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Vesicular stomatitis Indiana virus (VSIV) of genus Vesiculovirus , species Indiana Vesiculovirus (formerly as Vesicular stomatitis virus , VSV) causes a disease in livestock that is very similar to the foot and mouth disease, thereby an outbreak may lead to significant economic loss. Long-read sequencing (LRS) -based approaches already reveal a hidden complexity of the transcriptomes in several viruses. This technique has been utilized for the sequencing of the VSIV genome, but our study is the first for the application of this technique for the profiling of the VSIV transcriptome. Since LRS is able to sequence full-length RNA molecules, it thereby provides more accurate annotation of the transcriptomes than the traditional short-read sequencing methods. The objectives of this study were to assemble the complete transcriptome of using nanopore sequencing, to ascertain cell-type specificity and dynamics of viral gene expression, and to evaluate host gene expression changes induced by the viral infection. We carried out a time-course analysis of VSIV gene expression in human glioblastoma and primate fibroblast cell lines using a nanopore-based LRS approach and applied both amplified and direct cDNA sequencing (as well as cap-selection) for a fraction of samples. Our investigations revealed that, although the VSIV genome is simple, it generates a relatively complex transcriptomic architecture. In this study, we also demonstrated that VSIV transcripts vary in structure and exhibit differential gene expression patterns in the two examined cell types.

Viral transcriptomes that are determined using first- and second-generation sequencing techniques are incomplete. Due to the short read length, these methods are inefficient or fail to distinguish between transcript isoforms, polycistronic RNAs, and transcriptional overlaps and readthroughs. Additionally, these approaches are insensitive for the identification of splice and transcriptional start sites (TSSs) and, in most cases, transcriptional end sites (TESs), especially in transcript isoforms with varying transcript ends, and in multi-spliced transcripts. Long-read sequencing is able to read full-length nucleic acids and can therefore be used to assemble complete transcriptome atlases. Although vaccinia virus (VACV) does not produce spliced RNAs, its transcriptome has a high diversity of TSSs and TESs, and a high degree of polycistronism that leads to enormous complexity. We applied single-molecule, real-time, and nanopore-based sequencing methods to investigate the time-lapse transcriptome patterns of VACV gene expression.

In the last couple of years, the implementation of long-read sequencing (LRS) technologies for transcriptome profiling has uncovered an extreme complexity of viral gene expression. In this study, we carried out a systematic analysis on the pseudorabies virus transcriptome by combining our current data obtained by using Pacific Biosciences Sequel and Oxford Nanopore Technologies MinION sequencing with our earlier data generated by other LRS and short-read sequencing techniques. As a result, we identified a number of novel genes, transcripts, and transcript isoforms, including splice and length variants, and also confirmed earlier annotated RNA molecules. One of the major findings of this study is the discovery of a large number of 5'-truncations of larger putative mRNAs being 3'-co-terminal with canonical mRNAs of PRV. A large fraction of these putative RNAs contain in-frame ATGs, which might initiate translation of N-terminally truncated polypeptides. Our analyses indicate that CTO-S, a replication origin-associated RNA molecule is expressed at an extremely high level. This study demonstrates that the PRV transcriptome is much more complex than previously appreciated.