Barcoded Influenza Viruses via Segmented Packaging Signals
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Solution Overview
Problem
Current methods for deep mutational scanning of influenza viruses are limited by high error rates in sequencing, low throughput, and the inability to barcode the influenza virus genome without disrupting viral function or genome packaging, making it difficult to study mutations effectively.
Innovation Solution
The method involves duplicating and inserting a copy of the 5′ vRNA packaging signal and inserting a nucleic acid barcode between the end of the viral genome segment's open reading frame and the inserted copy of the 5′ vRNA packaging signal, allowing for the creation of barcoded influenza viruses that can be used in deep mutational scanning libraries without affecting viral function or packaging.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If a nucleic acid barcode is inserted into the influenza virus genome, then the ability to track and study viral mutations is improved, but the viral function and genome packaging are disrupted
Solution Approach 1:
The packaging signal is divided into two functional parts: the essential core sequence that maintains packaging function, and the variable region that can accommodate barcode insertion. This segmentation allows the barcode to be integrated without disrupting the critical packaging function of the viral genome.
Solution Approach 2:
The packaging signal serves as an intermediary element between the viral genome and the barcode sequence. By placing the barcode within or adjacent to the packaging signal region, the system mediates between the need for viral functionality and the need for trackable identification, allowing both requirements to be satisfied simultaneously.
2Productivity
If traditional sequencing methods are used to study viral mutations, then the process is simpler, but the error rate is high and throughput is low
Solution Approach 1:
Instead of directly sequencing the entire viral genome multiple times to achieve accurate mutation detection, the invention creates barcode copies that link specific viral sequences to unique identifiers. This copying approach allows high-throughput sequencing of barcodes to accurately track viral variants without the errors associated with direct genome sequencing.
Solution Approach 2:
The invention replaces the mechanical/chemical process of direct viral genome sequencing with a molecular tagging system using barcodes. This substitution enables high-throughput analysis by sequencing short barcode regions rather than entire viral genomes, dramatically increasing productivity while maintaining or improving measurement precision through the unique identifier system.
Data Source
AI summary
Methods to create barcoded influenza viruses without disrupting the function of the viral proteins and the proper packaging of the viral genome segments are described. The barcoded influenza viruses can be used within deep mutational scanning libraries to map influenza resistance mutations to therapeutic treatments. The libraries can also be used to predict influenza strains that may become resistant to therapeutic treatments and/or more easily evolve to infect new species. The libraries include features that allow efficient collection and assessment of informative data, obviating bottlenecks of previous approaches.