was also confirmed by Northern blot analysis with probes der ived from these regions. To examine possible divergence at the virus genome level, PCR analysis was carried out. The BamHI-H fragment-specific 132 bp repeats were present at variable copy number, ranging from 2 to more than 3 0 copies in both CVI988/Rispens and QT-CVI DNAs. PCR assays with primers ma pping at the U-S/IRS junction identified CVI988/Rispens-specific insertion of 116 bp in the region upstream of the ICP4 open reading frame (ORF). PCR analysis was positive also for DNA from non-infected QT35 cells and was con sistent with the observation of Yamaguchi et al. (J Virol. 74, 10176-10186, 2000) who have found that QT35 cells carry a latent MDV-1 genome. It is li kely, that adaptation of CVI988/Rispens to QT35 cells resulted in reactivat ion of an endogenous MDV-1 or at least in induction of expression of virule nce-related transcripts that have consequently led to QT-CVI pathogenicity for chickens.