Cells and methodology to generate non-segmented negative-strand RNA viruses

摘要

The present invention relates to recombinant cells as well as to methods for the generation of non-segmented negative-sense single-stranded RNA viruses (NNV or mononegavirales) from cloned deoxyribonucleic acid (cDNA), especially from measles virus and in particular from attenuated strains such as those approved for vaccination, in particular from the attenuated Schwarz measles virus and various recombinant Schwarz measles-based viruses expressing heterologous sequences. Such rescued viruses can be used, after amplification, as vaccines for immunization against measles and/or against the heterologous peptides or proteins expressed.

主权项

1. A method to produce an infectious non-segmented negative-strand RNA virus, comprising:
a) providing a cell line comprising:
i) a stably integrated retroviral vector comprising a coding sequence for an RNA polymerase;ii) a stably integrated retroviral vector comprising a coding sequence for a nucleoprotein (N) of a non-segmented negative-strand RNA virus; andiii) a stably integrated retroviral vector comprising a coding sequence for a polymerase cofactor phosphoprotein (P) of a non-segmented negative-strand RNA virus;wherein the cell line stably expresses the RNA polymerase, N and P proteins; b) transfecting the cell line of a) with (i) a vector comprising the coding sequence for a RNA polymerase large protein (L) of a non-segmented negative-strand RNA virus, and (ii) a vector comprising a cDNA clone of a non-segmented negative-strand RNA virus; c) combining transfected cells of b) with cells competent to sustain the replication and production of the non-segmented negative-strand RNA virus to form a co-culture; and d) recovering the infectious non-segmented negative-strand RNA virus from the co-culture; wherein the method does not comprise continuous selection.