Split Fluorescent Reporter Protein for Viral Protease Detection
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Solution Overview
Problem
Current virus detection methods often require visual signs of infection or disrupt the sample integrity, and existing methods are not suitable for detecting viruses independently of their replication pathway without interfering with viral components or genome integrity.
Innovation Solution
Development of genetically modified fluorescent proteins that act as reporter proteins, specifically activated by viral proteases, allowing for the detection of viral infections in biological samples without prior purification of viral components, using a split fluorescent protein system that reconstitutes upon protease cleavage.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional virus detection methods (plaque assays, TCID50, PCR) are used, then virus detection capability is achieved, but sample integrity is disrupted and viral components or genome are damaged
Solution Approach 1:
The patent introduces a reporter protein as an intermediary that detects viral proteases indirectly. The reporter protein contains a cleavage site that is processed by viral proteases, leading to fluorescence emission. This intermediary system allows detection without directly contacting or damaging the virus particles themselves, thus maintaining sample integrity while achieving reliable detection.
Solution Approach 2:
The patent replaces mechanical/disruptive detection methods (plaque assays, TCID50) with a biochemical detection mechanism based on fluorescence emission. Instead of physically disrupting samples to observe cytopathic effects or extract viral components, the system uses a fluorescent reporter that emits light when processed by viral proteases, enabling non-invasive detection that preserves sample integrity.
2Reliability
If visual signs of infection (cytopathic effects) are used for detection, then virus infection can be detected, but detection timing is delayed until cell death occurs
Solution Approach 1:
The patent implements preliminary action by detecting viral protease activity early in the infection cycle, before cell death and cytopathic effects occur. The reporter protein is designed to be processed by viral proteases as soon as they are activated during infection, allowing fluorescence emission to be detected at early time points. This enables detection of viral infection before the late-stage cell death that characterizes conventional CPE-based methods.
3Measurement precision
If detection methods interfere with viral replication pathway, then detection sensitivity is improved, but virus detection becomes specific to certain replication mechanisms
Solution Approach 1:
The patent achieves universality by designing a reporter protein system that detects a common functional element (viral protease activity) shared across diverse virus families. The reporter contains a cleavage site that is recognized by proteases from various viruses (e.g., coronaviruses, flaviviruses, alphaviruses), making the detection system broadly applicable regardless of specific viral replication pathways or virus types, thereby maintaining high sensitivity across different viral infections.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Enables sensitive and specific detection of viral infections in biological samples, allowing for early detection within a few hours post-infection without disrupting the sample, and is applicable to various viruses regardless of their replication pathway.
Implementation Method 1
the invention relates to a reporter protein designed for specific activation by viral components, in particular by viral proteins, such as viral protease... Development of genetically modified fluorescent proteins that act as reporter proteins, specifically activated by viral proteases... using a split fluorescent protein system that reconstitutes upon protease cleavage
Data Source
AI summary
The invention relates to novel means and processes for the detection of a virus in a biological sample comprising cells infected by the virus. In particular, the invention relates to a fluorescent reporter protein designed as a recombinant inactive form of flipGFP suitable for specific activation by viral components in particular by viral proteins, such as viral protease, wherein the viral component recognizes a cleavage site inserted in the recombinant flipGFP. The fluorescent reporter protein is suitable for use in an in vitro method of detection of virus infection in a biological sample when the virus is related to the viral components activating the inactive form of flipGFP into an active fluorescent flipGFP in a biological sample, especially a sample comprising cells, in particular unaltered cells.