Abstract
This study systematically characterizes the genetic diversity, phylogenetic relationships, recombination events, and structural variations of human adenovirus serotypes 40 and 41 (HAdV-F40/41) circulating in Shandong Province, China, between 2017 and 2019. A total of 2,221 stool samples were collected from patients presenting with acute gastroenteritis in Yantai. Enteric adenoviruses were identified using real-time quantitative PCR targeting the hexon gene. Whole-genome sequencing was performed via nanopore and Illumina platforms, enabling high-resolution phylogenetic reconstruction with IQ-TREE and the identification of recombination events using RDP5 and SimPlot. Evolutionary dynamics were inferred through Bayesian molecular clock analysis, while AlphaFold3-based structural modeling was used to assess the impact of genomic changes on the hexon protein. HAdV-F40/41 was detected in 94 samples (4.23%) with year-to-year variability in prevalence. Phylogenetic analysis revealed the co-circulation of multiple genetically distinct lineages, while recombination mapping identified key breakpoints, notably within the hexon and pVII genes. The estimated recent divergence of dominant lineages suggests ongoing adaptive evolution. Structural modeling of the recombinant strain SD376 identified 14 amino acid substitutions and three deletions relative to the reference strain OP378826.1, without substantial conformational alteration. These findings provide critical insights into the genomic plasticity and evolutionary potential of HAdV-F40/41, underscoring the importance of continuous genomic surveillance. The observed mutations and recombination patterns may facilitate immune escape and viral persistence, reinforcing the urgent need for improved monitoring strategies and the development of targeted vaccines. IMPORTANCE: Human adenovirus serotypes 40 and 41 (HAdV-F40/41) are among the leading viral causes of pediatric acute gastroenteritis worldwide, yet their genetic diversity and evolutionary dynamics remain poorly characterized in many regions, including China. In this study, we systematically investigated HAdV-F40/41 strains circulating in Yantai, Shandong Province, from 2017 to 2019. Using a combination of real-time PCR, nanopore sequencing, and next-generation sequencing, we elucidated the phylogenetic relationships, recombination events, and structural features of circulating strains. Our results reveal extensive genetic variability and the emergence of recombinant lineages with altered antigenic profiles, underscoring the role of adaptive evolution and potential immune escape. The identification of key mutations and recombination hotspots in hexon and pVII genes provides important molecular markers for surveillance and risk assessment. These findings enhance our understanding of HAdV-F40/41 evolution and highlight the urgent need for continuous genomic monitoring and targeted vaccine development to mitigate the public health burden of adenoviral gastroenteritis.