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Rescue of canine distemper virus from CDNA

A technology for canine distemper and virus, applied in the field of producing canine distemper virus as attenuated and/or infectious virus, immunogenic composition, immunogenic or therapeutic composition, which can solve the problem of inability to use susceptible Issues with animal populations, inability to grow efficiently, no minireplicon or viral rescue systems have been reported

Inactive Publication Date: 2003-11-12
AMERICAN CYANAMID CO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] A live attenuated CDV vaccine has been achieved for the control of this disease in the domestic dog population, but additional vaccine research is still needed
Three circulating vaccines cannot be used in all susceptible animal populations (4, 18, 52)
Additionally, some strains of canine distemper virus do not grow efficiently in tissue culture systems
Despite the fact that a modified genome sequence has been available since 1998 (GenBank accession number AF014953, incorporated herein by reference), no minireplicon or viral rescue system has been reported

Method used

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  • Rescue of canine distemper virus from CDNA
  • Rescue of canine distemper virus from CDNA
  • Rescue of canine distemper virus from CDNA

Examples

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example 1

[0145] Materials and methods

[0146] Cells and viruses

[0147] HEp2, A549, Vero, B95-8 and chicken embryo fibroblast (CEF) cells are maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS). HeLa suspension cells were grown in modified minimal essential medium (SMEM) supplemented with 5% FBS. The laboratory-modified Onderstepoort CDV strain (17) was propagated in HeLa cells according to the previously disclosed method (46). The second laboratory-modified Onderstepoort strain was provided by Dr. Martin Billeter of Zurich University and was propagated in B95-8 cells. Propagation of the recombinant attenuated vaccinia virus strain MVA / T7 (obtained from Dr. B. Moss, National Institute of Health, Bethesda, MD; Wyatt et al., 1995; reference 61) designed to express the T7 RNA polymerase gene in CEF cells ). Titrate the MVA / T7 mother liquor on CEFs. A laboratory modified Edmonston strain of measles virus (MV) grown in HeLa suspension cells (55...

example 2

[0179] General method of transient expression analysis and virus rescue by CAT assay

[0180] The microreplicon transfection is performed by several methods. For experiments using CDV microreplicon as RNA for transfection, 293 cells were transfected with Lipofectace (Life Technologies). The microreplicon RNA is prepared in vitro by T7 RNA polymerase using pCDV-CAT DNA (Figure 1B) as a transcription template (2). The RNA was synthesized and purified using the reagents and methods in the Megascript kit (Ambion). In the compensated microreplicon experiment provided by CDV infection (Figure 2A), the components of the RNA transfection mixture were prepared in two test tubes. One test tube contains 20 micrograms of purified microreplicon RNA and 100 microliters of serum-free OptiMEM (Life Technologies). The second test tube was prepared with 100 microliters of serum-free OptiMEM and 9-12 microliters of Lipofectace (Life Technologies). Then mix the contents of the two test tubes and incu...

example 3

[0185] CDV microreplicon expression. For the development of a virus rescue system, it is important to use the microreplicon reporting system for transient expression studies. By analyzing the transient expression of the microreplicon reporting gene, the transfection parameters can be evaluated relatively quickly to determine the optimal conditions, and it is still A valuable tool for determining whether N, P, and L expression vectors can guide the synthesis of functional proteins.

[0186] 3.1 Save the CDV microreplicon by virus

[0187] 3.1.1 The microreplicon experiment tested whether the CDV-CAT microreplicon can exert its ability to be rescued by virus compensation. The CDV-CAT microreplicon RNA (20 micrograms) synthesized in vitro was transferred to a 60 mm petri dish of 293 cells. At the beginning of transfection, the cells were also infected with a similar CDV at a multiplicity of infection of approximately 2. About 24 hours after transfection, when about 70% of the cells h...

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Abstract

This invention relates to a method for recombinantly producing via rescue canine distemper virus, a nonsegmented, negative-sense, single-stranded RNA virus, and immunogenic compositions formed therefrom. Additional embodiments relate to methods of producing the canine distemper virus as an attenuated and / or infectious viruses. The recombinant viruses can be prepared from cDNA clones, and, accordingly, viruses having defined changes, including nucleotide / polynucleotide deletions, insertions, substitutions and rearrangements, in the genome can be obtained.

Description

Invention field [0001] The present invention relates to a method for recombinantly producing canine fever virus, a non-segmented negative single-stranded RNA virus, and an immunogenic composition produced by the method. Other embodiments relate to methods of producing canine fever virus as an attenuated and / or infectious virus. The recombinant virus is prepared by cDNA cloning, and accordingly a virus with specific changes in its genome can be obtained. The present invention also relates to the use of the recombinant virus produced by the method as a vector to express foreign genetic information, such as foreign genes, for various purposes, including gene therapy and cell use for pathogens other than canine fever virus. Targeted immunogenic or therapeutic composition. Background of the invention [0002] Coated negative single-stranded RNA viruses have a single organization and can express. The genomic RNA of the negative single-stranded virus functions as tw...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K35/76A61K39/00A61K39/175A61P31/12A61P31/14C12N15/09C07K14/12C07K14/13C12N1/15C12N1/19C12N1/21C12N5/10C12N7/00C12N7/02C12N15/40C12N15/45
CPCC12N2760/18422A61K2039/5256C12N7/00C07K14/005C12N2760/18451A61P31/12A61P31/14C12N15/11
Inventor C·L·帕克斯M·S·西胡P·瓦尔皮塔G·R·科瓦克斯S·A·乌德姆
Owner AMERICAN CYANAMID CO
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