DNA transfection system for the generation of infectious influenza virus
The present invention is based on the development of a dual promoter system (preferably a RNA pol I-pol II system) for the efficient intracellular synthesis of viral RNA. The resultant minimal plasmid-based system may be used to synthesize any RNA virus, preferably viruses with a negative single stranded RNA genome. The viral product of the system is produced when the plasmids of the system are introduced into a suitable host cell. One application of the system is production of attenuated, reassortant influenza viruses for use as antigens in vaccines. The reassortant viruses generated by cotransfection of plasmids may comprise genes encoding the surface glycoproteins hemagglutinin and neuramimidase from an influenza virus currently infecting the population and the internal genes from an attenuated influenza virus. An advantageous property of the present invention is its versatility; the system may be quickly and easily adapted to synthesize an attenuated version of any RNA virus. Attenuated or inactivated RNA viruses produced by the present invention may be administered to a patient in need of vaccination by any of several routes including intranasally or intramuscularly.
1. A composition which generates infectious influenza viruses from cloned viral cDNA comprising a set of plasmids wherein each plasmid comprises one viral genomic segment, and wherein viral cDNA corresponding to the viral genomic segment is inserted between an RNA polymerase I (pol I) promoter and a regulatory element for the synthesis of vRNA or cRNA with an exact 3′ end, which results in expression of vRNA or cRNA, which are in turn inserted between an RNA polymerase II (pol II) promoter and a polyadenylation signal, which results in expression of viral mRNA and a corresponding viral protein, wherein the expression of the full set of vRNAs or cRNAs and viral proteins results in assembly of an infectious influenza virus.
2. The composition of claim 1 , wherein the regulatory element for the synthesis of vRNA or cRNA with an exact 3′ end is an RNA polymerase I (pol I) terminator sequence.
3. The composition of claim 1 , wherein the regulatory element for the synthesis of vRNA or cRNA with an exact 3′ end is a ribozyme sequence.
4. The composition of claim 2 , wherein the pol I promoter is proximal to the polyadenylation signal and the pol I terminator sequence is proximal to the pol II promoter.
5. The composition of claim 2 , wherein the pol I promoter is proximal to the pol II promoter and the pol I terminator sequence is proximal to the polyadenylation signal.
6. The composition of claim 1 , wherein the influenza virus is an influenza A virus.
7. The composition of claim 1 , wherein the influenza virus is an influenza B virus.
8. The composition of claim 6 , wherein the viral genomic segment (i) encodes a protein selected from the group consisting of a viral polymerase complex protein, M protein and NS protein; and (ii) is derived from a strain well adapted to grow in cell culture or from an attenuated strain, or both.
9. The composition of claim 6 , wherein the viral genomic segment comprises hemagglutinin (HA) gene, or neuramimidase (NA) gene, or both; wherein said genes are from a pathogenic influenza virus.
10. The composition of claim 6 wherein said composition comprises one or more plasmids having a map selected from the group consisting of pHW241-PB2, pHW242-PB1, pHW243-PA, pHW244-HA, pHW245-NP, pHW246-NA, pHW247-M, and pHW248-NS.
11. The composition of claim 6 , wherein said composition comprises one or more plasmids having a map selected from the group consisting of pHW181-PB2, pHW182-PB1, pHW183-PA, pHW184-HA, pHW185-NP, pHW186-NA, pHW187-M, and pHW188-NS.
12. A host cell comprising the composition of claim 1 .
13. A host cell comprising the composition of claim 7 .
14. A host cell comprising the composition of claim 6 .
15. A host cell comprising the composition of claim 9 .
16. A method for producing an infectious influenza virus virion, which method comprises culturing the host cell of claim 12 under conditions that permit production of viral proteins and vRNA or cRNA, whereby an infectious influenza virus is produced.
17. A method for producing a pathogenic influenza virion, which method comprises culturing the host cell of claim 15 under conditions that permit production of viral proteins and vRNA or cRNA, whereby a pathogenic infectious influenza virus is produced.
18. A method for generating an attenuated influenza virus, which method comprises:
(a) mutating one or more viral genes in the composition of claim 1 ; and
(b) determining whether infectious influenza viruses produced by the composition upon introduction into a suitable host cell are attenuated.