Subclade B3 and D3 enterovirus D68 among children with severe community-acquired pneumonia in China, 2021 to 2024

Enterovirus D68 (EV-D68) is an emerging pathogen that causes severe respiratory infections in children worldwide and has the potential to re-outbreak. The aim of this study was to assess EV-D68 detection and genetic variability in pediatric patients with community-acquired pneumonia (CAP) during and after the COVID-19 pandemic in China. Respiratory samples, including bronchoalveolar lavage fluid (BALF), were collected from hospitalized children with CAP in Shanghai, Shandong, Fujian, Zhejiang and Jiangsu province between January 1, 2021 and December 31, 2024. Samples were screened for EV-D68 using real-time RT-PCR, and clinical characteristics of positive cases and virus strain sequences were analyzed. Of 2,842 cases, 0.7% (21/2,842) positive with EV-D68. The median age of these patients was 5.0 years (IQR: 3.6-8.0 years), with 42.9% (9/21) presented severe CAP. Of the 21 identified virus strains, 15 belonged to subclade B3 and 6 to subclade D3. In 2024, a total of 14 strains were detected, including 6 from the newly emerged subclade D3. Seven strains, 5 of which were subclade D3, were found in BALF samples from severe CAP patients. One child with severe CAP exhibited subclade D3 EV-D68 viremia. Notable mutations in the VP1 protein included H99D, K168E, and E169K. Our research showed that the EV-D68 continued to spread among children in eastern China before and after the COVID-19 pandemic. From 2021 to 2024, subclades B3 and D3 predominated, with increase detections in 2024 and association with pediatric severe CAP.