**Abstract**

Human diseases caused by single-stranded, positive-sense RNA viruses, are among the deadliest of the 21st Century. In particular, there are two notable standouts: human immunodeficiency virus (HIV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Detection of these disease-causing viral transcripts, by next-generation RNA sequencing (RNA-Seq), represents the most immediate opportunity for advances in diagnostic, therapeutic, and preventive applicability in infectious diseases (e.g., AIDS and COVID-19). Moreover, RNA-Seq technologies add significant value to public health studies by first, providing real-time surveillance of known viral strains, and second, by the augmentation of epidemiological databases, construction of annotations and classifications of novel sequence variants. This chapter intends to recapitulate the current knowledge of HIV and SARS-CoV-2 transcriptome architecture, pathogenicity, and some features of the host immune response. Additionally, it provides an overview of recent advances in diagnostic sequencing methodologies and discusses the future challenges and prospects on the utilization of RNA-Seq technologies.

**Keywords:** Next generation RNA Sequencing, RNA-Seq, RNA viruses, human immunodeficiency virus, severe acute respiratory syndrome coronavirus, HIV transcriptome, SARS-CoV-2 transcriptome
