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, alphavi-ruses produce truncated, defective viral RNAs featuring large deletions during repli-cation. 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 chikun-gunya virus (CHIKV) 181/clone 25, intracellular virus, and P2 virions and analyzed samples for D-RNA expression, followed by diversity and differential expression anal-yses. 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. Impor-tantly, these trends were likewise observed in a murine model of CHIKV AF15561 in-fection, 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. To-gether, 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 tem-plate. IMPORTANCE Our understanding of viral defective RNAs (D-RNAs), or truncated viral genomes, comes largely from passaging studies in tissue culture under artificial conditions and/or packaged viral RNAs. Here, we show that specific populations of al-phavirus D-RNAs arise de novo and that they are not packaged into virions, thus im-posing a transmission bottleneck and impeding their prior detection. This raises important questions about the roles of D-RNAs, both in nature and in tissue culture, during viral infection and whether their influence is constrained by packaging re-quirements. Further, during the course of these studies, we found a novel type of al-phavirus D-RNA that is enriched intracellularly; dubbed subgenomic D-RNAs (sgD-RNAs), they are defined by deletion boundaries between the capsid-E3 region and the E1-3' untranslated region (UTR) and are common to chikungunya, Mayaro, Sind-bis, and Aura viruses. These sgD-RNAs are enriched intracellularly and do not appear to be selectively packaged, and additionally, they may exist as subgenome-derived transcripts.