Like single-stranded RNA viruses, SARS-CoV-2 hijacks the host transcriptional machinery for its own replication. Numerous traditional differential gene expression-based investigations have examined the diverse clinical symptoms caused by SARS-CoV-2 infection. The virus, on the other hand, also affects the host splicing machinery, causing host transcriptional dysregulation, which can lead to diverse clinical outcomes. Hence, in this study, we performed host transcriptome sequencing of 125 hospital-admitted COVID-19 patients to understand the transcriptomic differences between the severity sub-phenotypes of mild, moderate, severe, and mortality. We performed transcript-level differential expression analysis, investigated differential isoform usage, looked at the splicing patterns within the differentially expressed transcripts (DET), and elucidated the possible genome regulatory features. Our DTE analysis showed evidence of diminished transcript length and diversity as well as altered promoter site usage in the differentially expressed protein-coding transcripts in the COVID-19 mortality patients. We also investigated the potential mechanisms driving the alternate splicing and discovered a compelling differential enrichment of repeats in the promoter region and a specific enrichment of SINE (Alu) near the splicing sites of differentially expressed transcripts. These findings suggested a repeat-mediated plausible regulation of alternative splicing as a potential modulator of COVID-19 disease severity. In this work, we emphasize the role of scarcely elucidated functional role of alternative splicing in influencing COVID-19 disease severity sub-phenotypes, clinical outcomes, and its putative mechanism. IMPORTANCE The wide range of clinical symptoms reported during the COVID-19 pandemic inherently highlights the numerous factors that influence the progression and prognosis of SARS-CoV-2 infection. While several studies have investigated the host response and discovered immunological dysregulation during severe infection, most of them have the common theme of focusing only up to the gene level. Viruses, especially RNA viruses, are renowned for hijacking the host splicing machinery for their own proliferation, which inadvertently puts pressure on the host transcriptome, exposing another side of the host response to the pathogen challenge. Therefore, in this study, we examine host response at the transcript-level to discover a transcriptional difference that culminates in differential gene-level expression. Importantly, this study highlights diminished transcript diversity and possible regulation of transcription by differentially abundant repeat elements near the promoter region and splicing sites in COVID-19 mortality patients, which together with differentially expressed isoforms hold the potential to elaborate disease severity and outcome.

Over the past 8 years, multiple studies examined the phenomenon of isoform switching in human cancers and discovered that isoform switching is widespread, with hundreds to thousands of such events per cancer type. Although all of these studies used slightly different definitions of isoform switching, which in part led to a rather poor overlap of their results, they all leveraged transcript usage, a proportion of the transcript\'s expression in the total expression level of the parent gene, to detect isoform switching. However, how changes in transcript usage correlate with changes in transcript expression is not sufficiently explored. In this article, we adopt the most common definition of isoform switching and use a state-of-the-art tool for the analysis of differential transcript usage, SatuRn, to detect isoform switching events in 12 cancer types. We analyze the detected events in terms of changes in transcript usage and the relationship between transcript usage and transcript expression on a global scale. The results of our analysis suggest that the relationship between changes in transcript usage and changes in transcript expression is far from straightforward, and that such quantitative information can be effectively used for prioritizing isoform switching events for downstream analyses.

Tetracapsuloides bryosalmonae is a malacosporean endoparasite that infects a wide range of salmonids and causes proliferative kidney disease (PKD). Brown trout serves as a carrier host whereas rainbow trout represents a dead-end host. We thus asked if the parasite adapts to the different hosts by changing molecular mechanisms. We used fluorescent activated cell sorting (FACS) to isolate parasites from the kidney of brown trout and rainbow trout following experimental infection with T. bryosalmonae. The sorted parasite cells were then subjected to RNA sequencing. By this approach, we identified 1120 parasite transcripts that were expressed differentially in parasites derived from brown trout and rainbow trout. We found elevated levels of transcripts related to cytoskeleton organisation, cell polarity, peptidyl-serine phosphorylation in parasites sorted from brown trout. In contrast, transcripts related to translation, ribonucleoprotein complex biogenesis and subunit organisation, non-membrane bounded organelle assembly, regulation of protein catabolic process and protein refolding were upregulated in rainbow trout-derived parasites. These findings show distinct molecular adaptations of parasites, which may underlie their distinct outcomes in the two hosts. Moreover, the identification of these differentially expressed transcripts may enable the identification of novel drug targets that may be exploited as treatment against T. bryosalmonae. We here also describe for the first time how FACS based isolation of T. bryosalmonae cells from infected kidney of fish fosters research and allows to define differentially expressed parasite transcripts in carrier and dead-end fish hosts.