Reverse genetic system for rift valley fever virus and uses thereof
View Patent ↗The present invention describes a reverse genetic system for Phlebovirus such as Rift Valley fever virus. This system comprised of RNA expression plasmids and protein expression plasmids. Additionally, the present invention also discloses the modification of this system to generate a recombinant virus that expresses a non-viral foreign gene. Furthermore, the present invention discloses the use of this system in the development of anti-Rift Valley fever virus vaccines, screening of antivirals testing for anti RVF immune response and developing marker vaccines for Rift Valley fever virus. We also claim the utility of this approach to other phleboviruses .
1. A reverse genetic composition for a Phlebovirus , comprising:
at least one anti-viral sense RNA expression plasmid that encodes a non-viral gene inserted in place of a non-structural protein (NSs) open reading frame (ORF) in the Phlebovirus Small (S) segment, such that said non-structural protein (NSs) open reading frame (ORF) for said Phlebovirus is deleted but the N gene is not deleted;
at least one anti-viral sense RNA expression plasmid that encodes a Phlebovirus Large (L) segment;
at least one anti-viral sense RNA expression plasmid that encodes a Phlebovirus Medium (M) segment;
at least one viral protein expression plasmid that expresses a Phlebovirus L protein;
at least one viral protein expression plasmid that expresses a Phlebovirus N protein; and
at least one viral protein expression plasmid that expresses Phlebovirus envelope proteins.
2. The composition of claim 1 , wherein the non-viral gene expresses a reporter protein.
3. The composition of claim 2 , wherein the reporter protein is a Renilla luciferase or a green fluorescent protein.
4. The composition of claim 1 , wherein the Phlebovirus is a Rift Valley fever virus, a Toscana virus, a Sandfly fever virus or a Punta Toro virus.
5. The composition of claim 4 , wherein the Rift Valley fever virus strain is selected from the group consisting of MP-12, modified MP-12, ZH501, ZH548, SA75, and SPB 9800523.
6. An isolated cell comprising and expressing the plasmids of the reverse genetic system of claim 1 .
7. The isolated cell of claim 6 , wherein the host cell is a T7 polymerase expressing cell.
8. The isolated cell of claim 7 , wherein the host cell is a BHK cell.
9. The composition of claim 2 , wherein the M-segment encoded by anti-viral sense RNA expression plasmids comprise partial deletions in the sequence encoding the L segment, wherein said partial deletion does not abolish the replication of said Phlebovirus in host cells.
10. The composition of claim 2 , wherein the MM segment encoded by anti-viral sense RNA expression plasmids comprise partial deletions in the sequence encoding the M segment, wherein said partial deletion does not abolish the replication of said Phlebovirus in host cells.