Method for producing artificial recombinant rotavirus

a technology of rotavirus and recombinant rotavirus, which is applied in the direction of dsrna viruses, antibody medical ingredients, peptide sources, etc., can solve the problems of lag behind the rg system, high risk of rotavirus infection and rotavirus disease development in children aged 6 months to 2 years, and high production efficiency

Pending Publication Date: 2022-11-03
OSAKA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patent describes a new method for making artificial viruses, specifically those in the family Reoviridae and rotavirus. The method is faster and more efficient than previous methods, and can be used to create rotavirus without needing a helper virus. The resulting viruses can also have mutations introduced into them. These new methods and viruses could be useful for making vaccines or studying virus infections.

Problems solved by technology

Infants aged from 6 months to 2 years are at high risk of rotavirus infection and rotavirus disease development.
However, the development of RG systems for Reoviridae viruses, which have a 10 to 12 segmented double-stranded RNA (dsRNA) genome, lags behind that of RG systems for other RNA viruses due to the complexity of their segmented genome.
However, the helper virus-dependent RG systems have disadvantages in that a potent means of separating the virus of interest from the helper virus is required; that mutation can be introduced only into limited types of segment genes (VP4 gene, NSP2 gene); and that production efficiency is low.

Method used

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  • Method for producing artificial recombinant rotavirus
  • Method for producing artificial recombinant rotavirus
  • Method for producing artificial recombinant rotavirus

Examples

Experimental program
Comparison scheme
Effect test

example 1

nt of Reverse Genetics System for Mammalian Orthoreovirus

[0093]Mammalian orthoreovirus (MRV) has been extensively studied as a model virus of the family Reoviridae. An entirely plasmid-based reverse genetics (RG) system for MRV is the first system developed in the family Reoviridae (Non Patent Literature 3). For the purpose of improving RG systems for the family Reoviridae, it was examined whether the use of a FAST protein encoded by a group of fusogenic reoviruses and a capping enzyme encoded by vaccinia virus could enhance the efficiency of artificial recombinant virus production.

Materials and Methods

(1) Viruses

[0094]Mammalian orthoreovirus strain type 1 Lang (hereinafter referred to as “MRV T1L”) was used. MRV T1L can be purchased from ATCC (ATCC VR-230). The gene names and GenBank accession numbers of 10 individual RNA genome segments of MRV T1L are shown in Table 1.

TABLE 1Sequences of genome segments of MRV T1LGene nameGenBank ACCESSIONSEQ ID NOL1M247341L2AF3780032L3AF1298203M1...

example 2

nt of Rotavirus Reverse Genetics System

Materials and Methods

(1) Virus

[0105]Simian rotavirus strain SA11 was used. The present inventors previously determined and registered the nucleotide sequences of all 11 RNA genome segments of this virus strain. The names and GenBank accession numbers of the 11 individual RNA genome segments of the simian rotavirus strain SA11 (hereinafter referred to as “SA11”) used in the experiment below are shown in Table 2.

TABLE 2Sequences of genome segments of simian rotavirus SA11GenomeGenBankSEQ IDsegmentCoding proteinACCESSIONNOSegment 1VP1 (RNA-dependent LC17856411RNA polymerase)Segment 2VP2 (RNA-binding protein)LC17856512Segment 3VP3 (Guanylyltransferase)LC17856613Segment 4VP4 (Hemagglutinin, spike protein)LC17856714Segment 5NSP1 (Immune suppressive factor)LC17857015Segment 6VP6 (Inner capsid)LC17856816Segment 7NSP3 (Translation enhancer)LC17857217Segment 8NSP2 (NTPase)LC17857118Segment 9VP7(Outer capsid)LC17856919Segment 10NSP4 (Enterotoxin)LC1785732...

example 3

n of Artificial Recombinant Rotavirus Having a Deletion Mutation

[0115]An experiment was performed to examine the feasibility of the production of an artificial recombinant rotavirus having a partial deletion mutation in NSP1, a suppressive factor against host innate immune responses.

Materials and Methods

(1) Preparation of Plasmid Having a Deletion Mutation in NSP1 Gene

[0116]A plasmid having a mutated NSP1 gene (see FIG. 4), which had a 299-base deletion at positions 1192 to 1490 of the NSP1 gene (SEQ ID NO: 15), was prepared from pT7-NSP1SA11 as a template using KOD-Plus-Mutagenesis Kit (trade name, Toyobo) and specific primers for the gene. This plasmid (designated as pT7-NSP1SA11ΔC108) expresses an NSP1 protein having a deletion of C-terminal 108 amino acids of the native NSP1.

(2) Production of Artificial Recombinant Viruses and Confirmation of Mutation

[0117]A deletion mutant of rotavirus (rsSA11 / NSP1ΔC108) was produced in the same manner as in Example 2 except that pT7-NSP1SA11ΔC...

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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.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]This application is a divisional application of U.S. patent application Ser. No. 16 / 336,735, filed on Mar. 26, 2019, which is the U.S. National Phase application of PCT International Application No. PCT / JP2017 / 034783, filed on Sep. 26, 2017, designating the United States of America and published in the Japanese language, which is an International Application of and claims the benefit of priority to Japanese Patent Application No. 2016-188881, filed on Sep. 27, 2016 and Japanese Patent Application No. 2017-068323, filed on Mar. 30, 2017. The disclosures of the above-referenced applications are hereby expressly incorporated by reference in their entireties.REFERENCE TO SEQUENCE LISTING[0002]A Sequence Listing submitted as an ASCII text file via EFS-Web is hereby incorporated by reference in accordance with 37 U.S.C. § 1.52(e). The name of the ASCII text file for the Sequence Listing is SeqList-IWAT007-003D1.txt, the date of creation of the ...

Claims

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Application Information

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Patent Type & AuthorityApplications(United States)
IPC IPC(8): A61K39/15A61P31/14C07K14/14C12N15/86C12N15/09
CPCA61K39/15A61P31/14C07K14/14C12N15/86C12N15/09C12N7/00C12N2720/12351C12N2720/12051C12N2720/12022C07K14/005A61K39/12C12N2720/12034C12N2720/12334A61K2039/5254C12Q1/701
InventorKOBAYASHI, TAKESHIKANAI, YUTA
OwnerOSAKA UNIV