Method for producing artificial recombinant rotavirus

Patent metadata

Field	Value
Publication number	US-12109262-B2
Application number	US-202217805419-A
Country	US
Kind code	B2
Filing date	Jun 3, 2022
Priority date	Sep 27, 2016
Publication date	Oct 8, 2024
Grant date	Oct 8, 2024

Abstract

Provided is a method for producing an artificial recombinant virus of the family Reoviridae, the method comprising the steps of:
(1) introducing a FAST protein expression vector and/or a capping enzyme expression vector into host cells; (2) introducing a vector containing expression cassettes for individual RNA genome segments of a virus or introducing a set of single-stranded RNA transcripts from the expression cassettes into host cells; and (3) culturing the host cells.

The method of the present invention allows more efficient production of an artificial recombinant virus of the family Reoviridae as compared with conventional methods and allows artificial recombinant rotavirus production without using a helper virus.

First claim

The invention claimed is:

1. A method for producing an artificial recombinant virus of the family Reoviridae, the method comprising:
(1) introducing a FAST protein expression vector into host cells;
(2) introducing a vector containing a set of expression cassettes for individual RNA genome segments of a virus or introducing a set of single-stranded RNA transcripts of the RNA genome segments from the expression cassettes into host cells; and
(3) culturing the host cells.

2. The method according to claim 1 , wherein step (1) further comprises introducing a capping enzyme expression vector into the host cells.

3. The method according to claim 1 , wherein the artificial recombinant virus has a mutation introduced in at least one of the RNA genome segments and/or a foreign gene inserted in at least one of the RNA genome segments.

4. The method according to claim 1 , wherein the FAST protein is Nelson Bay reovirus p10, Avian reovirus p10, Broome reovirus p13, Reptilian reovirus p14, Baboon reovirus p15, grass carp reovirus p16 or Atlantic salmon reovirus p22.

5. The method according to claim 2 , wherein the capping enzyme expression vector encodes a capping enzyme of a DNA or RNA virus which replicates in the cytoplasm of host cells.

6. The method according to claim 1 , wherein the expression cassette for an RNA genome segment comprises an RNA polymerase promoter, a DNA encoding the RNA genome segment and a DNA encoding a self-cleaving ribozyme.

7. The method according to claim 6 , wherein the RNA polymerase promoter is a T7 promoter, and the host cells are recombinant T7 RNA polymerase-expressing cells.

8. The method according to claim 6 , wherein the ribozyme is a hepatitis D virus ribozyme.

9. The method according to claim 1 , wherein the host cells are co-cultured with highly virus-susceptible cells, wherein the highly virus-susceptible cells are cells selected from the group consisting of BHK cells, MA104 cells, COS7 cells, CV1 cells, Vero cells, L929 cells, 293T cells, and A549 cells.

10. The method according to claim 1 , wherein the artificial recombinant virus of the family Reoviridae is an artificial recombinant rotavirus.

11. The method according to claim 10 , comprising overexpressing a rotavirus NSP2 gene product and/or a rotavirus NSP5 gene product in the host cells.

12. The method according to claim 10 , wherein the artificial recombinant rotavirus expresses a foreign gene, and wherein a vector containing an expression cassette for an RNA genome segment encoding NSP1 which cassette has an insertion of the foreign gene in an NSP1 gene and a 100- to 1550-base deletion in the NSP1 gene is used instead of a vector containing an expression cassette for an RNA genome segment encoding NSP1.

13. A method for promoting viral replication, comprising infecting host cells expressing a FAST protein with a virus of the family Reoviridae and culturing the host cells.

14. The method according to claim 13 , wherein the virus of the family Reoviridae is a virus of the genus Orthoreovirus or Rotavirus.

15. The method according to claim 13 , wherein the FAST protein is selected from Nelson Bay reovirus p10, Avian reovirus p10, Broome reovirus p13, Reptilian reovirus p14, Baboon reovirus p15, grass carp reovirus p16 or Atlantic salmon reovirus p22.

Assignees

• Univ Osaka

Inventors

• Kobayashi Takeshi
• Kanai Yuta

Classifications

• C12N15/09

  Recombinant DNA-technology · CPC title

• C12N15/86

  Viral vectors · CPC title

• C07K14/14

  Reoviridae, e.g. rotavirus, bluetongue virus, Colorado tick fever virus · CPC title

• A61P31/14

  for RNA viruses · CPC title

• C12Q1/701

  Specific hybridization probes · CPC title