Highly infectious rubella virus clones and methods of production

Abstract

Highly infectious rubella virus cDNA clones that are chimeric constructs of an infectious cDNA clone having a low specific infectivity and nucleic acid molecule fragments from a second rubella virus genome, wherein portions of the nucleotide sequence of the infectious cDNA clone having low specific infectivity have been replaced with the corresponding cDNA fragments derived from the second rubella virus genome.

We claim:  
     
         1 . An isolated DNA molecule comprising a chimeric construct that encodes a highly infectious rubella virus.  
     
     
         2 . The isolated DNA molecule of  claim 1 , wherein the rubella virus has a specific infectivity of greater than 0.5 plaques/μg of transcript.  
     
     
         3 . The isolated DNA molecule of  claim 1 , wherein the rubella virus has a specific infectivity of approximately 10 4  plaques/μg of transcript.  
     
     
         4 . The isolated DNA molecule of  claim 1 , wherein the chimeric construct comprises a DNA molecule encoding an infectious rubella virus having low infectivity in which portions of the DNA molecule have been replaced with one or more corresponding DNA fragments of a second rubella virus genome.  
     
     
         5 . The isolated DNA molecule of  claim 4 , wherein the second rubella virus genome has a minimal number mutations that adversely affect infectivity.  
     
     
         6 . The isolated DNA molecule of  claim 4 , wherein the molecule comprises a structural protein open reading frame portion and a non-structural protein open reading frame portion and wherein the DNA fragments replace all or a portion of the structural protein open reading frame region of the DNA molecule encoding the infectious rubella virus having low infectivity.  
     
     
         7 . The isolated DNA molecule of  claim 4 , wherein the molecule comprises a structural protein open reading frame portion and a non-structural protein open reading frame portion and wherein the DNA fragments replace all or a portion of the non-structural protein open reading frame region of the DNA molecule encoding the infectious rubella virus having low infectivity.  
     
     
         8 . The isolated DNA molecule of  claim 4 , wherein the DNA fragments replace portions of the DNA molecule encoding the infectious rubella virus having low infectivity selected from the group consisting of fragment I, fragment II, and fragment III.  
     
     
         9 . The isolated DNA molecule of  claim 8 , wherein fragment I is a nucleic acid molecule between restriction endonuclease cleavage sites HindIII and KpnI, fragment II is a nucleic acid molecule between restriction endonuclease cleavage sites NheI and BglII, and fragment III is a nucleic acid molecule between restriction endonuclease cleavage sites NheI and BglII.  
     
     
         10 . The isolated DNA molecule of  claim 8 , wherein the DNA molecule encoding the infectious rubella virus having low infectivity has the sequence set forth in SEQ ID NO. 1 and fragment I replaces nucleotides 1 to 1723 of SEQ ID NO. 1, fragment II replaces nucleotides 2800 to 5352 of SEQ ID NO. 1, and fragment III replaces nucleotides 5353 to 9734 of SEQ ID NO. 1.  
     
     
         11 . The isolated DNA molecule of  claim 1  further comprising a vector, wherein the vector enables replication of the nucleotide sequence.  
     
     
         12 . The isolated DNA molecule of  claim 11 , wherein the vector is a bacterial plasmid.  
     
     
         13 . The isolated DNA molecule of  claim 12 , further comprising a vector, wherein the vector enables replication of the DNA molecule.  
     
     
         14 . The isolated DNA molecule of  claim 13 , wherein the vector is a bacterial plasmid.  
     
     
         15 . A method of producing a highly infectious rubella virus comprising the steps of: 
 a. inserting a chimeric DNA molecule into a plasmid, wherein the chimeric construct comprises a DNA molecule encoding an infectious rubella virus having low infectivity in which portions of the DNA molecule have been replaced with one or more corresponding DNA fragments of a second rubella virus genome;    b. transcribing the DNA into linear RNA;    c. transfecting cells with the nucleic acid transcript; and    d. recovering highly infectious rubella virus from the transfected cells.    
     
     
         16 . The method of  claim 15 , wherein the molecule comprises a structural protein open reading frame portion and a non-structural protein open reading frame portion and wherein the DNA fragments replace all or a portion of the structural protein open reading frame region of the DNA molecule encoding the infectious rubella virus having low infectivity.  
     
     
         17 . The method of  claim 15 , wherein the molecule comprises a structural protein open reading frame portion and a non-structural protein open reading frame portion and wherein the DNA fragments replace all or a portion of the non-structural protein open reading frame region of the DNA molecule encoding the infectious rubella virus having low infectivity.  
     
     
         18 . The method of  claim 15 , wherein the DNA fragments replace portions of the DNA molecule encoding the infectious rubella virus having low infectivity selected from the group consisting of fragment I, fragment II, and fragment III.  
     
     
         19 . The method of  claim 18 , wherein fragment I is a nucleic acid molecule between restriction endonuclease cleavage sites HindIII and KpnI, fragment II is a nucleic acid molecule between restriction endonuclease cleavage sites NheI and BglII, and fragment III is a nucleic acid molecule between restriction endonuclease cleavage sites NheI and BglII.  
     
     
         20 . The method of  claim 18 , wherein the DNA molecule encoding the infectious rubella virus having low infectivity has the sequence set forth in SEQ ID NO. 1 and fragment I replaces nucleotides 1 to 1723 of SEQ ID NO. 1, fragment II replaces nucleotides 2800 to 5352 of SEQ ID NO. 1, and fragment III replaces nucleotides 5353 to 9734 of SEQ ID NO. 1.