Infectious clone of human parvovirus B19 and methods
AssigneesThe United States of America, as represented by the Secretary, Department of Health and Human Services, Institut National de Rechesche Scientifique
InventorsKevin Edward Brown, Ning Zhi, Peter Tijssen, Zoltan Zadori
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Abstract
The invention relates to infectious clones of parvovirus B19, methods of cloning infectious B19 clones, and methods of cloning viral genomes that have secondary DNA structures that are unstable in bacterial cells. A B19 infectious clone and methods of producing B19 infectious clones are useful for producing infectious virus. Infectious virus is useful for identifying and developing therapeutically effective compositions for treatment and/or prevention of human parvovirus B19 infections.
Claims10
A method for cloning a parvovirus B19 viral genome comprising:
- (a) introducing a vector comprising all or a portion of a parvovirus B19 genome into a prokaryotic cell that is deficient in at least one recombinase enzyme, wherein the method uses a full length inverted terminal repeat (ITR) at the 5′ end and the 3′ end of the genome, wherein the ITR located at the 5′ end comprises SEQ ID NO:1 or SEQ ID NO:2, and wherein the ITR located at the 3′ end comprises SEQ ID NO:1 or SEQ ID NO:2;
- (b) incubating the cells at about 25° C. to 35° C.;
- and
- (c) recovering the vector from the prokaryotic cells.
An isolated cell comprising an infectious clone of parvovirus comprising the parvovirus B19 ITR sequence set forth in SEQ ID NO:
- 1 and/or SEQ ID NO:
- 2.
A method for producing an infectious virus of parvovirus B19, comprising:
- introducing a vector comprising an infectious clone of parvovirus B19 into a population of cells, wherein the infectious clone of parvovirus B19 comprises a parvovirus B19 genome, wherein the genome comprises a full length inverted terminal repeat (ITR) at the 5′ end and the 3′ end, wherein the ITR located at the 5′ end comprises SEQ ID NO:1 or SEQ ID NO:2, wherein the ITR located at the 3′ end comprises SEQ ID NO:1 or SEQ ID NO:2;
- and wherein the vector is present in at least about 15% of the cells;
- and
- incubating the cells under conditions to allow for viral replication.
Dependencies are inferred from the claim text.