Plasmid-Based TiLV Reverse Genetics Without Fish Promoter Data
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Solution Overview
Problem
The establishment of a reverse genetics system (RGS) for tilapia lake virus (TiLV) is hindered by the lack of knowledge about fish RNA polymerase I promoter, unknown functions of TiLV genes and proteins, and the complexity of its ten-segment genome, which impedes understanding of its pathogenesis and development of vaccines or antiviral therapeutics.
Innovation Solution
A plasmid-based reverse genetics system is developed, utilizing bi-directional expression plasmids to express TiLV genomic segments in Vero and fish-derived cells, enabling the rescue of recombinant and reporter TiLVs.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If a bidirectional transcription system is used to express TiLV genomic segments, then viral gRNAs and mRNAs can be generated, but knowledge of the fish RNA polymerase I promoter is required which is currently unavailable
Solution Approach 1:
The patent introduces mammalian RNA polymerase I promoters (such as the human H1 promoter) as intermediaries to drive transcription of TiLV cDNA segments in fish cells. This mediator approach bypasses the need for native fish RNA polymerase I promoter sequences, enabling bidirectional transcription to generate both viral gRNAs and mRNAs simultaneously.
Solution Approach 2:
The patent changes the promoter parameter from unknown fish-specific sequences to well-characterized mammalian promoter sequences. This parameter substitution allows the transcription system to function without requiring prior knowledge of fish RNA polymerase I promoter architecture, while still achieving the desired bidirectional transcription in fish-derived cells.
2Reliability
If all ten TiLV genomic segments are cloned and expressed, then complete viral replication can be achieved, but the complexity of the procedure increases significantly
Solution Approach 1:
The patent merges the expression of all ten TiLV genomic segments into a single plasmid construct containing multiple cDNA segments arranged in tandem. This consolidation allows simultaneous introduction of all viral genes into host cells through a single transfection event, followed by co-transcription to generate the complete set of viral RNAs needed for virus rescue.
Solution Approach 2:
The patent segments the TiLV genome into ten individual cDNA blocks within the plasmid, each flanked by transcription start and stop signals. This segmentation enables independent cloning of each viral gene while maintaining the ability to transcribe all segments bidirectionally from a single plasmid, balancing complexity with completeness.
3Loss of information
If the functions of TiLV genes and proteins are unknown, then basic virology studies cannot be performed, but this lack of knowledge prevents development of targeted vaccines or therapeutics
Solution Approach 1:
The reverse genetics system enables TiLV to serve its own functional analysis needs. By allowing precise manipulation and expression of individual viral genes, the system generates the necessary information about gene functions that can then be used to develop targeted vaccines and therapeutics, creating a self-sustaining research platform.
Data Source
AI summary
The present disclosures relate to the establishment of a plasmid-based reverse genetic system for TiLV and use of the system to rescue recombinant and reporter TiLVs. The present disclosures also relate to the plasmids used for the rescue of TiLV and the recombinant and reporter TiLVs generated using the methods disclosed herein.