Transcriptional architecture of African swine fever virus

African swine fever virus (ASFV), a large double-stranded DNA virus, poses one of the most significant infectious disease threats to the global swine industry. ASFV transcription is particularly complex because of its unique host-independent transcription system. Using nanopore sequencing technology, we accurately identified the viral transcription start and termination sites. Combining direct RNA sequencing with experimental verification strongly supports the presence of long multicistronic RNAs. By combining this information with the viral genome annotation, we identified six novel transcriptional genes with potential functions. Furthermore, we observed transcriptional "read-through" events, in which transcription initiated at a common start site continues beyond typical termination sites, producing multiple RNA transcripts of different lengths. Additionally, via direct RNA nanopore sequencing, we identified m6A and m5C methylation modifications on viral transcripts, which are predominantly distributed at both termini of the viral genome. Compared to unmodified transcripts, modified RNAs have shorter poly(A) tails, indicating a correlation between RNA modification and poly(A) tail length. Further investigation of ASFV transcriptional architecture, "read-through" events, RNA modifications, and candidate genes may improve our understanding of the viral life cycle and pathogenicity and inform the identification of potential antiviral targets.