Reverse genetics systems
View Patent ↗The invention provides various reverse genetics systems for producing segmented RNA viruses, wherein the systems do not require bacteria for propagation of all of their expression constructs.
1. A non-bacterial DNA construct comprising a promoter and coding sequences for expressing at least two different genome segments of a segmented RNA virus, wherein the construct lacks both a bacterial origin of replication and a bacterial selection marker, wherein the at least two segments are selected from the group of segments consisting of PB2, PB1, PA, HA, NP, NA, M and NS, wherein the construct is linear, and, wherein the promoter is operatively linked for transcription in an eukaryotic host cell, wherein the eukaryotic host cell is a mammalian cell or avian cell.
2. A eukaryotic host cell comprising the non-bacterial construct of claim 1 .
3. A method for producing a segmented RNA virus, comprising a step of culturing a host cell of claim 2 such that expression of the RNA virus segments occurs to produce the virus.
4. The host cell of claim 2 , wherein the cell is a MDCK cell.
5. The non-bacterial DNA construct of claim 1 , wherein the coding sequences are for influenza virus A or B genome segments PB2, PB1, PA, NP, and NS.
6. A method for producing a segmented RNA virus, comprising
a) transfecting a host cell with the construct of claim 1 , and
b) culturing the host cell such that expression of the RNA virus segments occurs to produce the virus;
wherein the host cell is a mammalian or avian host cell.
7. A non-bacterial DNA construct comprising a promoter and coding sequences for expressing all eight genome segments of an influenza A or B virus, wherein the construct lacks both a bacterial origin of replication and a bacterial selection marker, wherein the promoter is operatively linked for transcription in an eukaryotic host cell, wherein the eukaryotic host cell is a mammalian cell or avian cell, and wherein the construct is linear.
8. A eukaryotic host cell comprising the non-bacterial construct of claim 7 .
9. A method for producing a segmented RNA virus, comprising a step of culturing a host cell of claim 8 such that expression of the RNA virus segments occurs to produce the virus.
10. The host cell of claim 8 , wherein the cell is a MDCK cell.