The development and use of cutting edge next generation sequencing methodologies to study RNA viruses

With new next generation sequencing technologies and methodologies being published often, our ability to sequence viruses and host transcriptomes (in response to viral infection) has expanded – driving virology forward. Here we show the development and use of novel methodologies to study RNA viruses from their impact on the host, to variations in viral genomes, to how context of an infection may alter disease outcomes. We have published methods such as Tiled Click-Seq (TCS) and poly-A Click-Seq (PAC-Seq) with corresponding pipelines (Virus Recombination Mapper and Differential Poly-A Cluster [DPAC]) to study variation in viral genomes and transcriptomic changes respectively. We have also used single nuclei RNA sequencing (snRNA-Seq) for a more granular look at transcriptomic changes using the package Seurat in R. As the 2015-2016 Zika virus (ZIKV) outbreak in S. America was associated with development of microcephaly in infants born to expectant mothers infected early in pregnancy we wanted to study the transfer of ZIKV from mother to fetus. As this involves placental infection, we extracted total cellular RNA from ZIKV infected (or mock-infected) human placental (JEG3) cells and used it to construct PAC-Seq libraries. Subsequent DPAC analysis provided data on differential gene expression, alternative poly-adenylation (APA), and use of alternative terminal exons. We found that up-regulated poly-A sites (PASs) lacked the sequences for canonical poly-adenylation (AAUAAA ~20 nts upstream of the PAS or a GU region just downstream) that were found in down-regulated PASs. Here we present a potential mechanism for the large-scale APA occurring in response to ZIKV infection in JEG3 cells. Microcephaly, and other CNS issues, can be symptoms of ZIKV infection we wanted to look at the brain as well. As opioid overdose deaths have increased in recently in the U.S. (coinciding with the COVID-19 pandemic) we investigated the potential impact of opioid use on severity of neurological disease from RNA virus infection, looking at expression of genes involved in SARS-CoV-2 or ZIKV infection in the mesolimbic pathway, using both snRNA-Seq and PAC-Seq. Our results suggest that opioid use may exacerbate symptoms of these infections by up-regulating inflammation and down-regulating anti-viral pathways in this brain region.