Hypoxia and HIF-1α promote lytic de novo KSHV infection.

IMPORTANCE: The current view is that the default pathway of Kaposi's sarcoma-associated herpesvirus (KSHV) infection is the establishment of latency, which is a prerequisite for lifelong infection and viral oncogenesis. This view about KSHV infection is supported by the observations that KSHV latently infects most of the cell lines cultured in vitro in the absence of any environmental stresses that may occur in vivo. The goal of this study was to determine the effect of hypoxia, a natural stress stimulus, on primary KSHV infection. Our data indicate that hypoxia promotes euchromatin formation on the KSHV genome following infection and supports lytic de novo KSHV infection. We also discovered that hypoxia-inducible factor-1α is required and sufficient for allowing lytic KSHV infection. Based on our results, we propose that hypoxia promotes lytic de novo infection in cells that otherwise support latent infection under normoxia; that is, the environmental conditions can determine the outcome of KSHV primary infection.

Evaluation of microhardness of short fiber-reinforced composites inside the root canal after different light curing methods - An in vitro study.

OBJECTIVES: Short fiber-reinforced composite (SFRC) materials make it possible to reinforce root canal treated teeth with individualized, directly layered intraradicular posts (the Bioblock technique). The question arises, however, as to whether the photopolymerization of the material is sufficient deep within the root canal space and if it can be improved through different light-conducting options. Our study aimed to investigate the hardness of intraradicular SFRC material applied using the Bioblock technique and cured with various illumination methods, as measured through nanoindentation. MATERIALS AND METHODS: For this investigation, thirty plastic artificial teeth that had undergone root canal treatment were selected. These teeth were randomly divided into six study groups (Group 1-6; each group consisting of 5 teeth). The restoration procedures involved the use of SFRC or conventional composite materials, placed 6 mm apically from the root canal orifice. In Group 1 and 2, a conventional composite was used, whereas in Group 3-6, SFRC was employed for interradicular reinforcement (with a layered technique in Group 3 and 4 and a bulk-fill technique in Group 5 and 6). A modified light source was utilized for photopolymerization in Group 2, 4, and 6, whereas in Group 3 and 5, the polymerization light was directed through a prefabricated glass fiber posts. The control group (Group 1) utilized conventional composite material with a standard light-curing method. Following embedding and sectioning, the hardness of the composite materials was measured at 2 mm intervals within the root canal (1st, 2nd, 3rd measurements, in the coronal to apical direction). RESULTS: During the 1st measurement, light curing conducted through the glass fiber posts (Group 3 and 5) led to markedly higher hardness levels compared to the groups restored with conventional composite (control group with p = 0.002, p = 0.001, and Group 2 with p = 0.043, p = 0.034, respectively). In the 2nd measurement, only Group 5 demonstrated significantly greater hardness in comparison to the control group (p = 0.003) and Group 2 (p = 0.015). However, in the 3rd measurement, no statistically significant differences were observed among the groups. CONCLUSION: light curing through the glass fiber post provides outstanding hardness for the SFRC material in the apical layer in the root canal.

KSHV 3.0: a state-of-the-art annotation of the Kaposi's sarcoma-associated herpesvirus transcriptome using cross-platform sequencing.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a large, oncogenic DNA virus belonging to the gammaherpesvirus subfamily. KSHV has been extensively studied with various high-throughput RNA-sequencing approaches to map the transcription start and end sites, the splice junctions, and the translation initiation sites. Despite these efforts, the comprehensive annotation of the viral transcriptome remains incomplete. In the present study, we generated a long-read sequencing data set of the lytic and latent KSHV transcriptome using native RNA and direct cDNA-sequencing methods. This was supplemented with Cap Analysis of Gene Expression sequencing based on a short-read platform. We also utilized data sets from previous publications for our analysis. As a result of this combined approach, we have identified a number of novel viral transcripts and RNA isoforms and have either corroborated or improved the annotation of previously identified viral RNA molecules, thereby notably enhancing our comprehension of the transcriptomic architecture of the KSHV genome. We also evaluated the coding capability of transcripts previously thought to be non-coding by integrating our data on the viral transcripts with translatomic information from other publications.IMPORTANCEDeciphering the viral transcriptome of Kaposi's sarcoma-associated herpesvirus is of great importance because we can gain insight into the molecular mechanism of viral replication and pathogenesis, which can help develop potential targets for antiviral interventions. Specifically, the identification of substantial transcriptional overlaps by this work suggests the existence of a genome-wide interference between transcriptional machineries. This finding indicates the presence of a novel regulatory layer, potentially controlling the expression of viral genes.

Mobilisation and analyses of publicly available SARS-CoV-2 data for pandemic responses.

The COVID-19 pandemic has seen large-scale pathogen genomic sequencing efforts, becoming part of the toolbox for surveillance and epidemic research. This resulted in an unprecedented level of data sharing to open repositories, which has actively supported the identification of SARS-CoV-2 structure, molecular interactions, mutations and variants, and facilitated vaccine development and drug reuse studies and design. The European COVID-19 Data Platform was launched to support this data sharing, and has resulted in the deposition of several million SARS-CoV-2 raw reads. In this paper we describe (1) open data sharing, (2) tools for submission, analysis, visualisation and data claiming (e.g. ORCiD), (3) the systematic analysis of these datasets, at scale via the SARS-CoV-2 Data Hubs as well as (4) lessons learnt. This paper describes a component of the Platform, the SARS-CoV-2 Data Hubs, which enable the extension and set up of infrastructure that we intend to use more widely in the future for pathogen surveillance and pandemic preparedness.

Hybrid sequencing discloses unique aspects of the transcriptomic architecture in equid alphaherpesvirus 1.

This study employed both short-read sequencing (SRS, Illumina) and long-read sequencing (LRS Oxford Nanopore Technologies) platforms to conduct a comprehensive analysis of the equid alphaherpesvirus 1 (EHV-1) transcriptome. The study involved the annotation of canonical mRNAs and their transcript variants, encompassing transcription start site (TSS) and transcription end site (TES) isoforms, in addition to alternative splicing forms. Furthermore, the study revealed the presence of numerous non-coding RNA (ncRNA) molecules, including intergenic and antisense transcripts, produced by EHV-1. An intriguing finding was the abundant production of chimeric transcripts, some of which potentially encode fusion polypeptides. Moreover, EHV-1 exhibited a greater incidence of transcriptional overlaps and splicing compared to related viruses. It is noteworthy that many genes have their unique TESs along with the co-terminal transcription ends, a characteristic scarcely seen in other alphaherpesviruses. The study also identified transcripts that overlap the replication origins of the virus. Moreover, a novel ncRNA, referred to as NOIR, was found to intersect with the 5'-ends of longer transcript isoform specified by the major transactivator genes ORF64 and ORF65, surrounding the OriL. These findings together imply the existence of a key regulatory mechanism that governs both transcription and replication through, among others, a process that involves interference between the DNA and RNA synthesis machineries.

KSHV 3.0: A State-of-the-Art Annotation of the Kaposi's Sarcoma-Associated Herpesvirus Transcriptome Using Cross-Platform Sequencing.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a large, oncogenic DNA virus belonging to the gammaherpesvirus subfamily. KSHV has been extensively studied with various high-throughput RNA-sequencing approaches to map the transcription start and end sites, the splice junctions, and the translation initiation sites. Despite these efforts, the comprehensive annotation of the viral transcriptome remains incomplete. In the present study, we generated a long-read sequencing dataset of the lytic and latent KSHV transcriptome using native RNA and direct cDNA sequencing methods. This was supplemented with CAGE sequencing based on a short-read platform. We also utilized datasets from previous publications for our analysis. As a result of this combined approach, we have identified a number of novel viral transcripts and RNA isoforms and have either corroborated or improved the annotation of previously identified viral RNA molecules, thereby notably enhancing our comprehension of the transcriptomic architecture of the KSHV genome. We also evaluated the coding capability of transcripts previously thought to be non-coding, by integrating our data on the viral transcripts with translatomic information from other publications.

