On September 11, 2024, two classes at an elementary school in the Jingkai area reported multiple fever cases. Public health professionals from the local CDC immediately conducted an on-site epidemiological investigation. The school housed 189 classes with 7,368 students and 739 faculty and staff members. Overall sanitary conditions were satisfactory, and full-time school physicians were available onsite. All reported cases occurred exclusively among students; no teachers or staff members were affected. The two affected classes were situated on the east side of the same floor within the same building, positioned adjacent to the girls’ restroom where ventilation was observed to be inadequate.
Epidemiological investigation revealed that the index case developed symptoms on September 9, including fever (reaching 38.6 °C), body aches, and nasal congestion. Between September 9 and 11, a total of 15 students from the 2 classes developed fever, with maximum recorded temperatures reaching 39.9 °C. Among these cases, 4 students experienced headache, fatigue, and other systemic symptoms; 3 reported cough and sore throat; 2 had muscle aches; 1 presented with nasal congestion; and 5 exhibited only fever. No hospitalizations, severe cases, or fatalities occurred. The temporal distribution of cases demonstrated a concentrated pattern: 5 cases on September 9, 6 cases on September 10, and 4 cases on September 11. Of the 15 affected individuals, 6 were male and 9 were female, with all students being 6 years old.
A total of 14 throat swab samples were collected from the identified cases. Total viral nucleic acid was extracted using a nucleic acid extraction and purification kit (DA0623, Da’an, Guangzhou) with a nucleic acid extractor (Smart32, Da’an, Guangzhou). Comprehensive screening for respiratory pathogens was performed using a nucleic acid detection kit targeting 22 respiratory pathogens (Beijing Kangrun Gino) in combination with a PCR amplification instrument (TianLong Gentier 96R, Xi’an Tianlong). The screening panel included Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), influenza A (H1N1) pdm09 and A(H3N2), influenza B viruses, human respiratory syncytial virus, HAdV, human metapneumovirus, human rhinovirus, human parainfluenza virus (HPIV I-IV), human coronavirus (HCoV-NL63, -229E, -OC43, and -HKU1), human bocavirus, human enterovirus, Mycoplasma pneumoniae, Streptococcus pneumoniae, group A streptococcus, Bordetella pertussis, Haemophilus influenzae, Klebsiella pneumoniae, Legionella pneumoniae, Aspergillus, Chlamydia pneumoniae, Chlamydia psittaci, Cryptococcus, and Pneumocystis. HAdV-specific genes were detected in 10 of the 14 samples, while no other pathogens were identified, establishing HAdV as the causative agent of the outbreak.
Six viral isolates were successfully obtained by inoculating the human epidermoid larynx carcinoma cell line (HEp-2 cells) with HAdV-positive samples. Cytopathic effects were monitored daily, and viral material was harvested when greater than 75% of the HEp-2 cells exhibited cytopathic changes. The six viral isolates were subsequently stored at −70 °C for further analysis.
For preliminary genotyping, we amplified and sequenced the Loop2 region of the hexon gene from all six viral strains following national CDC protocols (3) (Table 1). For definitive genotyping, we amplified and sequenced the complete penton base gene (1,635 bp), hexon gene (2,935 bp), and fiber gene (960 bp) (4) (Table 1). All PCR products underwent bidirectional sequencing to ensure accuracy and reliability. We assembled and processed sequence data using Sequencher v5.0 software (Genecode，USA). Phylogenetic trees were constructed using both Neighbor-Joining and Maximum Likelihood (ML) methods in MEGA12, with bootstrap values set to 1,000 replicates for statistical validation. Reference sequences were obtained from the GenBank database.
Table 1.
Primers used to amplify the target genome.
Sequence analysis of the hexon Loop2 region revealed 100% identity among the six viral strains, all clustering with the HAdV-B3 genotype. Further analysis of the complete penton base gene, hexon gene, and fiber gene demonstrated 100% sequence identity among the six isolates. Phylogenetic analysis indicated that the penton base gene was closely related to HAdV-B7 (Figure 1), while the hexon gene and fiber gene were closely related to HAdV-B3 (Figure 1). According to the International Adenovirus Working Group guidelines for HAdV genotype identification (http://hadvwg.gmu.edu), this recombinant profile (P7H3F3) corresponds to the genotype HAdV-B114.
Phylogenetic tree of HAdV final genotyping based on the penton base gene, hexon gene, and fiber gene.
Note:
Abbreviation: HAdV=human adenovirus.
Further analysis of amino acid sequence differences between the six HAdV-B114 strains revealed that the penton base demonstrated 99.2% amino acid homology with the HAdV-B7 reference strain, the hexon showed 99% amino acid homology with the HAdV-B3 reference strain, and the fiber exhibited 96.8% amino acid homology with the HAdV-B3 reference strain (Table 2). BLAST analysis further confirmed that the penton base gene, hexon gene, and fiber gene of the six HAdV strains exhibited the highest homology with the GenBank reference OR853835 for HAdV-B114 — at 99.9%, 100% and 99.8%, respectively. Therefore, the causative agent of this outbreak was conclusively identified as the novel recombinant HAdV type HAdV-B114.
Table 2.
Variations in amino acids of human adenovirus type 114 (HAdV-114) strains.
