Plasmid-Based Lassa Virus Reverse Genetics.

Several mammarenaviruses cause hemorrhagic fever (HF) disease in humans and pose a significant public health problem in their endemic regions. The Old World (OW) mammarenavirus Lassa virus (LASV) is estimated to infect several hundred thousand people yearly in West Africa, resulting in high numbers of Lassa fever (LF) cases, a disease associated with high morbidity and mortality. No licensed vaccines are available to combat LASV infection, and anti-LASV drug therapy is limited to the off-label use of ribavirin whose efficacy remains controversial. The development of reverse genetics approaches has provided investigators with a powerful approach for the investigation of the molecular, cell biology and pathogenesis of mammarenaviruses. The use of cell-based minigenome systems has allowed examining the cis- and trans-acting factors involved in viral genome replication and gene transcription, assembly, and budding, which has facilitated the identification of several anti-mammarenavirus candidate drugs. Likewise, it is possible now to rescue infectious recombinant mammarenaviruses from cloned cDNAs containing predetermined mutations in their genomes to investigate virus-host interactions and mechanisms of viral pathogenesis. Reverse genetics have also allowed the generation of mammarenaviruses expressing foreign genes to facilitate virus detection, to identify antiviral drugs, and to generate live-attenuated vaccine (LAV) candidates. Likewise, reverse genetics techniques have allowed the generation of single-cycle infectious, reporter-expressing mammarenaviruses to study some aspects of the biology of HF-causing human mammarenavirus without the need of high security biocontainment laboratories. In this chapter, we describe the experimental procedures to generate recombinant (r)LASV using state-of-the-art plasmid-based reverse genetics.