An efficient helper-virus-free method for rescue of recombinant paramyxoviruses and rhadoviruses from a cell line suitable for vaccine development.
Recovery of recombinant, negative-strand, nonsegmented RNA viruses from a genomic cDNA clone requires a rescue system that promotes de novo assembly of a functional ribonucleoprotein (RNP) complex in the cell cytoplasm. This is accomplished typically by cotransfecting permissive cells with multiple plasmids that encode the positive-sense genomic RNA, the nucleocapsid protein (N or NP), and the two subunits of the viral RNA-dependent RNA polymerase (L and P). The transfected plasmids are transcribed in the cell cytoplasm by phage T7 RNA polymerase (T7 RNAP), which usually is supplied by infection with a recombinant vaccinia virus or through use of a stable cell line that expresses the polymerase. Although both methods of providing T7 RNAP are effective neither is ideal for viral vaccine development for a number of reasons. Therefore, it was necessary to modify existing technology to make it possible to routinely rescue a variety of recombinant viruses when T7 RNAP was provided by a cotransfected expression plasmid. Development of a broadly applicable procedure required optimization of the helper-virus-free methodology, which resulted in several modifications that improved rescue efficiency such as inclusion of plasmids encoding viral glycoproteins and matrix protein, heat shock treatment, and use of electroporation. The combined effect of these enhancements produced several important benefits including: (1) a helper-virus-free methodology capable of rescuing a diverse variety of paramyxoviruses and recombinant vesicular stomatitis virus (rVSV); (2) methodology that functioned effectively when using Vero cells, a suitable substrate for vaccine production; and (3) a method that enabled rescue of highly attenuated recombinant viruses, which had proven refractory to rescue using published procedures.
Witko
Susan E
SE
Wyeth Vaccines Research, 401 North Middletown Road, Pearl River, NY 10965, USA.
Kotash
Cheryl S
CS
Nowak
Rebecca M
RM
Johnson
J Erik
JE
Boutilier
Lee Anne C
LA
Melville
Krista J
KJ
Heron
Sannyu G
SG
Clarke
David K
DK
Abramovitz
Aaron S
AS
Hendry
R Michael
RM
Sidhu
Mohinder S
MS
Udem
Stephen A
SA
Parks
Christopher L
CL
D000818:Animals; D002522:Chlorocebus aethiops; D004274:DNA, Recombinant; D004279:DNA, Viral; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D006378:Helper Viruses / Q000235:genetics; D009154:Mutation; D018098:Paramyxovirinae / Q000235:genetics / Q000302:isolation & purification*; D010957:Plasmids / Q000235:genetics; D012367:RNA, Viral / Q000378:metabolism; D014162:Transfection; D014613:Vaccines, Attenuated / Q000235:genetics; D014614:Vaccines, Synthetic / Q000235:genetics; D014709:Vero Cells; D014721:Vesicular stomatitis Indiana virus / Q000235:genetics / Q000302:isolation & purification*; D014764:Viral Proteins / Q000235:genetics; D014765:Viral Vaccines / Q000235:genetics*; D014779:Virus Replication / Q000235:genetics / Q000502:physiology
D004274:DNA, Recombinant; D004279:DNA, Viral; D012367:RNA, Viral; D014613:Vaccines, Attenuated; D014614:Vaccines, Synthetic; D014764:Viral Proteins; D014765:Viral Vaccines; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases