Time-course Profiling of Bovine Herpesvirus Type 1 and Host Cell Transcriptomes using Multiplatform Sequencing (original) (raw)
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Scientific Reports, 2017
Herpesvirus gene expression is co-ordinately regulated and sequentially ordered during productive infection. The viral genes can be classified into three distinct kinetic groups: immediate-early, early, and late classes. In this study, a massively parallel sequencing technique that is based on PacBio Single Molecule Real-time sequencing platform, was used for quantifying the poly(A) fraction of the lytic transcriptome of pseudorabies virus (PRV) throughout a 12-hour interval of productive infection on PK-15 cells. Other approaches, including microarray, real-time RT-PCR and Illumina sequencing are capable of detecting only the aggregate transcriptional activity of particular genomic regions, but not individual herpesvirus transcripts. However, SMRT sequencing allows for a distinction between transcript isoforms, including length-and splice variants, as well as between overlapping polycistronic RNA molecules. The non-amplified Isoform Sequencing (Iso-Seq) method was used to analyse the kinetic properties of the lytic PRV transcripts and to then classify them accordingly. Additionally, the present study demonstrates the general utility of long-read sequencing for the time-course analysis of global gene expression in practically any organism.
In-Depth Temporal Transcriptome Profiling of an Alphaherpesvirus Using Nanopore Sequencing
Viruses
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 th...
Multiple Long-read Sequencing Survey of Herpes Simplex Virus Lytic 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 transcript isoforms including polycistronic and multi-spliced RNA molecules, 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...
Transcriptomic study of Herpes simplex virus type-1 using full-length sequencing techniques
Scientific Data
Herpes simplex virus type-1 (HSV-1) is a human pathogenic member of the Alphaherpesvirinae subfamily of herpesviruses. The HSV-1 genome is a large double-stranded DNA specifying about 85 protein coding genes. The latest surveys have demonstrated that the HSV-1 transcriptome is much more complex than it had been thought before. Here, we provide a long-read sequencing dataset, which was generated by using the RSII and Sequel systems from Pacific Biosciences (PacBio), as well as MinION sequencing system from Oxford Nanopore Technologies (ONT). This dataset contains 39,096 reads of inserts (ROIs) mapped to the HSV-1 genome (X14112) in RSII sequencing, while Sequel sequencing yielded 77,851 ROIs. The MinION cDNA sequencing altogether resulted in 158,653 reads, while the direct RNA-seq produced 16,516 reads. This dataset can be utilized for the identification of novel HSV RNAs and transcripts isoforms, as well as for the comparison of the quality and length of the sequencing reads derived from the currently available longread sequencing platforms. The various library preparation approaches can also be compared with each other. Design Type(s) transcription profiling by high throughput sequencing design • parallel group design Measurement Type(s) total RNA Technology Type(s) RNA sequencing assay Factor Type(s) temporal_interval • assay material selection • cap analysis of gene expression assay • enzymatic amplification • size fractionation • nucleic acid library construction protocol • Technology Platform Sample Characteristic(s) Human alphaherpesvirus 1
Differentiation of bovine herpesvirus1 subtypes based on UL0.5 gene sequencing
VirusDisease, 2018
Infectious bovine rhinotracheitis/infectious pustular vulvovaginitis is one of the high economic importance diseases of cattle and caused by bovine herpesvirus1 (BoHV1). Based on the restriction endonuclease fingerprinting of viral DNA, the BoHV1 can be divided into three subtypes viz., BoHV1.1, 1.2a, and 1.2b. Since this method requires a pure viral DNA, it is time-consuming and labour intense. In the current study, the UL0.5 gene based PCR sequencing has been used for the subtyping of BoHV1. Out of five isolates, four had BoHV1-like signatures and one isolate had BoHV1.2-like signatures. Further, these viruses phylogenetically clustered under the respective subtypes. These results indicate that the UL 0.5 gene based PCR sequencing could be used as an alternate method of subtyping of BoHV1.
Virology, 2018
Bovine herpesvirus-1 (BoHV-1) is a major pathogen affecting cattle worldwide causing primarily respiratory illness referred to as infectious bovine rhinotracheitis (IBR), along with reproductive disorders including abortion and infertility in cattle. While modified live vaccines (MLVs) effectively induce immune response against BoHV-1, they are implicated in disease outbreaks in cattle. Current diagnostic methods cannot distinguish between MLVs and field strains of BoHV-1. We performed whole genome sequencing of 18 BoHV-1 isolates from Pennsylvania and Minnesota along with five BoHV-1 vaccine strains using the Illumina Miseq platform. Based on nucleotide polymorphisms (SNPs) the sequences were clustered into three groups with two different vaccine groups and one distinct cluster of field isolates. Using this information, we developed a novel SNP-based PCR assay that can allow differentiation of vaccine and clinical strains and help accurately determine the incidence of BoHV-1 and th...
Sequencing of bovine herpesvirus 4 v.test strain reveals important genome features
Virology Journal, 2011
Background Bovine herpesvirus 4 (BoHV-4) is a useful model for the human pathogenic gammaherpesviruses Epstein-Barr virus and Kaposi's Sarcoma-associated Herpesvirus. Although genome manipulations of this virus have been greatly facilitated by the cloning of the BoHV-4 V.test strain as a Bacterial Artificial Chromosome (BAC), the lack of a complete genome sequence for this strain limits its experimental use. Methods In this study, we have determined the complete sequence of BoHV-4 V.test strain by a pyrosequencing approach. Results The long unique coding region (LUR) consists of 108,241 bp encoding at least 79 open reading frames and is flanked by several polyrepetitive DNA units (prDNA). As previously suggested, we showed that the prDNA unit located at the left prDNA-LUR junction (prDNA-G) differs from the other prDNA units (prDNA-inner). Namely, the prDNA-G unit lacks the conserved pac-2 cleavage and packaging signal in its right terminal region. Based on the mechanisms of cle...
PLOS ONE, 2016
Whole transcriptome studies have become essential for understanding the complexity of genetic regulation. However, the conventionally applied short-read sequencing platforms cannot be used to reliably distinguish between many transcript isoforms. The Pacific Biosciences (PacBio) RS II platform is capable of reading long nucleic acid stretches in a single sequencing run. The pseudorabies virus (PRV) is an excellent system to study herpesvirus gene expression and potential interactions between the transcriptional units. In this work, non-amplified and amplified isoform sequencing protocols were used to characterize the poly(A +) fraction of the lytic transcriptome of PRV, with the aim of a complete transcriptional annotation of the viral genes. The analyses revealed a previously unrecognized complexity of the PRV transcriptome including the discovery of novel protein-coding and non-coding genes, novel mono-and polycistronic transcription units, as well as extensive transcriptional overlaps between neighboring and distal genes. This study identified non-coding transcripts overlapping all three replication origins of the PRV, which might play a role in the control of DNA synthesis. We additionally established the relative expression levels of gene products. Our investigations revealed that the whole PRV genome is utilized for transcription, including both DNA strands in all coding and intergenic regions. The genome-wide occurrence of transcript overlaps suggests a crosstalk between genes through a network formed by interacting transcriptional machineries with a potential function in the control of gene expression.
Genome of Bovine Herpesvirus 5
Journal of Virology, 2003
Here we present the complete genomic sequence of bovine herpesvirus 5 (BHV-5), an alphaherpesvirus responsible for fatal meningoencephalitis in cattle. The 138,390-bp genome encodes 70 putative proteins and resembles the ␣2 subgroup of herpesviruses in genomic organization and gene content. BHV-5 is very similar to BHV-1, the etiological agent of infectious bovine rhinotracheitis, as reflected by the high level of amino acid identity in their protein repertoires (average, 82%). The highest similarity to BHV-1 products (>95% amino acid identity) is found in proteins involved in viral DNA replication and processing (UL5, UL15, UL29, and UL39) and in virion proteins (UL14, UL19, UL48, and US6). Among the least conserved (<75%) are the homologues of immediate-early (IE) proteins BICP0, BICP4, and BICP22, the three proteins being longer in BHV-5 than in BHV-1. The structure of the BHV-5 latency-related (LR) region departs markedly from that of BHV-1 in both coding and transcriptional regulatory regions. Given the potential significance of IE genes and the LR region in virus-neuron interactions, it is likely these differences contribute to BHV-5 neuropathogenicity.
Search for the genome of bovine herpesvirus types 1, 4 and 5 in bovine semen
Bovine herpesvirus type 1 (BoHV-1) causes respiratory and reproductive disorders in cattle. Recently, bovine herpesvirus type 5 (BoHV-5) and bovine herpesvirus type 4 (BoHV-4) have been identified to be associated with genital disease. In this study, the presence of the genome of BoHV-1, BoHV-4 and BoHV-5 in bovine semen of Argentinean and international origin was analyzed by PCR assays. The most important finding of this study is the detection of the genome of BoHV-1 and BoHV-4 in semen of bulls maintained at artificial insemination centers. It is particularly relevant that BoHV-1 DNA was also identified in one sample of international origin suggesting the need for extensive quality control measures on international transport of bovine semen. Keyword: Artificial insemination, Bovine herpesviruses, Genome, Semen. _____________________________________________________________________________________________