A Retrospective Cross-sectional Study on Factors Associated with HIV Testing History among Non-elderly Adults in the United States

adequate virus samples for efficacy evaluation.However, obtaining viruses or clinical samples during outbreaks is challenging.Reverse genetics offers a powerful solution to this problem by enabling the rapid and efficient production of viruses.This technique enhances containment and response strategies, thereby accelerating the development of preventive measures.Materials & Methods: We established a reverse genetics system for RNA viruses, focusing on vesicular stomatitis virus (VSV), a prototypical negative-sense RNA virus.Using a rapid singletube method, we assembled the full-length cDNA clone of the VSV antigenome into a single plasmid.This involved amplifying the antigenome as five overlapping fragments, excluding the G protein genes, and integrating regulatory elements (T7 promoter, ribozyme, and T7 terminator) into a plasmid vector (pLinker).To ensure virus rescue, we cloned minigenome plasmids encoding essential accessory proteins (N, P, and L) with regulatory elements.Co-transfection of plasmids encoding VSV antisense genome, minigenome, and T7 polymerase into BHK-21 cells facilitated virus rescue.Virus titration was performed by infecting Vero-E6 cells with the collected supernatant.Results: To construct functional VSV full-length genome and minigenome plasmids, we integrated regulatory elements like leader, trailer, and ribozyme sequences ( < 200 bp each) into the larger genome via overlap PCR approach, with a 35 bp overlap between fragments.Deleting the G protein and replacing it with eGFP produced replication-incompetent VSV.Both full-length antigenome (VSV G/GFP) and minigenome (NP, L) were successfully cloned into a plasmid using our rapid cloning method.Co-transfection with T7 polymerase generated genome-length positive-sense RNA transcripts, resulting in successful rescue of VSV G/eGFP within 24-48 hours, confirmed by eGFP signal detection using fluorescence microscopy.Titration of VSV G/eGFP virus demonstrated the effectiveness of our reverse genetics system.Discussion: Our study constructed functional VSV genome and minigenome plasmids through the rapid overlap extension approach.Substituting the G protein with eGFP facilitated quick detection of the VSV G/GFP virus which is faster than the live virus infection studies.Similar approach can be used to introduce any genes such as viral glycoproteins which can be used as vaccine delivery system.Utilizing a rapid cloning method, we efficiently inserted the genome into a plasmid vector.This validation of our reverse genetics technology for recombinant VSV synthesis provides a solid foundation for future research and therapeutic breakthroughs. Conclusion:In conclusion, our study reveals an effective reverse genetics technique for Vesicular Stomatitis Virus, which enables the rapid synthesis and rescue of recombinant VSV variants.This technology shows promise for producing reverse genetics not only for VSV but also for other RNA viruses.