Rescue of very virulent and mosaic infectious bursal disease virus from cloned cDNA: VP2 is not the sole determinant of the very virulent phenotype

Many recent outbreaks of infectious bursal disease in commercial chicken fl ocks worldwide are due to the spread of very virulent strains of infectious bursal disease virus (vvIBDV). The molecular determinants for the enhanced virulence of vvIBDV compared to classical IBDV are unknown. The lack of a reverse genetics system to rescue vvIBDV from its cloned cDNA hampers the i dentification and study of these determinants. In this report we describe, fur the first time, the rescue of vvIBDV from its cloned cDNA, Two plasmids containing a T7 promoter and either the full-length A- or B-segment cDNA o f vvIBDV (D6948) were cotransfected into QM5 cells expressing T7 polymerase , The presence of vvIBDV could be detected after passage of the transfectio n supernatant in either primary bursa cells (in vitro) or embryonated eggs (in vivo), but not QM5 cells. Rescued vvIBDV (rD6948) appeared to have the same virulence as the parental isolate, D6948, Segment-reasserted IBDV, in which one of the two genomic segments originated from cDNA of classical att enuated IBDV CEF94 and the other from D6938, could also be rescued by using this system, Segment-reasserted virus containing the A segment of the clas sical attenuated isolate (CEF94) and the B segment of the very virulent iso late (D6948) is not released until 15 h after an in vitro infection, This i ndicates a slightly retarded replication, as the first release of CEF94 is already found at 10 h after infection, Next to segment reassortants, we gen erated and analyzed mosaic IBDVs (mIBDVs), In these mIBDVs we replaced the region of CEF94 encoding one of the viral proteins (pVP2, VP3, or VP4) by t he corresponding region of D6948, Analysis of these mIBDV isolates showed t hat tropism for non-B-lymphoid cells was exclusively determined by the vira l capsid protein VP2, However, the very virulent phenotype was not solely d etermined by this protein, since mosaic virus containing VP2 of vvIBDV indu ced neither morbidity nor mortality in young chickens.