# US 2013/0183740 A1 · Novel method for generation of rna virus > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2013/0183740 A1 - Document ID: US20130183740A1 - Status: Published application - Kind: A1 - Application number: 13/701,164 - Filed: 2011-06-06 - Published: 2013-07-18 - Earliest claimed: 2010-06-02 - Assignees: Avir Green Hills Biotechnology Research Development Trade AG - Inventors: Thomas Muster; Andrej Egorov; Markus Wolschek - Web page: https://patentgrep.com/patent/US20130183740A1 ## CPC - C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00) - C12N 7/02: Recovery or purification - C12N 15/86: Viral vectors - C07K 2319/50: containing protease site - C12N 2760/00011: Details - C12N 2760/00052: relating to complementing cells and packaging systems for producing virus or viral particles - C12N 2760/16111: Influenzavirus A, i.e. influenza A virus - C12N 2760/16134: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein - C12N 2760/16152: relating to complementing cells and packaging systems for producing virus or viral particles - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) ## 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. ## Claims ### Claim 1 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. ### Claim 2 (depends on 1) A method according to claim 1, wherein the host cell is transfected with linear constructs encoding a proteins selected from the group consisting of PB1, PB2, PA, NS, M, and NP. ### Claim 3 (depends on 1) A method according to claim 1, wherein progeny virus particles comprising HA protein derived from the helper virus are separated from candidate virus particles by treating progeny virus particles with a protease, and wherein the protease does not cleave HA protein derived from the helper virus but cleaves the HA protein of the candidate virus particles ### Claim 4 (depends on 1) A method according to claim 1, wherein the protease is selected from the group consisting of trypsin, elastase, chymotrypsin, and papain. ### Claim 5 (depends on 1) A method according to claim 1, wherein the HA protein of the helper virus is modified to be cleaved by a protease, and wherein said protease is not trypsin. ### Claim 6 (depends on 1) A method according to claim 1, wherein progeny virus particles comprising HA and NA proteins of helper virus origin are separated from candidate virus particles by providing low pH conditions. ### Claim 7 (depends on 1) A method according to claim 1, wherein progeny virus particles comprising HA and/or NA proteins of helper virus origin are separated from candidate virus particles by contacting the progeny virus particles with antibodies binding said HA and/or NA proteins. ### Claim 8 (depends on 1) A method according claim 1, wherein the helper virus NA protein comprises a deletion of at least one of N-terminal amino acids 1 to 6 in comparison to the sequence according to the numbering of SEQ ID NO:10. ### Claim 9 (depends on 1) A method according to claim 1, wherein the helper virus NA protein comprises a deletion of N-terminal amino acids 2 to 6 in comparison to the sequence according to the numbering of SEQ ID NO:10. ### Claim 10 (depends on 1) A method according to claim 1, wherein the helper virus comprises NA protein with reduced activity, or which lacks a functional NA protein. ### Claim 11 (depends on 1) A method according to claim 1, wherein the helper virus comprises the HEF protein of influenza C virus. ### Claim 12 (depends on 11) A method according to claim 11, wherein the HEF protein of the helper virus is modified to be cleaved by a protease, and wherein said protease is not trypsin. ### Claim 13 (depends on 1) A method according to claim 1, wherein the helper virus comprises the HA protein of a coronavirus. ### Claim 14 (depends on 1) A method according to claim 1, wherein said virus particle is an influenza virus particle. ### Claim 15 (depends on 1) A method according to claim 1, wherein said virus particle is an attenuated influenza virus particle. ### Claim 16 (depends on 1) A method according to claim 1, wherein said virus comprises a deletion or modification within the NS1 gene. ### Claim 17 (depends on 1) A method according to claim 1, wherein the helper virus comprises a modified or deleted NS gene and is growth deficient in interferon competent cells. ### Claim 18 (depends on 1) A method according to claim 1, wherein the helper virus contains at least 4 segments identical to the virus to be produced. ### Claim 19 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. ### Claim 20 (depends on 19) The helper virus according to claim 19, wherein the N-terminal amino acids 2 to 6 of said NA protein are deleted. ### Claim 21 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. ### Claim 22 (depends on 1) A method according to claim 1, wherein the absence of helper virus HA and NA proteins is determined by analysis of the nucleic acid or amino acid sequence of the HA and NA proteins. ### Claim 23 (depends on 18) A method according to claim 18, wherein the helper virus contains at least 6 segments identical to the virus to be produced.