Viruses encoding mutant membrane protein

1. An isolated recombinant influenza B virus comprising a knockout mutation of the NB protein gene.

2. The isolated recombinant virus of claim 1 wherein the NB protein gene comprises at least two mutations, one of which is not in the transmembrane domain.

3. The isolated recombinant virus of claim 1 wherein at least one mutation is at the codon for the initiator methionine.

4. The isolated recombinant virus of claim 3 wherein at least one mutation encodes a substitution at the codon for the initiator methionine.

5. The isolated recombinant virus of claim 3 wherein at least one mutation in the mutant NB protein gene is a stop codon for the codon for the initiator methionine.

6. The isolated recombinant virus of claim 3 wherein at least one mutation in the mutant NB protein gene is a stop codon in the coding region for the membrane protein.

7. The isolated recombinant virus of claim 1 wherein the mutant NB protein gene comprises a deletion of one or more nucleotides.

8. The isolated recombinant virus of claim 7 wherein the deletion alters the reading frame for the NB protein.

9. The isolated recombinant virus of claim 1 wherein the mutant NB protein gene comprises an insertion of one or more nucleotides.

10. The isolated recombinant virus of claim 9 wherein the insertion alters the reading frame for the NB protein.

11. The isolated recombinant virus of claim 1 wherein the mutant NB protein gene comprises a deletion of one or more nucleotides and encodes an amino acid substitution.

12. The isolated recombinant virus of claim 1 wherein the mutant NB protein gene comprises an insertion of one or more nucleotides and encodes an amino acid substitution.

13. The isolated recombinant virus of claim 1 which further comprises a heterologous immunogenic protein of a pathogen or a therapeutic protein.

14. The isolated recombinant virus of claim 1 which further comprises a heterologous immunogenic protein gene of a pathogen or a therapeutic protein gene.

15. The isolated recombinant virus of claim 1 wherein the mutation does not alter the in vitro replication of the virus but is associated with attenuation of the virus in vivo.

16. A vaccine comprising the isolated recombinant virus of claim 1 .

17. A method of preparing a recombinant influenza B virus comprising a knockout mutation of the NB protein gene comprising:

(i) contacting a host cell with a plurality of influenza vectors so as to yield recombinant influenza virus, wherein the plurality of vectors comprises: a) vectors to generate vRNA, comprising a vector comprising a promoter operably linked to an influenza virus PA cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus PB1 cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus PB2 cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus HA cDNA linked to a transcription termination sequence, a vector comprising promoter operably linked to an influenza virus NP cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus cDNA for NB and NA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus M cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus NS cDNA linked to a transcription termination sequence, wherein the sequence of the cDNA for NB and NA comprises a knockout mutation in the NB sequence, and b) vectors to express influenza virus proteins, comprising a vector comprising a promoter operably linked to a DNA segment encoding influenza virus PA, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus PB1, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus PB2, and a vector comprising a promoter operably linked to a DNA segment encoding influenza virus NP, and optionally one or more vectors selected from a vector comprising a promoter operably linked to a DNA segment encoding influenza virus HA, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus NA, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus M, and a vector comprising a promoter operably linked to a DNA segment encoding influenza virus NS2; and

(ii) isolating the virus.

18. The method of claim 17 wherein the mutant NB protein gene encodes at least one amino acid substitution.

19. The method of claim 17 wherein the NB protein gene comprises at least two mutations, one of which is not in the transmembrane domain.

20. The method of claim 19 wherein at least one mutation in the mutant NB protein gene is a stop codon for the codon for the initiator methionine.

21. The method of claim 19 wherein at least one mutation in the mutant NB protein gene is a stop codon in the coding region for the NB protein.

22. The method of claim 17 wherein at least one mutation is at the codon for the initiator methionine.

23. The method of claim 17 wherein the mutant NB protein gene comprises a deletion of one or more nucleotides.

24. The method of claim 23 wherein the deletion alters the reading frame for the NB protein.

25. The method of claim 17 wherein the mutant NB protein gene comprises an insertion of one or more nucleotides.

26. The method of claim 25 wherein the insertion alters the reading frame for the NB protein.

27. The method of claim 17 wherein the mutant NB protein gene encodes a substitution at the initiator methionine.

28. The method of claim 17 wherein the mutant NB protein gene comprises a deletion of one or more nucleotides and encodes at least one amino acid substitution.

29. The method of claim 17 wherein the mutant NB protein gene comprises an insertion of one or more nucleotides and encodes an amino acid substitution.

30. Isolated virus prepared by the method of claim 17 .

31. An isolated host cell contacted with the virus of claim 1 or 30 .

32. A method to immunize a vertebrate, comprising: contacting the vertebrate with an effective amount of the recombinant virus of claim 1 .

33. The method of claim 32 wherein the vertebrate is an avian.

34. The method of claim 32 wherein the vertebrate is a mammal.

35. The method of claim 32 wherein the vertebrate is a human.

36. A composition comprising a plurality of influenza B vectors, comprising: a) vectors to generate vRNA, comprising a vector comprising a promoter operably linked to an influenza virus PA cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus PB1 cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus PB2 cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus HA cDNA linked to a transcription termination sequence, a vector comprising promoter operably linked to an influenza virus NP cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus cDNA for NB and NA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus M cDNA linked to a transcription termination sequence, and a vector comprising a promoter operably linked to an influenza virus NS cDNA linked to a transcription termination sequence, wherein the sequence of the cDNA for NB and NA comprises a knockout mutation in the sequence for NB the presence of which in the mutant gene optionally yields a functional NA protein; and

b) vectors to express influenza virus proteins, comprising a vector comprising a promoter operably linked to a DNA segment encoding influenza virus PA, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus PB1, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus PB2, and a vector comprising a promoter operably linked to a DNA segment encoding influenza virus NP.

37. The composition of claim 36 further comprising a vector to generate vRNA comprising a promoter operably linked to a DNA fragment of interest in antisense orientation.

38. The composition of claim 37 wherein the vector comprises a DNA fragment which encodes an immunogenic polypeptide or peptide of a pathogen or a therapeutic protein.

39. The composition of claim 36 wherein the NB protein gene comprises at least two mutations, one of which is not in the transmembrane domain.

40. The composition of claim 39 wherein at least one mutation is in the codon for the initiator methionine.