Related Experiment Video
Updated: Mar 2, 2026
11:40
Simplified Reverse Genetics Method to Recover Recombinant Rotaviruses Expressing Reporter Proteins
Published on: April 17, 2020
9.5K
Rapid Reverse Genetics Systems for Rhabdoviruses: From Forward to Reverse and Back Again
Tobias Nolden1,2, Stefan Finke3
1Institute of Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut, Südufer 10, 17493, Greifswald, Insel Riems, Germany.
Methods in Molecular Biology (Clifton, N.J.)
|May 17, 2017
Summary
Researchers developed a new system for rapid cloning of non-segmented negative strand RNA viruses (NNSVs) to study their natural genetic diversity and pathogenesis. This advance improves reverse genetics approaches for RNA virus research.
Area of Science:
- Virology
- Molecular Biology
- Genetics
Background:
- Reverse genetics systems for non-segmented negative strand RNA viruses (NNSVs) have historically been limited by time-consuming cloning methods.
- Conventional ligase-based cloning of PCR subfragments restricts the generation of recombinant virus cDNA clones, hindering the study of natural viral genetic variability.
Purpose of the Study:
- To develop an improved reverse genetics system for rapid cloning of wild-type NNSV genome populations.
- To facilitate the investigation of natural virus functions and pathogenesis by overcoming limitations of existing cloning techniques.
Main Methods:
- Development of a novel system utilizing linear-to-linear homologous RecE/T recombination (LLHR) for inserting complete rhabdovirus cDNA populations into vector plasmids.
- LLHR allows for efficient cloning with limited prior sequence information.
Main Results:
- The new system enables rapid cloning of rhabdovirus cDNA populations.
- High cloning efficiencies were achieved.
- Direct generation of recombinant viruses from individual cDNA clones is now possible.
Conclusions:
- This LLHR-based system offers novel opportunities for forward and reverse genetics approaches to study natural rhabdovirus populations.
- The method overcomes previous limitations, allowing for a more comprehensive understanding of viral complexity and pathogenesis.
- This advance is crucial for studying RNA viruses in their natural context, minimizing cell culture adaptation effects.
Related Concept Videos
01:10Retrovirus Life Cycles
50.1K
Retroviruses have a single-stranded RNA genome that undergoes a special form of replication. Once the retrovirus has entered the host cell, an enzyme called reverse transcriptase synthesizes double-stranded DNA from the retroviral RNA genome. This DNA copy of the genome is then integrated into the host’s genome inside the nucleus via an enzyme called integrase. Consequently, the retroviral genome is transcribed into RNA whenever the host’s genome is transcribed, allowing the...
50.1K
01:29Viruses with RNA Genomes
1.1K
RNA viruses are categorized into positive-strand, negative-strand, or double-stranded groups based on their genomic structure and replication mechanisms. This classification dictates how they exploit host cellular machinery for protein synthesis and replication. Some RNA viruses also utilize reverse transcription as part of their life cycle, further diversifying their replication strategies.Positive-Strand RNA VirusesPositive-strand RNA viruses have genomes that function directly as messenger...
1.1K
02:33Retroviruses
15.1K
Retroviruses and retrotransposons both insert copies of their genetic elements into the genome of the host cell. Thus, the viral genes are passed on when the host genome is replicated or translated. A typical retroviral DNA sequence contains 3-4 genes that encode the different proteins required for its structural assembly and function as a molecular parasite. This DNA is transcribed into a single mRNA, which is very similar in structure to conventional mRNAs, i.e., it is capped at the 5’...
15.1K
00:57Viral Recombination
25.4K
Cells are sometimes infected by more than one virus at once. When two viruses disassemble to expose their genomes for replication in the same cell, similar regions of their genomes can pair together and exchange sequences in a process called recombination. Alternatively, viruses with segmented genomes can swap segments in a process called reassortment.
25.4K
02:46Genetic Screens
5.8K
Genetic screens are tools used to identify genes and mutations responsible for phenotypes of interest. Genetic screens help identify individuals or a group of people at risk of developing genetic diseases and help them with early intervention, targeted therapy, and reproductive options.
Forward genetic screens
Forward or “classical” genetic screens involve creating random mutations in an organism’s DNA using radiation, mutagens, or insertion of additional bases, which...
Forward genetic screens
Forward or “classical” genetic screens involve creating random mutations in an organism’s DNA using radiation, mutagens, or insertion of additional bases, which...
5.8K