Differential Alphavirus Defective RNA Diversity between Intracellular and Extracellular Compartments Is Driven by Subgenomic Recombination Events

Alphaviruses are positive-sense RNA arboviruses that can cause either a chronic arthritis or a potentially lethal encephalitis. Like other RNA viruses, alphaviruses produce truncated, defective viral RNAs featuring large deletions during replication. These defective RNAs (D-RNAs) have primarily been isolated from virions after high-multiplicity-of-infection passaging. Here, we aimed to characterize both intracellular and packaged viral D-RNA populations during early-passage infections under the hypothesis that D-RNAs arise de novo intracellularly that may not be packaged and thus have remained undetected. To this end, we generated next-generation sequencing libraries using RNA derived from passage 1 (P1) stock chikungunya virus (CHIKV) 181/clone 25, intracellular virus, and P2 virions and analyzed samples for D-RNA expression, followed by diversity and differential expression analyses. We found that the diversity of D-RNA species is significantly higher for intracellular D-RNA populations than P2 virions and that specific populations of D-RNAs are differentially expressed between intracellular and extracellular compartments. Importantly, these trends were likewise observed in a murine model of CHIKV AF15561 infection, as well as in vitro studies using related Mayaro, Sindbis, and Aura viruses. Additionally, we identified a novel subtype of subgenomic D-RNA that is conserved across arthritogenic alphaviruses. D-RNAs specific to intracellular populations were defined by recombination events specifically in the subgenomic region, which were confirmed by direct RNA nanopore sequencing of intracellular CHIKV RNAs. Together, these studies show that only a portion of D-RNAs generated intracellularly are packaged and D-RNAs readily arise de novo in the absence of transmitted template.