Rapid and efficient recovery of Sendai virus from cDNA: factors influencing recombinant virus rescue.
In a comparative study the factors influencing the recovery of recombinant Sendai viruses (SeV) from plasmid based cDNA were analysed systematically in order to establish an efficient and robust method for virus rescue. The amounts and ratios of transfected helper plasmids encoding the viral N, P and L proteins proved to be crucial for virus rescue, and they were optimised step-by-step for enhanced virus release. When the C open reading frame from the P gene was expressed at low level, virus rescue was generally possible but virus release could be improved when C gene expression was abolished completely. SeV particle formation could be increased greatly when the transcription initiation site for T7 polymerase in the cDNA was modified or when the genomic ribozyme instead of the antigenomic ribozyme of hepatitis delta virus was used for processing the 3'end of the viral RNA transcript. Heterologous helper viruses vTF7-3 and MVA-T7, which are necessary for T7 polymerase production in transfected cells, were compared for their use in SeV recovery and subsequent elimination of the helper virus from recombinant SeV. Interference with SeV replication was less severe with MVA-T7, and MVA-T7 was eliminated efficiently without the need for any inhibitors by serial passages in Vero cells. Optimal combination of all parameters led to a highly efficient generation of recombinant SeV from cDNA. Titres of the released virus particles are high enough to enable analysis of the recombinant SeV directly on test cells or propagation in cell cultures without the need for amplification in embryonated chicken eggs. The system is very robust and allows rapid generation of defined SeV mutants that require specialised host cells for propagation.
Leyrer
S
S
Max-Planck-Institut für Biochemie, Abteilung Virusforschung, Martinsried, Germany.
Neubert
W J
WJ
Sedlmeier
R
R
D000818:Animals; D002642:Chick Embryo; D002522:Chlorocebus aethiops; D018076:DNA, Complementary; D004279:DNA, Viral; D006367:HeLa Cells; D006801:Humans; D011995:Recombination, Genetic; D018108:Respirovirus / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology; D013997:Time Factors; D014709:Vero Cells; D014762:Viral Interference
D018076:DNA, Complementary; D004279:DNA, Viral