Gábor Torma

Long-read RNA sequencing (lrRNA-seq) enables direct reconstruction of full-length transcripts, yet existing annotation tools show variable performance across genomes and library chemistries, particularly for novel isoforms. We present LoRTIA Plus, a chemistry-agnostic suite for transcriptome annotation and reconstruction from lrRNA-seq data. LoRTIA Plus first detects and filters transcription start sites (TSSs), transcription end sites (TESs), and introns using adapter-aware and quality-based criteria, and evaluates read support before assembling high-confidence transcript models. We benchmarked LoRTIA Plus against bambu, FLAIR, IsoQuant, and NAGATA on KSHV transcriptomes with dense overlap, using a validated literature-supported boundary set, and on transcriptomes from three human cell lines from the Long Read RNA-seq Genome Annotation Assessment Project (LRGASP) sequenced with five long-read chemistries. On KSHV, LoRTIA Plus achieved the highest F1 scores for TSSs, TESs, and transcripts in both direct-cDNA and direct-RNA datasets by improving recall without sacrificing precision. Across human datasets, LoRTIA Plus consistently ranked among the top boundary annotators across all chemistries and was the best-performing tool in PCR-based libraries, while remaining highly competitive on native RNA. Junction- and isoform-level analyses show that LoRTIA Plus yields a rich, reproducible repertoire of novel isoforms and transcript boundaries from viral to human transcriptomes.

Here we discuss three veterinary alphaherpesviruses—pseudorabies virus, equid alphaherpesvirus 1, and bovine alphaherpesvirus 1—that were instrumental in uncovering the true extent of transcriptome complexity through long-read RNA sequencing, which earlier short-read approaches could not resolve. We focus on three major transcriptomic features whose discovery and characterization relied heavily on these viral models: (i) widespread transcriptional overlaps that complicate read assignment and may drive transcriptional interference; (ii) diverse transcript isoforms arising from alternative 5′ and 3′ transcript termini, as well as splicing; and (iii) non-coding RNAs clustered near replication origins that illuminate potential replication–transcription interactions on a shared nuclear template. Long-read viromics in these veterinary systems has additionally served as a stringent benchmark for transcript callers and annotation pipelines, because the extreme density of overlaps and co-terminal transcript families exposes errors that often go unnoticed in typical mammalian transcriptomes. This has made veterinary herpesvirus datasets disproportionately influential in shaping best practices for full-length isoform calling, transcript end mapping, and artifact-robust cDNA library handling. We also discuss animal gammaherpesviruses as proxies for human gammaherpesviruses, allowing experimental investigation of viral programs difficult to study in human infection. Finally, we describe pseudorabies virus applications as a retrograde transneuronal tracer.

Here we discuss three veterinary alphaherpesviruses—pseudorabies virus, equid alphaherpesvirus 1, and bovine alphaherpesvirus 1—that were instrumental in uncovering the true extent of transcriptome complexity through long-read RNA sequencing, which earlier short-read approaches could not resolve. We focus on three major transcriptomic features whose discovery and characterization relied heavily on these viral models: (i) widespread transcriptional overlaps that complicate read assignment and may drive transcriptional interference; (ii) diverse transcript isoforms arising from alternative 5′ and 3′ transcript termini, as well as splicing; and (iii) non-coding RNAs clustered near replication origins that illuminate replication–transcription interactions on a shared nuclear template. Long-read viromics in these veterinary systems has additionally served as a stringent benchmark for transcript callers and annotation pipelines, because the extreme density of overlaps and co-terminal transcript families exposes errors that often go unnoticed in typical mammalian transcriptomes. This has made veterinary herpesvirus datasets disproportionately influential in shaping best practices for full-length isoform calling, transcript end mapping, and artifact-robust cDNA library handling. We also discuss animal gammaherpesviruses as proxies for human gammaherpesviruses, allowing experimental investigation of viral programs difficult to study in human infection. Finally, we describe pseudorabies virus applications as a retrograde transneuronal tracer.

