Method for rapid generation of an infectious RNA virus

Patent metadata

Field	Value
Publication number	US-10407667-B2
Application number	US-201515315667-A
Country	US
Kind code	B2
Filing date	Jun 19, 2015
Priority date	Jun 20, 2014
Publication date	Sep 10, 2019
Grant date	Sep 10, 2019

Abstract

The present invention relates to a method for rapid generation of an infectious RNA virus that completely eliminates the need of constructing a full-length c DNA, which covers the entire viral genome, cloning and propagating such full length c DNA.

First claim

The invention claimed is:

1. A method for generating an infectious RNA virus comprising:
a) introducing a promoter of DNA-dependent RNA polymerase in position 5′ and optionally a terminator and a RNA polyadenylation sequence in position 3′ of the entire genome of a RNA virus;
b) amplifying the entire viral genome as prepared in step a) including said promoter and optionally said terminator and RNA polyadenylation sequence, in at least 2, 3, 4, 5 or 6 overlapping cDNA fragments;
c) transfecting said cDNA fragments into a host cell,
d) incubating the host cell of step c); and
e) recovering the infectious RNA virus from said incubated host cell.

2. The method of claim 1 , wherein said virus is a single stranded positive RNA virus.

3. The method of claim 1 , wherein:
said promoter of DNA-dependent RNA polymerase in position 5′ is the human cytomegalovirus promoter (pCMV) ; and/or
said optional terminator and RNA polyadenylation sequence is respectively the hepatitis delta ribozyme and the simian virus 40 polyadenylation signal (HDR/SV40pA).

4. The method of claim 1 , wherein step b) allows the production from 2 to 15 overlapping cDNA fragments.

5. The method of claim 1 , wherein said host cell is selected from the group consisting of SW13 and BHK-21, HEK 293 and Vero cell lines.

6. The method of claim 5 , wherein:
step (c) is a step of direct transfection of the cDNA fragments obtained in step (b) as such, and
said step (c) occurs directly after step (b).

7. The method of claim 1 , wherein step c) is a step of transfecting plasmids or vectors comprising a cDNA fragment obtained in step (b), wherein each cDNA fragment is included in an individual and separate plasmid or vector.

8. The method of claim 1 , wherein said method further comprises a step (b′) after step (b) and prior to step (c) of purification of the overlapping cDNA fragments.

9. The method of claim 1 , wherein step (d) of incubation lasts from 3 to 9 days.

10. The method of claim 1 , wherein the transfected cDNA fragments of step (c) spontaneously recombine in the host cells during the incubation step (d).

11. The method of claim 1 wherein said method is used for reverse genetic analysis.

12. The method of claim 2 , wherein said virus is a virus selected from the group consisting of flavivirus, alphavirus and enterovirus.

13. The method according to claim 12 , wherein said Flavivirus is selected from the group consisting of Japanese encephalitis viruses (JEV), West Nile virus (WNV); Dengue virus (DENV); Yellow fever virus (YFV); and Tick-borne encephalitis virus (TBEV).

14. The method according to claim 12 , wherein said alphavirus is Chikungunya.

15. The method of claim 12 , wherein said enterovirus is Coxsackie.

16. The method of claim 4 , wherein step b) allows the production of 3, 4, 5 or 6 overlapping cDNA fragments.

17. The method of claim 8 , wherein the purification step is through a chromatography column.

Assignees

• Univ Aix Marseille
• Inst Nat Sante Rech Med

Inventors

• Aubry Fabien
• Nougairéde Antoine
• Quérat Gilles
• De Lamballerie Xavier
• Gould Ernest Andrew
• De Fabritus Lauriane

Classifications

• C12N7/00 Primary

  Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00) · CPC title

• A61K2039/525

  Virus · CPC title

• C12N2770/24151

  Methods of production or purification of viral material · CPC title

• C12N2770/32651

  Methods of production or purification of viral material · CPC title

• C12N2770/24051

  Methods of production or purification of viral material · CPC title