Filovirus vectors and particles produced therefrom

Abstract

Cloned filovirus genomic cDNA and methods of using the cDNA are provided. Further provided are noninfectious lipid encapsulated filovirus-based particles.

What is claimed is: 
     
         1 . A method to prepare infectious filovirus from a cDNA in the absence of helper filovirus infection, comprising:
 providing a full-length infectious filovirus genomic cDNA;   modifying the full-length infectious filovirus genomic cDNA, thereby providing for a mutant filovirus genomic cDNA, wherein the modification comprises an internal deletion or a DNA fragment of interest that replaces or is inserted into a coding region for one or more filovirus proteins;   contacting a mammalian cell with a vector comprising the mutant filovirus genomic cDNA in an amount effective to yield infectious filovirus comprising mutant filovirus genomic RNA corresponding to the mutant filovirus genomic cDNA, wherein the mammalian cell expresses one or more filovirus proteins, the coding region of which in the mutant filovirus genomic cDNA has a deletion, is replaced by the DNA fragment of interest or the coding region of which is interrupted by the DNA fragment of interest, and the filovirus protein is a filovirus RNA transcriptase-polymerase, a filovirus NP, a filovirus VP30, or a filovirus VP35; and   isolating the infectious filovirus.   
     
     
         2 . A method to prepare infectious filovirus from a cDNA in the absence of helper filovirus infection, comprising:
 modifying a full-length infectious filovirus genomic cDNA, thereby providing for a mutant filovirus genomic cDNA, wherein the modification comprises an internal deletion or a DNA fragment of interest that replaces or is inserted into a coding region for one or more filovirus proteins;   contacting a mammalian cell with a vector comprising the mutant filovirus genomic cDNA in an amount effective to yield infectious filovirus comprising mutant filovirus genomic RNA corresponding to the mutant filovirus genomic cDNA, wherein the mammalian cell expresses one or more filovirus proteins, the coding region of which in the mutant filovirus genomic cDNA has a deletion, is replaced by the DNA fragment of interest or the coding region of which is interrupted by the DNA fragment of interest, and the filovirus protein is a filovirus RNA transcriptase-polymerase, a filovirus NP, a filovirus VP30, or a filovirus VP35; and   isolating the infectious filovirus.   
     
     
         3 . A method to prepare infectious filovirus from a cDNA in the absence of helper filovirus infection, comprising:
 providing a mutant filovirus genomic cDNA prepared by modifying a full-length infectious filovirus genomic cDNA, wherein the modification comprises an internal deletion or a DNA fragment of interest that replaces or is inserted into a coding region for one or more filovirus proteins;   contacting a mammalian cell with a vector comprising the mutant filovirus genomic cDNA in an amount effective to yield infectious filovirus comprising mutant filovirus genomic RNA corresponding to the mutant filovirus genomic cDNA, wherein the mammalian cell expresses one or more filovirus proteins, the coding region of which in the mutant filovirus genomic cDNA has a deletion, is replaced by the DNA fragment of interest or the coding region of which is interrupted by the DNA fragment of interest, and the filovirus protein is a filovirus RNA transcriptase-polymerase, a filovirus NP, a filovirus VP30, or a filovirus VP35; and   isolating the infectious filovirus.   
     
     
         4 . The method of  claim 1  wherein the mutant filovirus genomic cDNA has a deletion in the RNA transcriptase-polymerase, VP30 or VP35. 
     
     
         5 . Virus prepared by the method of  claim 1 . 
     
     
         6 . Virus prepared by the method of  claim 4 . 
     
     
         7 . The method of  claim 2  wherein the mutant filovirus genomic cDNA has a deletion in the RNA transcriptase-polymerase, VP30 or VP35. 
     
     
         8 . Virus prepared by the method of  claim 2 . 
     
     
         9 . Virus prepared by the method of  claim 7 . 
     
     
         10 . The method of  claim 3  wherein the mutant filovirus genomic cDNA has a deletion in the RNA transcriptase-polymerase, VP30 or VP35. 
     
     
         11 . Virus prepared by the method of  claim 3 . 
     
     
         12 . Virus prepared by the method of  claim 10 .