System producing infectious recombinant influenza virus
The invention provides compositions and methods useful to prepare segmented, negative strand RNA viruses, e.g., orthomyxoviruses such as influenza A viruses, entirely from cloned cDNAs and in the absence of helper virus.
1. A recombinant influenza viral system comprising:
a) expression vectors comprising vector comprising a promoter operably linked to an influenza virus PA antisense cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus PB1 antisense cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus PB2 antisense cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus HA antisense cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus NP antisense cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus M antisense cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus NA antisense cDNA linked to a transcription termination sequence, and a vector comprising a promoter operably linked to an influenza virus NS antisense cDNA linked to a transcription termination sequence, and
(b) protein expression vectors, comprising a vector comprising a promoter operably linked to a DNA segment encoding an influenza virus PA polypeptide, a vector comprising a promoter operably linked to a DNA segment encoding an influenza virus PB1 polypeptide, a vector comprising a promoter operably linked to a DNA segment encoding an influenza virus PB2 polypeptide, and a vector comprising a promoter operably linked to a DNA segment encoding an influenza virus NP polypeptide;
wherein upon expression of vectors of (a) and (b) in a host cell, functional viral ribonucleoproteins (RNP) comprising the genomic vRNA segments of recombinant influenza virus are formed, and
wherein said system does not comprise a helper virus.
2. An isolated host cell comprising the system of claim 1 .
3. The isolated host cell of claim 2 , wherein the host cell is capable of producing an infectious recombinant influenza virus.
4. A method for producing an infectious influenza virus comprising culturing the isolated host cell of claim 2 under conditions that permit the production of the recombinant influenza virus.
5. The isolated host cell of claim 2 , wherein the host cell is augmented to express at least one DNA molecule encoding a protein selected from the group consisting of PA, PB1, PB2, HA, NP, M1, M2, NA, NS1 and NS2.
6. An immunogenic composition comprising an infectious influenza virus produced by the system of claim 1 .
7. The system of claim 1 , comprising an expression vector encoding a reporter gene.
8. The system of claim 1 , wherein at least one expression vector of (a) comprises a mutated influenza gene.
9. The system of claim 1 , wherein at least one expression vector of (a) comprises a FLAG epitope.
10. The system of claim 1 , wherein expression vectors of (a) comprise an RNA polymerase I (polI) promoter and a pol I termination sequence.
11. An immunogenic composition comprising an infectious recombinant influenza virus produced by the system of claim 1 .