Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Defective interfering virus

A technology of viruses and virions, applied in virology and virus infection in animals, prevention and/or treatment of influenza A, interference with defective viruses, can solve the problem of low yield of DI virus

Active Publication Date: 2009-06-10
UNIVERSITY OF WARWICK
View PDF3 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0048] The inventors have tried to produce cloned DI influenza A virus by passaging in embryonated chicken eggs, but the yield of DI virus obtained was too low

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Defective interfering virus
  • Defective interfering virus
  • Defective interfering virus

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0190] Preparation of cloned DI virus

[0191] The methods used in the specific examples are described below and are subject to variation, see also the materials and methods described in Duhaut & Dimmock (2003), supra.

[0192] HEK293T cells were transfected by known methods with the set of plasmids necessary for the preparation of infectious A / PR8 / 34 virus (H1N1).

[0193] The transfected cells are then co-cultured with MDCK cells to amplify infectious virus. Tissue culture fluid containing the infectious virus and any DI virus present was collected, centrifuged at low speed to remove debris, and stored at -70°C. The presence of virus was shown by agglutination of chicken erythrocytes (see below). Virus titers were recorded as hemagglutination units (HAU) per milliliter of virus.

[0194] Tissue culture fluid (500 μl) was then injected into the allantoic cavity of 10-day-old embryonated chicken eggs to boost the concentration of infectious virus and infer the presence of D...

Embodiment 1

[0255] Example 1 - Production of cloned DI virus 220 / PR8

[0256] Previous work known in the art teaches that production of DI virus is optimal when inoculating embryonated chicken eggs with a large inoculum (eg, up to 2 ml per egg). The explanation given is that cells inhabited by non-infectious DI virus also need to receive infectious (helper) virus so that the former can replicate. Furthermore, the presence of DI virus normally increased when passage was repeated two or three times. Eventually, the amount of DI virus produced exceeds the amount of infectious virus present, and overall virus production declines due to the lack of helper virus function.

[0257] Unexpectedly, the inventors found that inoculation of embryonated chicken eggs with routine, i.e., standard amounts of cloned influenza A virus, failed to yield the expected amount of DI virus material; the gap was so large that it affected further laboratory studies, Including in vivo animal studies; not to menti...

Embodiment 2

[0280] Example 2 - Production of protective virus 244 / PR8

[0281] The primary protective RNA used (segment 1; RNA244) was spontaneously generated during transfection / co-cultivation of a plasmid encoding infectious A / PR / 8 / 34 (Subbarao, K. et al. 2003 Virology 305 : 192-200). The DNA mixture for transfection of 293T cells contained 0.5 μg of each of the 8 A / PR8 gene segments (under the control of the Poll promoter), 0.5 μg of various PB1 and PB2 expression plasmids, 0.1 μg of PA expression plasmids, and 1 μg of NP expression plasmids and Fugene (Roche).

[0282]To optimize the transfection of the 244 iRNA plasmid, the 244 RNA was cloned into the Poll expression plasmid pPOLI-SapIT (Subbarao (2003) supra) in order to express the vRNA sense transcript. Different amounts of 244 plasmid (0-0.5 μg) were transfected into 293T cells as described above. After 24 hours, 293T cells were trypsinized, mixed with MDCK cells and replated. Culture supernatants were harvested after 7 day...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

Cloned, i.e. defined, defective interfering (DI) influenza A virus is produced in embryonated hens eggs using a method which generates large quantities of DI virus material. Cloned DI virus is then used in tests on mice and ferrets given a lethal challange of wild-type influenza A virus. When cloned DI influenza A virus is co-administered with a lethal dose of virulent influenza A virus, mice areprotected compared to a control of inactivated cloned DI influenza A virus. Mice which survived the administration of cloned DI influenza A virus and infective challange virus are three weeks later still protected against lethal challange with infective virus. Control mice which received only cloned DI influenza A virus and no lethal challange are not protected three weeks later on lethal challange with infective virus. A therapeutic benefit of administering cloned DI influenza a virus is found when the administration takes place in less than 48 hours after challange with infective virus. Cloned DI influenza A virus of one subtype is found to act in vivo as an effective antiviral against the same or any other sub-type of influenza A virus. The antiviral effect has been found to have both a therapeutic and a prophylactic application against influenza A infection in humans, mammals and birds.

Description

field of invention [0001] The present invention relates to virology and the prevention and / or treatment of viral infections in animals (including birds and humans), especially influenza A. The invention also relates to the field of antiviral therapy. The present invention further relates to a process for the production of interference deficient (DI) viruses, ie "DI viruses", useful as active agents for the prophylaxis and / or treatment of viral infections. A "DI virus" is a defined, usually cloned, "interfering defect" virus. A "interfering virus" is generally a defective virus that interferes with the normal replication and infection cycle of a non-defective virus. (DI influenza virus preparations are known in the art, but there is genetic heterogeneity.) Background technique [0002] Orthomyxoviridae are RNA viruses that infect vertebrates. This family includes those viruses that cause influenza. [0003] Influenza is a viral infection of the respiratory system charact...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): A61K39/145C12N15/09C12N15/44C12N7/04C07K14/11
CPCA61K39/145A61K2039/543A61K2039/525A61K2039/5252C12N2760/16134C12N7/00A61K2039/542A61K39/12C12N7/04C12N15/09
Inventor 奈杰尔·迪默克
Owner UNIVERSITY OF WARWICK
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products