In-depth Temporal Transcriptome Profiling of Monkeypox and Host Cells using Nanopore Sequencing.

The recent human Monkeypox outbreak underlined the importance of studying 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. It will enable the in-depth characterization of the transcriptomic architecture of the monkeypox virus, and may even make possible to annotate novel host transcripts. Moreover, our direct cDNA and native RNA sequencing reads will allow the estimation of gene expression changes of both the virus and the host cells during the infection. Overall, our study will lead to a deeper understanding of the alterations caused by the viral infection on a transcriptome level.

Identification of herpesvirus transcripts from genomic regions around the replication origins.

Long-read sequencing (LRS) techniques enable the identification of full-length RNA molecules in a single run eliminating the need for additional assembly steps. LRS research has exposed unanticipated transcriptomic complexity in various organisms, including viruses. Herpesviruses are known to produce a range of transcripts, either close to or overlapping replication origins (Oris) and neighboring genes related to transcription or replication, which possess confirmed or potential regulatory roles. In our research, we employed both new and previously published LRS and short-read sequencing datasets to uncover additional Ori-proximal transcripts in nine herpesviruses from all three subfamilies (alpha, beta and gamma). We discovered novel long non-coding RNAs, as well as splice and length isoforms of mRNAs. Moreover, our analysis uncovered an intricate network of transcriptional overlaps within the examined genomic regions. We demonstrated that herpesviruses display distinct patterns of transcriptional overlaps in the vicinity of or at the Oris. Our findings suggest the existence of a 'super regulatory center' in the genome of alphaherpesviruses that governs the initiation of both DNA replication and global transcription through multilayered interactions among the molecular machineries.

Whole genomes from bacteria collected at diagnostic units around the world 2020.

The Two Weeks in the World research project has resulted in a dataset of 3087 clinically relevant bacterial genomes with pertaining metadata, collected from 59 diagnostic units in 35 countries around the world during 2020. A relational database is available with metadata and summary data from selected bioinformatic analysis, such as species prediction and identification of acquired resistance genes.

In-Depth Temporal Transcriptome Profiling of an Alphaherpesvirus Using Nanopore Sequencing.

In this work, a long-read sequencing (LRS) technique based on the Oxford Nanopore Technology MinION platform was used for quantifying and kinetic characterization of the poly(A) fraction of bovine alphaherpesvirus type 1 (BoHV-1) lytic transcriptome across a 12-h infection period. Amplification-based LRS techniques frequently generate artefactual transcription reads and are biased towards the production of shorter amplicons. To avoid these undesired effects, we applied direct cDNA sequencing, an amplification-free technique. Here, we show that a single promoter can produce multiple transcription start sites whose distribution patterns differ among the viral genes but are similar in the same gene at different timepoints. Our investigations revealed that the circ gene is expressed with immediate-early (IE) kinetics by utilizing a special mechanism based on the use of the promoter of another IE gene (bicp4) for the transcriptional control. Furthermore, we detected an overlap between the initiation of DNA replication and the transcription from the bicp22 gene, which suggests an interaction between the two molecular machineries. This study developed a generally applicable LRS-based method for the time-course characterization of transcriptomes of any organism.

High temporal resolution Nanopore sequencing dataset of SARS-CoV-2 and host cell RNAs.

