Stabilizing Influenza Virus Mutations for Fluorescent Reporter Expression
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Solution Overview
Problem
Current methods for analyzing immune responses to influenza virus infections in vivo are limited by attenuated viruses that do not accurately reflect natural infections, and the stability of fluorescent reporter proteins is often compromised, making it difficult to study the dynamics of virus infection and pathogenesis effectively.
Innovation Solution
Generation of influenza viruses expressing fluorescent proteins, such as GFP, BFP, RFP, or YFP, with specific stabilizing mutations that enhance genetic stability, allowing for stable expression and live imaging of virus-infected cells, and enabling the analysis of virus spread and differential gene expression in mouse lungs.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If viruses are generated to express fluorescent reporter proteins for analyzing immune responses, then the ability to monitor virus infection and pathogenesis is improved, but the viral genetic stability is compromised leading to attenuated viruses that do not accurately reflect natural infections
Solution Approach 1:
The patent applies parameter changes by introducing specific point mutations (S31N, T380A, E712D) at defined positions in viral proteins to optimize the balance between fluorescent protein expression stability and viral replication fidelity. These parameter changes in amino acid sequences resolve the contradiction by enhancing genetic stability without compromising monitoring capability
Solution Approach 2:
The patent creates a composite viral system that integrates fluorescent reporter genes with mutated viral proteins containing stabilizing mutations. This composite structure combines the monitoring function of fluorescent proteins with the enhanced stability provided by the mutated viral components, resolving the contradiction between measurement precision and genetic stability
2Adaptability or versatility
If heterologous gene sequences are inserted into viral gene segments, then the functionality for tracking and analysis is enhanced, but the replication stability and fidelity of the virus is reduced
Solution Approach 1:
The patent uses parameter changes by introducing specific stabilizing mutations (S31N, T380A, E712D) in viral proteins to counterbalance the destabilizing effect of heterologous gene insertions. These parameter changes restore replication stability while preserving the tracking functionality provided by the heterologous genes
Solution Approach 2:
The patent applies preliminary anti-action by pre-introducing stabilizing mutations into the viral genome before inserting heterologous genes. This preliminary modification creates a more stable genetic background that can tolerate the presence of heterologous sequences, preventing replication instability before it occurs
Data Source
AI summary
The disclosure provides for an isolated recombinant influenza virus having at least one of: a PA gene segment encoding PA with a residue at position 443 that is not arginine, a PB1 gene segment encoding PB1 with a residue at position 737 that is not lysine, a PB2 gene segment encoding PB2 with a residue at position 25 that is not valine or a residue at position 712 that is not glutamic acid, a NS gene segment encoding a NS1 with a residue at position 167 that is not proline, a HA gene segment encoding a HA with a residue at position 380 that is not threonine, or any combination thereof, and methods of making and using the virus.