The African Swine Fever Virus Transcriptome

Abstract

African swine fever virus (ASFV) causes hemorrhagic fever in domestic pigs, presenting the biggest global threat to animal farming in recorded history. Despite the importance of ASFV, little is known about the mechanisms and regulation of ASFV transcription. Using RNA sequencing methods, we have determined total RNA abundance, transcription start sites, and transcription termination sites at single-nucleotide resolution. This allowed us to characterize DNA consensus motifs of early and late ASFV core promoters, as well as a polythymidylate sequence determinant for transcription termination. Our results demonstrate that ASFV utilizes alternative transcription start sites between early and late stages of infection and that ASFV RNA polymerase (RNAP) undergoes promoter-proximal transcript slippage at 5′ ends of transcription units, adding quasitemplated AU- and AUAU-5′ extensions to mRNAs. Here, we present the first much-needed genome-wide transcriptome study that provides unique insight into ASFV transcription and serves as a resource to aid future functional analyses of ASFV genes which are essential to combat this devastating disease. IMPORTANCE African swine fever virus (ASFV) causes incurable and often lethal hemorrhagic fever in domestic pigs. In 2020, ASF presents an acute and global animal health emergency that has the potential to devastate entire national economies as effective vaccines or antiviral drugs are not currently available (according to the Food and Agriculture Organization of the United Nations). With major outbreaks ongoing in Eastern Europe and Asia, urgent action is needed to advance our knowledge about the fundamental biology of ASFV, including the mechanisms and temporal control of gene expression. A thorough understanding of RNAP and transcription factor function, and of the sequence context of their promoter motifs, as well as accurate knowledge of which genes are expressed when and the amino acid sequence of the encoded proteins, is direly needed for the development of antiviral drugs and vaccines.

Combined short and long-read sequencing reveals a complex transcriptomic architecture of African swine fever virus

“…We detected 16 TSS isoforms of which 14 were longer and 2 shorter than the canonical transcripts. Only a single TSS isoform (MGF 300-2R) was identified by Cackett and colleagues [29] (they detected altogether 8 TSS variants), but two of our isoforms were present in their CAGE-seq non-primary TSS list (D345L, C147L).…”

“…The genome size of the various ASFV strains can significantly vary [33]. We used ASFV_HU_2018 isolate ( [31]; which is probably the same as strain Georgia 2007/1 [6]) and compared it to the strain Ba71V used for transcriptome analyis by Cackett and colleaguses in a very recent publication [29]. We obtained that Ba71V genome lacks 15,143 bps (22 genes), which is present in our ASFV_HU_2018 strain ( Supplementary Table S1).…”

“…We obtained 69.01 bp length for an average 5'-untranslated region (UTR) length, and 369.31 bp length for the average 3'-UTR lengths. We obtained the TSS of 92 and the TES of 57 earlier annotated TSSs and TESs [29].…”

“…Here we demonstrate the same phenomenon in ASFV transcripts ( Fig.2a). We compared our results with the CAGE analysis published by Cackett and colleagues [29] ( Supplementary Table S3). In total, we discovered 19 novel putative mRNAs with shorter in-frame ORFs ( Supplementary Table S2), of which 3 (A137R, CP312R, MGF 100-1L) have also been detected by Cackett and co-workers.…”

“…Although this study has provided valuable information about highly expressed ASFV genes, it has not supplied sufficient data to construct a detailed transcriptional map of the virus. In a very recent study, Cackett and co-workers reported an SRS analysis on ASFV transcriptome [29]. The authors also carried out cap analysis gene expression sequencing (CAGE) and 3'-RNA-Seq.…”