BACKGROUND: Recent studies have disclosed the genome, transcriptome, and epigenetic compositions of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the effect of viral infection on gene expression of the host cells. It has been demonstrated that, besides the major canonical transcripts, the viral genome also codes for noncanonical RNA molecules. While the structural characterizations have revealed a detailed transcriptomic architecture of the virus, the kinetic studies provided poor and often misleading results on the dynamics of both the viral and host transcripts due to the low temporal resolution of the infection event and the low virus/cell ratio (multiplicity of infection [MOI] = 0.1) applied for the infection. It has never been tested whether the alteration in the host gene expressions is caused by aging of the cells or by the viral infection. FINDINGS: In this study, we used Oxford Nanopore's direct cDNA and direct RNA sequencing methods for the generation of a high-coverage, high temporal resolution transcriptomic dataset of SARS-CoV-2 and of the primate host cells, using a high infection titer (MOI = 5). Sixteen sampling time points ranging from 1 to 96 hours with a varying time resolution and 3 biological replicates were used in the experiment. In addition, for each infected sample, corresponding noninfected samples were employed. The raw reads were mapped to the viral and to the host reference genomes, resulting in 49,661,499 mapped reads (54,62 Gbs). The genome of the viral isolate was also sequenced and phylogenetically classified. CONCLUSIONS: This dataset can serve as a valuable resource for profiling the SARS-CoV-2 transcriptome dynamics, the virus-host interactions, and the RNA base modifications. Comparison of expression profiles of the host gene in the virally infected and in noninfected cells at different time points allows making a distinction between the effect of the aging of cells in culture and the viral infection. These data can provide useful information for potential novel gene annotations and can also be used for studying the currently available bioinformatics pipelines.

Transcriptome dataset of six human pathogen RNA viruses generated by nanopore sequencing.

Long-read sequencing (LRS) approaches shed new light on the complexity of viral (Kakuk et al., 2021 [1]; Boldogkoi et al., 2019 [2]; Depledge et a., 2019 [3]), bacterial (Yan et al., 2018 [4]) and eukaryotic (Tilgner et al., 2014 [5]) transcriptomes. Emerging RNA viruses are zoonotic (Woolhouse et al., 2016 [6]) and create public health problems, e.g. influenza pandemic caused by H1N1 virus in (Fraser et al., 2009 [7]), as well as the current SARS-CoV-2 pandemic (Kim et al., 2020 [8]). In this study, we carried out nanopore sequencing for generating transcriptomic data valuable for structural and kinetic profiling of six important human pathogen RNA viruses, the H1N1 subtype of Influenza A virus (IVA), the Zika virus (ZIKV), the West Nile virus (WNV), the Crimean-Congo hemorrhagic fever virus (CCHFV), the Coxsackievirus [group B serotype 5 (CVB5)] and the Vesicular stomatitis Indiana virus (VSIV), and the response of host cells upon viral infection. The raw sequencing data were filtered during basecalling and only high quality reads (Qscore ≥ 7) were mapped to the appropriate viral and host genomes. Length distribution of sequencing reads were assessed and statistics of data were plotted by the ReadStat.4 python script. The datasets can be used to profile the transcriptomic landscape of RNA viruses, provide information for novel gene annotations, can serve as resource for studying the virus-host interactions, and for the analysis of RNA base modifications. These datasets can be used to compare the different sequencing techniques, library preparation approaches, bioinformatics pipelines, and to analyze the RNA profiles of viruses with small RNA genomes.

Time course profiling of host cell response to herpesvirus infection using nanopore and synthetic long-read transcriptome sequencing.

Third-generation sequencing is able to read full-length transcripts and thus to efficiently identify RNA molecules and transcript isoforms, including transcript length and splice isoforms. In this study, we report the time-course profiling of the effect of bovine alphaherpesvirus type 1 on the gene expression of bovine epithelial cells using direct cDNA sequencing carried out on MinION device of Oxford Nanopore Technologies. These investigations revealed a substantial up- and down-regulatory effect of the virus on several gene networks of the host cells, including those that are associated with antiviral response, as well as with viral transcription and translation. Additionally, we report a large number of novel bovine transcript isoforms identified by nanopore and synthetic long-read sequencing. This study demonstrates that viral infection causes differential expression of host transcript isoforms. We could not detect an increased rate of transcriptional readthroughs as described in another alphaherpesvirus. According to our knowledge, this is the first report on the use of LoopSeq for the analysis of eukaryotic transcriptomes. This is also the first report on the application of nanopore sequencing for the kinetic characterization of cellular transcriptomes. This study also demonstrates the utility of nanopore sequencing for the characterization of dynamic transcriptomes in any organisms.

