Evolutionary trajectory and spread of respiratory syncytial virus group A in neonatal cohorts in Pakistan amidst the COVID-19 pandemic.

A collaborative effort by the National Institute of Health, Pakistan and the World Health Organization initiated RSV surveillance in children under 2 years old in 2019 in Pakistan. This study aimed to characterise RSV disease, assess genetic diversity of circulating subtypes, and reconstruct the evolutionary history and transmission dynamics during the COVID-19 pandemic. Nasal swabs were collected weekly from five sites across three seasons (2019-2022). A subset of samples was sequenced and studied using the BEAST phylodynamic framework. Multivariate logistic regression was performed to explore associations between patient characteristics and infection odds. Out of 758 samples collected, 18.7% tested positive for RSV, mostly RSV-A (93.6%). Influenza A and B positivity rates were 4.2% and 6.3%, respectively. RSV positivity increased from 13% in 2019-2020 to 32.1% in 2020-2021, likely due to the wide spread of respiratory infection and limited implementation of preventive measures. These rates later dropped to 10.0% (2021-22), suggesting a possible re-implementation of such measures. Mutational analysis revealed a unique P376S mutation within the F-4-2 epitopic region of the F gene of one Pakistani virus, which contributes to viral protein oligomerization. The phylodynamic inference revealed predominant circulation of RSV-A over a wider geographical region, reflecting a combination of continual viral entry, with three introductions in 2020, and one each in 2017 and 2018, and one event of persistence across seasons. The observed introductions likely stemmed from Pakistani nationals returning from Europe to their home country. Combined RSV surveillance and phylodynamic modeling effectively uncovers RSV transmission patterns and provides valuable insights for the development and deployment of RSV vaccination and treatment strategies.