Limited adaptive mutations but deterministic genome formula variation in bluetongue virus

Arthropod-borne viruses or arboviruses alternate between vertebrates and arthropods, facing distinct selection pressures in each host. Surprisingly, mechanisms facilitating host alternation remain unstudied. Here, we investigate two phenomena potentially facilitating host alternation in bluetongue virus (BTV): adaptive mutations and genome formula variation (GFV; i.e., differences in segment copy numbers). First, we searched for mutations indicative of host adaptation in cattle, sheep, and midges infected during an epizootic. No mutation was fixed in all samples of a given host. Thus, adaptive mutations may not be a major mechanism facilitating host alternation in the studied epizootic. Second, we investigated the relationship between GFV and a proxy of viral fitness, namely, head infection in midges. We analyzed the genome formula (GF) in Culicoides imicola midges, either with or without head infection 10 days after oral inoculation. GFs differed between dissemination groups, suggesting a correlation between GF and head infection. Finally, we investigated the mechanisms underlying GF generation. Currently, two scenarios have been proposed: random generation followed by selection or directed generation through molecular mechanisms. To limit the influence of selection, we analyzed the GF after a single replication cycle in cell culture. GFs converged toward specific values across replicates. Moreover, GFs differed between arthropod and vertebrate cells. Thus, GF generation does not seem purely stochastic and is probably influenced by host-specific mechanisms. Overall, our findings highlight GFV as a mechanism potentially facilitating host alternation of BTV, and they do not strongly support an involvement of mutations in the same phenomenon. IMPORTANCE Arthropod-borne viruses are major public health concerns worldwide. These viruses follow a complex cycle involving an alternation between arthropods and vertebrates. Surprisingly, little is known about the mechanisms that could facilitate this alternation. Understanding such mechanisms could aid the development of antiviral strategies. Here, using bluetongue virus as a model, we investigated two potential mechanisms facilitating alternation. These mechanisms are adaptive mutations and genome formula variation (GFV). We found limited evidence for host-specific parallel mutations in natural populations from a single epizootic. In contrast, we observed a potential link between GFV and viral fitness. Moreover, our results suggest that molecular mechanisms generate host-specific genome formulas from the first round of cell infection.