Caviid gammaherpesvirus 1 (CaGHV-1), formerly known as the guinea pig herpes-like virus, is an oncogenic gammaherpesvirus with a sequenced genome but an as-yet uncharacterized transcriptome. Using nanopore long-read RNA sequencing, we annotated the CaGHV-1 genome and constructed a detailed transcriptomic atlas. Our findings reveal diverse viral mRNAs and non-coding RNAs, along with mapped promoter elements for each viral gene. We demonstrated that the CaGHV-1 RTA lytic cycle transcription factor activates its own promoter, similar to Kaposi’s sarcoma-associated herpesvirus (KSHV), and that the CaGHV-1 ORF50 promoter responds to RTA proteins from other gammaherpesviruses, highlighting the evolutionary conservation of RTA-mediated transcriptional mechanisms. Additionally, our analysis uncovered extensive transcriptional overlap within the viral genome, suggesting a role in regulating global gene expression. Given its tumorigenic properties, broad host range, and non-human pathogenicity, this work establishes CaGHV-1 as a promising small animal model for investigating human gammaherpesvirus pathogenesis. IMPORTANCE: The molecular underpinnings of gammaherpesvirus pathogenesis remain poorly understood, partly due to limited animal models. This study provides the first comprehensive transcriptomic atlas of CaGHV-1, highlighting both coding and non-coding RNAs and revealing regulatory elements that drive viral gene expression. Functional studies of the CaGHV-1 RTA transcription factor demonstrated its ability to self-activate and cross-activate promoters from homologous gammaherpesviruses, reflecting conserved mechanisms of transcriptional control. These findings solidify CaGHV-1 as a unique and versatile small animal model, offering new opportunities to investigate gammaherpesvirus replication, transcriptional regulation, and tumorigenesis in a controlled experimental system.

A member of the Varicellovirus genus of herpesviruses, equid alphaherpesvirus 1 (EHV-1) was subjected to timescale profiling in this research. We employed cap analysis gene expression sequencing (CAGE-Seq) on Illumina platform to determine the transcript start sites alongside long-read direct cDNA sequencing (dcDNA-Seq) on Oxford Nanopore Technology platform to detect full-length viral transcripts. Samples were collected at nine distinct stages of the viral lifecycle, with triplicates taken at each stage. We also applied protein synthesis inhibition to determine the immediate-early gene expression of the virus. Analysis of the time-course expression of the viral transcripts using dcDNA-Seq allowed their kinetic categorization. Moreover, the EHV-1 transcriptome was reannotated using CAGE-Seq, long-read dcDNA-Seq, and our previous data obtained from native RNA sequencing.

In the last couple of years, the rapid advances and decreasing costs of sequencing technologies have revolutionized transcriptomic research. Long-read sequencing (LRS) techniques are able to detect full-length RNA molecules in a single run without the need for additional assembly steps. LRS studies have revealed an unexpected transcriptomic complexity in a variety of organisms, including viruses. A number of transcripts with proven or putative regulatory role, mapping close to or overlapping the replication origins (Oris) and the nearby transcription activator genes, have been described in herpesviruses. In this study, we applied both newly generated and previously published LRS and short-read sequencing datasets to discover additional Ori-proximal transcripts in nine herpesviruses belonging to all of the three subfamilies (alpha, beta and gamma). We identified novel long non-coding RNAs (lncRNAs), as well as splice and length isoforms of mRNAs and lncRNAs. Furthermore, our analysis disclosed an intricate meshwork of transcriptional overlaps at the examined genomic regions. Our results suggest the existence of a ‘super regulatory center’, which controls both the replication and the global transcription through multilevel interactions between the molecular machineries.

The recent Monkeypox outbreak showed the importance of studying the basic biology of orthopoxviruses. However, the transcriptome of its causative agent has not been investigated before neither with short-, nor with long-read sequencing approaches. This Oxford Nanopore long-read RNA-Sequencing dataset fills this gap. Our direct cDNA and native RNA sequencing data enable the in-depth characterization of the transcriptomic architecture and dynamics of the gene expressions of monkeypox virus; and also the deeper understanding of the changes it causes in the host cells on a transcriptome level.

In this study, a structural profiling of equid alphaherpesvirus 1 (EHV-1) transcriptome was carried out using next-generation (Illumina) and third-generation (Oxford Nanopore Technologies) sequencing platforms. We annotated the canonical mRNA molecules and their isoforms, including transcript start and end site isoforms, and splice variants. Additionally, a number of putative 5′-truncated mRNAs containing shorter in-frame ORFs were detected. We also demonstrated that EHV-1 produces a high number of non-coding transcripts, including antisense and intergenic RNAs. One of the most remarkable features of the EHV-1 is the generation of abundant fusion transcripts some of which encoding chimeric polypeptides. We observed a higher number of splicing and transcriptional overlaps than in related viruses. Additionally, we found that many upstream genes of tandem gene clusters have their own transcript end sites (TESs) besides the co-terminal TESs, which is rare in other alphaherpesviruses. We show here that the replication origins (OriS and OriL) of the virus are co-localized with promoter sequences and overlap with specific RNA molecules. Furthermore, we discovered a novel non-coding RNA (designated as NOIR) that overlaps the 5′-ends of the longer transcript variants encoded by the two main transactivator genes ORF64 and 65 bracketing the OriL. These all suggest the existence of a central regulatory system which controls the genome-wide transcription and the replication through a mechanism based on the interference between the machineries carrying out the synthesis of DNA and RNA.