Recombinant RSV reporter virus

Patent metadata

Field	Value
Publication number	US-10100372-B2
Application number	US-201515319642-A
Country	US
Kind code	B2
Filing date	Jun 18, 2015
Priority date	Jun 18, 2014
Publication date	Oct 16, 2018
Grant date	Oct 16, 2018

Abstract

An RSV reporter strain that can be used for high-throughput drug discovery is disclosed. Also disclosed is a recombinant RSV vector that contains an RSV genome encoding the disclosed RSV reporter strain operably linked to an expression control sequence. Also disclosed is an infectious RSV virion produce by expression of the disclosed recombinant RSV vector in a host cell. Also disclosed is a recombinant nucleic acid that comprises an RSV F protein having an escape mutation operably linked to a heterologous expression control sequence. Also disclosed are methods of screening for antiviral agents using the disclosed RSV reporter strains.

First claim

What is claimed is:

1. A recombinant respiratory syncytial virus (RSV) vector, comprising an RSV genome encoding an infectious RSV virion operably linked to an expression control sequence, wherein the RSV genome comprises a nucleic acid encoding a fusion (F) protein having a D401E mutation, a D489E mutation, or combination thereof that allows a RSV virus produced by expression of the RSV vector to escape from GPAR-3710 inhibition, wherein the fusion (F) protein mutation positions correspond to SEQ ID NO. 1, wherein the RSV genome further comprises a nucleic acid encoding a luciferase, and wherein the RSV genome further comprises a nucleic acid encoding a small-molecule assisted shutoff (SMASh) tag wherein the SMASh tag comprises a protease cleavage site, hepatitis C virus-derived NS3 protease domain, and a degron domain.

2. The recombinant RSV vector of claim 1 , wherein the mutation is a D401E mutation.

3. The recombinant RSV vector of claim 1 , wherein the mutation is a D489E mutation.

4. The recombinant RSV vector of claim 1 , wherein the RSV genome is derived from an RSV line19 (L19) strain or an RSV A2 strain.

5. The recombinant RSV vector of claim 1 , wherein the RSV genome is derived from a chimeric A2 strain, wherein the nucleic acid encoding the F protein is derived from an RSV L19 strain.

6. The recombinant RSV vector of claim 1 , wherein the recombinant RSV vector further comprises a firefly luciferase.

7. The recombinant RSV vector of claim 1 , wherein the recombinant RSV vector further comprises a Renilla luciferase.

8. The recombinant RSV vector of claim 1 , wherein the vector comprises a bacterial artificial chromosome backbone.

9. An infectious RSV virion produced by expression of the recombinant RSV vector of claim 1 in a host cell.

10. A method of screening for antiviral agents, comprising
(a) contacting a culture comprising the infectious RSV virion of claim 9 with a candidate agent; and
(b) assaying the culture for RSV levels or activity;
wherein a decrease in RSV levels or activity is an indication that the candidate agent is an effective antiviral agent for RSV.

11. The method of claim 10 , wherein the RSV genome comprises a nucleic acid encoding a first luciferase, wherein step (b) comprises contacting the culture with a substrate for the first luciferase, and assaying the culture for bioluminescence, wherein a decrease in bioluminescence from the first luciferase activity is an indication that the candidate agent is an effective antiviral agent for RSV.

12. The method of claim 11 , wherein the culture further comprises an infectious influenza virion encoded by a recombinant influenza vector comprising an influenza genome encoding a second luciferase that has a substrate distinct from the first luciferase,
wherein the infectious influenza virus virion and the infectious RSV virion have comparable growth kinetics,
wherein the method further comprises contacting the culture with a substrate for the second luciferase and assaying the culture for bioluminescence,
wherein a decrease in bioluminescence from the second luciferase activity is an indication that the candidate agent is an effective antiviral agent for influenza,
wherein a decrease in bioluminescence from both the first luciferase activity and second luciferase activity is an indication that the candidate agent is an effective pan-antiviral agent.

13. The method of claim 11 , wherein the mutation is a D489E mutation.

14. The method of claim 11 , wherein the first luciferase comprises firefly luciferase, wherein the second luciferase comprises nano-luciferase, gaussia luciferase, or Renilla luciferase.

15. The method of claim 11 , wherein the influenza genome is derived from strain WSN-33 (H1N1).

16. A recombinant nucleic acid, comprising a respiratory syncytial virus (RSV) fusion (F) protein having a D401E mutation, a D489E mutation, or a combination thereof, operably linked to a heterologous expression control sequence, wherein the D401E mutation, the D489E mutation, or combination thereof allows an RSV virus produced by expression of the recombinant nucleic acid to escape from GPAR-3710 inhibition wherein the RSV F protein mutation positions correspond to SEQ ID NO. 1.

Assignees

• Georgia State Univ And Research Foundation Inc
• Univ Georgia State Res Found

Inventors

• Plemper Richard K
• Yan Dan

Classifications

• C12Q1/70 Primary

  involving virus or bacteriophage {(immunoassay for viruses G01N33/56983)} · CPC title

• C12N9/0069

  acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13) · CPC title

• C12Y113/12007

  Photinus-luciferin 4-monooxygenase (ATP-hydrolysing) (1.13.12.7), i.e. firefly-luciferase · CPC title

• C12Y113/12005

  Renilla-luciferin 2-monooxygenase (1.13.12.5), i.e. renilla-luciferase · CPC title

• C12N2760/18543

  viral genome or elements thereof as genetic vector · CPC title