An efficient helper-virus-free method for rescue of recombinant paramyxoviruses and rhadoviruses from a cell line suitable for vaccine development
| Autor: | J. Erik Johnson, Stephen A. Udem, R. Michael Hendry, Cheryl S. Kotash, Christopher L. Parks, Aaron S. Abramovitz, Krista J. Melville, Sannyu G. Heron, Susan E. Witko, Mohinder S. Sidhu, David K. Clarke, Rebecca M. Nowak, Lee Anne C. Boutilier |
|---|---|
| Rok vydání: | 2006 |
| Předmět: |
viruses
DNA Recombinant Biology Transfection Vaccines Attenuated Virus Replication Recombinant virus Vesicular stomatitis Indiana virus law.invention Viral Proteins chemistry.chemical_compound law Virology RNA polymerase Chlorocebus aethiops medicine Animals T7 RNA polymerase Vero Cells Polymerase Vaccines Synthetic Viral matrix protein Viral Vaccine Viral Vaccines DNA-Directed RNA Polymerases chemistry Helper virus DNA Viral Mutation Recombinant DNA biology.protein Paramyxovirinae RNA Viral Helper Viruses Plasmids medicine.drug |
| Zdroj: | Journal of Virological Methods. 135:91-101 |
| ISSN: | 0166-0934 |
| DOI: | 10.1016/j.jviromet.2006.02.006 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full Text from ScienceDirect