ABSTRACT Chikungunya virus (CHIKV) is an alphavirus spread by mosquitos that causes persistent arthritis in approximately 25% of people two years after initial infection. Pain is the predominant symptom in CHIKV arthritis. There is currently no standard evidence-based treatment for CHIKV chronic arthritis. The main goal of our U01 is to determine the efficacy of 6 months of methotrexate treatment versus placebo on clinical manifestations and synovial mechanisms in chronic CHIKV arthritis in Colombia in a Phase III randomized controlled trial in order to guide the evidence-based treatment of CHIKV arthritis. Our main goal of this NOSI application is to expand our current specific aims to quantify pain in a clinical trial that will accumulate a rich trove of clinical and omics data along with tissue-based analysis to determine pathways and genes associated with the unique pain state of chikungunya viral arthritis and methotrexate treatment in an underrepresented Latino population. Our central hypothesis of this NOSI application is that nociceptive, neuropathic and nociplastic pain play a significant role in CHIKV arthritis morbidity via mechanisms involving inflammatory cytokines (IL-1 & IL-6), proinflammatory neuropeptides (calcitonin gene-related peptide (CGRP) and substance P (SP)), the neurotrophin nerve growth factor (NGF) and gene pathways involving inflammation, the GABAergic, adrenergic, serotonergic and hypothalamic-pituitary-adrenal systems. This hypothesis will be evaluated in expansion of our 2 specific aims. In the Parent Aim 1, we will determine the efficacy of oral methotrexate treatment versus placebo for 6 months in chronic CHIKV arthritis. In Expanded Aim 1.1, we propose to enhance our outcome measures with additional functional pain metrics that characterize nociceptive, neuropathic and nociplastic pain phenotyping in a Latin American chronic chikungunya arthritis cohort, including assessments of social determinants of health as modifiers of pain severity and response to treatment. In Parent Aim 2, we will determine the effect of methotrexate on synovial inflammation by obtaining synovial biopsy samples before and during treatment. In Expanded Aim 2.1, we propose to determine the effect of methotrexate on the bidirectional interaction between tissue damage and pain mechanisms via analysis of peripheral blood and synovial tissue using immunoassays and immunohistochemical staining for CGRP, SP and NGF, cytokine profiling, and single-cell RNAseq transcriptomic and ATACseq chromatic state analyses to determine if they correlate with the clinical pain state and to define markers that are associated with resolution of pain states. Impact: This work will define the diverse pain phenotypes and predictive biomarkers in CHIKV arthritis, the mechanisms of CHIKV arthritis pain and the utility of methotrexate in the treatment of CHIKV arthritis pain.