Novel method for generation of rna virus
AssigneeAvir Green Hills Biotechnology Research Development Trade AG
InventorsThomas Muster, Andrej Egorov, Markus Wolschek
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Abstract
The present invention provides a method for generating negative-stranded segmented RNA viruses using linear expression constructs in the presence of helper virus which comprises at least one amino acid modification within the N-terminal cyto plasmic region of the NA protein.
Claims23
A method for production of negative stranded segmented RNA virus particles, comprising the steps of:
- a) providing a linear expression construct free of any amplification and/or selection sequences, which construct comprises an RNA polymerase I (polI) promoter and a polI termination signal, both inserted between an RNA polymerase II (polII) promoter and a polyadenylation signal, which construct further comprises a HA and/or a NA gene segment inserted between the polI promoter and the polI termination signal,
- b) transfecting a host cell with said linear expression construct,
- c) infecting said host cell with a helper virus having helper virus HA and/or NA proteins, wherein said NA protein comprises at least one amino acid modification within the N-terminal cytoplasmic domain,
- d) cultivating said host cell to propagate virus particles,
- e) selecting the virus particles, wherein said virus particles comprise:
- (i) the HA and/or NA proteins derived from the linear expression construct, but do not comprise
- (ii) the helper virus HA and NA proteins, or segments thereof,
- wherein said selection is based on phenotypic, genotypic or antigenic properties of the HA and/or NA proteins.
A helper virus comprising a HA protein with an elastase cleavage site and a NA protein with at least one amino acid modification within the N-terminal cytoplasmic domain.
A helper virus, wherein HA and NA proteins of the helper virus originate from influenza A/New Caledonia/20/99 strain and wherein the HA protein comprises an elastase cleavage site and the NA protein comprises a deletion of the terminal amino acids 2 to 6.
Dependencies are inferred from the claim text.