Meta-analytic approach for transcriptome profiling of herpes simplex virus type 1.

In this meta-analysis, we re-analysed and compared herpes simplex virus type 1 transcriptomic data generated by eight studies using various short- and long-read sequencing techniques and different library preparation methods. We identified a large number of novel mRNAs, non-coding RNAs and transcript isoforms, and validated many previously published transcripts. Here, we present the most complete HSV-1 transcriptome to date. Furthermore, we also demonstrate that various sequencing techniques, including both cDNA and direct RNA sequencing approaches, are error-prone, which can be circumvented by using integrated approaches. This work draws attention to the need for using multiple sequencing approaches and meta-analyses in transcriptome profiling studies to obtain reliable results.

Extended Applications of the Depth-Sensing Indentation Method.

The depth-sensing indentation method has been applied for almost 30 years. In this review, a survey of several extended applications developed during the last three decades is provided. In depth-sensing indentation measurements, the load and penetration depth data are detected as a function of time, in most cases at controlled loading rates. Therefore, beside the determination of hardness and Young's modulus, different deformation mechanisms and many other dynamic characteristics and phenomena, such as the dynamic elastic modulus, load-induced phase transition, strain rate sensitivity, etc. can be studied. These extended applications of depth-sensing indentation measurements are briefly described and reviewed.

Time-course profiling of bovine alphaherpesvirus 1.1 transcriptome using multiplatform sequencing.

Long-read sequencing (LRS) has become a standard approach for transcriptome analysis in recent years. Bovine alphaherpesvirus 1 (BoHV-1) is an important pathogen of cattle worldwide. This study reports the profiling of the dynamic lytic transcriptome of BoHV-1 using two long-read sequencing (LRS) techniques, the Oxford Nanopore Technologies MinION, and the LoopSeq synthetic LRS methods, using multiple library preparation protocols. In this work, we annotated viral mRNAs and non-coding transcripts, and a large number of transcript isoforms, including transcription start and end sites, as well as splice variants of BoHV-1. Our analysis demonstrated an extremely complex pattern of transcriptional overlaps.

Multiple Long-Read Sequencing Survey of Herpes Simplex Virus Dynamic Transcriptome.

Long-read sequencing (LRS) has become increasingly important in RNA research due to its strength in resolving complex transcriptomic architectures. In this regard, currently two LRS platforms have demonstrated adequate performance: the Single Molecule Real-Time Sequencing by Pacific Biosciences (PacBio) and the nanopore sequencing by Oxford Nanopore Technologies (ONT). Even though these techniques produce lower coverage and are more error prone than short-read sequencing, they continue to be more successful in identifying polycistronic RNAs, transcript isoforms including splice and transcript end variants, as well as transcript overlaps. Recent reports have successfully applied LRS for the investigation of the transcriptome of viruses belonging to various families. These studies have substantially increased the number of previously known viral RNA molecules. In this work, we used the Sequel and MinION technique from PacBio and ONT, respectively, to characterize the lytic transcriptome of the herpes simplex virus type 1 (HSV-1). In most samples, we analyzed the poly(A) fraction of the transcriptome, but we also performed random oligonucleotide-based sequencing. Besides cDNA sequencing, we also carried out native RNA sequencing. Our investigations identified more than 2,300 previously undetected transcripts, including coding, and non-coding RNAs, multi-splice transcripts, as well as polycistronic and complex transcripts. Furthermore, we found previously unsubstantiated transcriptional start sites, polyadenylation sites, and splice sites. A large number of novel transcriptional overlaps were also detected. Random-primed sequencing revealed that each convergent gene pair produces non-polyadenylated read-through RNAs overlapping the partner genes. Furthermore, we identified novel replication-associated transcripts overlapping the HSV-1 replication origins, and novel LAT variants with very long 5' regions, which are co-terminal with the LAT-0.7kb transcript. Overall, our results demonstrated that the HSV-1 transcripts form an extremely complex pattern of overlaps, and that entire viral genome is transcriptionally active. In most viral genes, if not in all, both DNA strands are expressed.