Molecular characteristics of human adenovirus isolated from the 2024 influenza-like illness outbreaks in Suzhou City, China

Abstract

Background: Human adenoviruses (HAdV) comprises a genetically diverse group of double-stranded DNA viruses strongly associated with influenza-like illness (ILI) and acute respiratory illnesses (ARI) outbreaks. This study aimed to elucidate the molecular characteristics of HAdV implicated during ILI outbreaks in Suzhou City, Jiangsu Province in 2024.

Methods: Throat swab samples were gathered from settings with ILI outbreaks in Suzhou between March 2024 and January 2025. These samples were analyzed using rapid multi-pathogen detection and real-time PCR. HAdV-positive samples were inoculated into human epidermoid larynx carcinoma cell line (Hep2) for HAdV isolation, followed by whole-genome sequencing (WGS) using the MiSeq platform. Maximum likelihood (ML) phylogenetic trees for the whole genome, hexon, penton base and fiber genes were constructed using IQ-TREE v2.3.6 software. Amino acid variation analysis was performed with BioEdit 7.7.1 software, while recombination detection and visualization were conducted using Recombination Detection Program version 4.101 (RDP4) and SimPlot 3.5.1 software.

Results: Of 488 outbreak samples collected, 53 (10.86%) tested positive for HAdV, making it the second most prevalent respiratory pathogen. These HAdV-related respiratory outbreaks occurred during the spring-summer and autumn-winter transitions, showing no significant regional differences but a slightly higher positive rate in males than in females (13.83% vs. 7.66%, p = 0.028). Twenty-nine HAdV-positive samples were selected for viral isolation and genome sequencing. Phylogenetic analysis revealed that most isolates clustered with recently circulating HAdVB3 strains, while several clustered with HAdVB7 or previously reported HAdVB3/7 recombinant genotypes. Compared with the HAdVB3 reference sequence (OQ518322.1), multiple nonsynonymous amino acid substitutions were identified in antigenic determinant domains (loop 1 and loop 2) of the hexon protein. Besides, four substitution sites were detected in each of the penton base and fiber proteins. Recombination analysis identified seven strains as HAdVB3/7 recombinants, displaying three distinct recombination patterns. Notably, the strain SZTC20241129 carried HAdVB7-derived insertional fragments spanning partial E2B gene region, 52/55 kDa protein, protein IIIa precursor (pIIIa), and the penton base. In contrast, the other recombinant strains (SZZJ20240513, SZTC20241133, SZZJ20240515, SZZJ20240516, SZCS20241240 and SZCS20241242) possessed an HAdVB7-derived genomic backbone along with multiple HAdVB3-derived insertional fragments, encompassing a range of structural and regulatory genes.

Conclusions: HAdV was one of the leading causative agents of ILI outbreaks in Suzhou in 2024, with HAdVB3 being the predominant genotype (22/29, 75.9%). Strikingly, HAdVB3/7 recombinant strains with P7H7F3 and P7H3F3 genetic constituents were also detected in a subset of cases (7/29, 24.1%). The genetic diversity of circulating HAdV continues to expand, driven by the emergence of novel amino acid substitutions and intertypic recombination events. These findings underscore the importance of sustained molecular surveillance to track viral evolution and inform effective public health strategies aimed at reducing the risk of severe clinical outcomes in vulnerable populations.

Keywords: Genome sequencing; Human adenovirus; Phylogenetic analysis; Recombination; Variation.

Fig. 1

The geographical distribution of HAdV outbreak samples in this study

Fig. 2

Phylogenetic analysis of HAdV strains isolated from 2024 ILI outbreaks in Suzhou. The trees for whole genome (A), hexon (B), penton base (C) and fiber gene (D) were constructed by the maximum likelihood (ML) method with IQ-TREE v2.3.6. The strains obtained in this study were highlighted in bold and marked with red circles (). Numbers above branches indicate bootstrap values, with those greater than 75% were shown

Fig. 3

Amino acid variation analysis of hexon (A), penton base (B) and fiber (C) sequences obtained in this study. The loop 1 domain of hexon (amino acids 131–314) and knob domain of fiber (amino acids 124–319) were highlighted in pink, while the loop 2 domain of hexon (amino acids 405–454) and RGD loop of penton base (amino acids 299–362) were colored in yellow

Fig. 4

Genome recombination analysis of the three representative recombinant strains (A-C) and reference strain NICED/23 − 01/1914 (D) by SimPlot software. The 29 whole genome sequences obtained in this study shared high homology in nucleotide sequence and recombination pattern. Thus, the bootscan analysis results of three representative recombinant strains SZTC20241129 (A), SZZJ20240513 (B) and SZZJ20240516 (C) were shown and the recombination patterns of the remaining recombinant strains resembled any one of the three strains above

Fig. 5

Phylogenetic analysis of potential recombinant regions of strain SZZJ20240513. The trees for the Region 1 (18,257 − 25,218 nucleotide location of SZZJ20240513), Region 2 (27,927 − 33,987 nucleotide location of SZZJ20240513) and Region 3 (25,219 − 27,926 nucleotide location of SZZJ20240513) were constructed by the maximum likelihood (ML) method using IQ-TREE v2.3.6. The HAdVB3 and HAdVB7 reference strains were colored in black and red, respectively. The corresponding fragments in the strain SZZJ20240513 was highlighted in bold and labeled with the red solid circle (). Numbers above branches indicate bootstrap values, with those greater than 70% were shown