PROJECT SUMMARY Respiratory viruses such as SARS-CoV-2 and influenza are common pathogens that cause seasonal illness and can reach pandemic proportions. The early human response to these viruses are in the nasal cavity and nasopharyngeal regions. Defining biomarkers of disease trajectory is important for opting for treatment and monitoring decisions. We hypothesize that the nasopharyngeal tRNA profiles can be used to predict symptom severity resulting from respiratory virus infection. We will apply our newly developed RNA sequencing technology to explore the human molecular signatures and factors that contribute to disease trajectory. These profiles can include the host tRNA composition, fragmentation patterns, and epitranscriptomic modification in the upper respiratory tract. Aim 1 will perform tRNA sequencing of nasopharyngeal samples to identify infection signatures of host tRNA using banked nasopharyngeal swabs with documented SARS-CoV-2, influenza A, and influenza B infections. Together with known patient outcomes, we will dissect tRNA signatures in nasopharyngeal swabs and map the relationship between nasal tRNA profiles and viral infection severity. Since the nasopharyngeal region is highly populated with immune cells, including secret RNases to counter viral infections, we will quantify the ribonuclease activity and associate these results with tRNA signatures. Aim 2 will develop qPCR assays of tRNA-based biomarkers. We developed an algorithm that optimizes primer design for nasopharyngeal samples based on tRNA sequencing results. Our approach also detects changes in tRNA modification fractions. We will further develop this assay to enable its implementation to nasopharyngeal swab samples for rapid, routine and economic tRNA biomarker analysis. These results will expand our insights on how human tRNA profiles can be biomarkers for the pathology of respiratory viruses in general.