Seroprevalence and molecular characterisation of human Respiratory Syncytial Virus (RSV) infections in children at a tertiary care centre in rural Maharashtra, India.

Respiratory syncytial virus (RSV) is the foremost viral cause of acute lower respiratory infections (ALRIs) in children under five years of age, yet surveillance data from rural India remain conspicuously scarce. This cross-sectional study simultaneously characterised RSV seroprevalence and active molecular infection in two non-overlapping paediatric cohorts from Karad, Maharashtra, India (September 2021-August 2023). In Cohort 1 (serological study, n = 290; ≥246 required by a prior calculation), RSV-specific immunoglobulin M (IgM) antibodies were detected in 182 children (62.8%) by enzyme-linked immunosorbent assay (ELISA; indirect format with rheumatoid factor pre-absorption), with the highest positivity in the 2-3-year age group. This rate is interpreted as an indicator of high community-level RSV exposure rather than a precise incidence estimate, given the known limitations of single-timepoint IgM testing in previously exposed children. In Cohort 2 (molecular study, n = 283; ≥233 required), RSV-B was detected by real-time reverse transcription polymerase chain reaction (RT-PCR) in 7 of 283 (2.47%) symptomatic children presenting with acute respiratory infection (ARI) or severe ARI (SARI); no RSV-A was identified. The low detection rate is consistent with pandemic-era suppression of respiratory virus transmission. All seven RSV-B strains belonged to the GB5 genotype and formed a phylogenetically distinct local sub-cluster within global GB5 (82% bootstrap support), carrying shared amino acid substitutions in glycoprotein G (A74V, I252T, 165del) and fusion protein F (F12I, S190N, S211N, S389P), numbered relative to the GISAID reference sequence EPI_ISL_1653999 (F precursor). Substitutions S190N and S211N map to the apex region of prefusion F at or adjacent to antigenic site Ø-the primary binding epitope for nirsevimab-though their functional impact on neutralisation was not assessed in vitro and requires dedicated experimental evaluation. A novel predicted N-linked glycosylation site at position F180 was identified in one strain (KIMS-102/2022), with potential but unconfirmed implications for local immune evasion. RSV-B illness was predominantly mild. These findings provide the first combined serological and molecular RSV dataset from rural Maharashtra and underscore the need for region-specific surveillance as RSV prophylactics become available in India.