Reverse Genetics of RNA Viruses

Kessler, Susanne, García-Sastre, Adolfo, Schwemmle, Martin, Ciminski, Kevin, Susanne Kessler, Adolfo García-Sastre, Martin Schwemmle, Kevin Ciminski

New World fruit bats were recently found to harbor two distinct and previously unknown influenza A viruses (IAVs) of the subtypes H17N10 and H18N11. Although viral genome sequences were detected in the liver, intestine, lung, and kidney of infected bats and the complete genome sequences have been isolated from their rectal swab samples, all attempts to isolate an infectious virus from bats in nature have failed. The lack of an infectious bat IAV isolate was overcome by reverse genetic approaches that led to the generation of an infectious virus in vitro. Using such synthetic bat IAVs enabled the identification of their unconventional cell entry via major histocompatibility complex II (MCH-II) molecules and their ability to replicate in mice, ferrets, and bats. Importantly, we also showed that these synthetic recombinant bat IAVs are not able to reassort with conventional IAVs, preventing the acquisition of enhanced transmission properties in non-bat species by reassortment with conventional IAVs. As authentic viruses are key for understanding the molecular biology of bat IAVs, in this chapter, we describe our recently established reverse genetics protocol for generating H17N10 and H18N11 in vitro. This step-by-step protocol starts with cloning of cDNA copies of the viral RNA segments into reverse genetics plasmids, followed by the generation of a highly concentrated stock and finally a method to determine viral titers.