Respiratory virus infection dynamics and genomic surveillance to detect seasonal influenza subtypes in wastewater: A longitudinal study in Bengaluru, India

Recent global pandemics have been caused by respiratory viruses in humans and animals with zoonotic spillover potential. Respiratory viruses, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), respiratory syncytial virus (RSV), influenza A virus (IAV), and influenza B virus (IBV), share overlapping ecology and similar symptoms. However, respiratory disease surveillance is often passive, relying on clinical specimen testing. Wastewater surveillance has been used for early detection of SARS-CoV-2 variants and can differentiate between respiratory virus infections and SARS-CoV-2 peaks at the community level. In this retrospective longitudinal study covering four SARS-CoV-2 Omicron waves, we conducted monthly sampling for 28 months (812 samples) between August 2021 and December 2023 at 28 sewershed sites in Bengaluru (~11 million inhabitants), India. Using RT-qPCR kits, we quantified SARS-CoV-2 RNA concentrations, IAV, IBV, and RSV to understand community viral infections. We found 86% of samples positive for SARS-CoV-2, while positivity rates for influenza virus and RSV were lower (37% for IAV, 16% for IBV, and 15% for RSV) and this pattern was consistent across sites. We observed a seasonal increase in influenza viruses during the monsoon, peaking in October, with mean IAV viral loads of 755 copies/person/day in 2021, 2000 copies/person/day in 2022, and 1749 copies/person/day in 2023. IAV was present in January and February but absent the rest of 2022. However, IAV viral load was detected throughout 2023 (except in June). IBV showed a similar trend, peaking in October, with mean viral loads of 616.56 copies/person/day in 2021, 323.37 copies/person/day in November 2022, and 373.37 copies/person/day in September 2023. RSV displayed a shorter transmission window, peaking at around 2000 copies/person/day in October. Using genomic data, we provide evidence of changes in the relative abundance of influenza subtypes and SARS-CoV-2 variants, identifying all eight segments of influenza virus genomes and emerging SARS-CoV-2 variants in wastewater samples. Wastewater surveillance provides data on the diversity and relative abundance of respiratory viruses in urban Bengaluru that would not be reported otherwise. Under the One Health framework, wastewater surveillance can offer early warning signs and enhance traceability of infectious diseases in wildlife and humans.