Deep sequencing of serially passaged Sudan virus in guinea pigs uncovers adaptive mutations

Orthoebolaviruses are non-segmented, negative sense RNA viruses that make up the genus Orthoebolavirus in the family Filoviridae. Sudan virus (SUDV) is a highly pathogenic orthoebolavirus that causes severe disease in humans. In the last 45 years, SUDV has been responsible for several outbreaks in eastern Africa, particularly in Sudan and Uganda, with an average case fatality rate of approximately 50 %. Despite having caused numerous outbreaks, including a recent outbreak in 2022, no licensed therapeutics or prophylactics currently exist for Sudan virus disease. Small animal models like mice, hamsters, and guinea pigs have paved the way for the initial evaluation of filovirus countermeasures; however, since filoviruses are apathogenic in immunocompetent rodents, they must first be adapted through serial passaging. As a result of this process, viruses acquire genomic changes that may contribute to increased virulence and lethality. Currently, only a single immunocompetent small animal model exists for SUDV, where the virus was serially passaged in guinea pigs until uniform lethality was observed. To better understand the serial passaging process, we used next-generation sequencing to identify and quantify the mutations that arose throughout the adaptation process in guinea pigs. We identified 7 nonsynonymous and 9 synonymous mutations that were present at frequencies near 100 % at the end of serial passaging. The glycoprotein and virion protein (VP) 40 harboured many of the substitutions, most of which were nonsynonymous, while VP35 and VP24 each maintained a single nonsynonymous mutation. These results are consistent with the previously determined genome sequence, obtained by Sanger sequencing, with two notable exceptions: we identified a novel mutation in VP40, but we were unable to confirm the mutation in the genome leader, due to poor sequence coverage. This analysis has allowed us to identify adaptive hotspots within the viral genome, which may hint at the molecular determinants contributing to pathogenicity.