Infectious clone of human parvovirus B19 and methods
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.
1. 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.
2. The method of claim 1 , wherein the ITR located at the 5′ end or the 3′ end comprises SEQ ID NO:1.
3. The method of claim 1 , wherein the ITR located at the 5′ end or the 3′ end comprises SEQ ID NO:2.
4. The method of claim 1 , wherein the viral genome further comprises a nucleic acid sequence encoding at least one or all of a parvovirus B19 VP1, a parvovirus B19 VP2, a parvovirus B19 nonstructural protein, or a parvovirus B19 11-kpa protein.
5. The method of claim 1 , wherein the viral genome is a full length parvovirus B19 genome.
6. 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.
7. 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.
8. The method of claim 7 , wherein the cells are eukaryotic cells.
9. The method of claim 7 , wherein introducing the vector into the population of cells is conducted by electric current.
10. The method of claim 9 , wherein the cells are exposed to an electrical pulse comprising a field strength of about 2kV/cm to about 10kV/cm, a duration of at least about usec, and a current of at least about 1 A followed by a current flow of about 1 A to about 3 A for at least 10 msec.