Head-to-tail sequences have been reported in human bocavirus (HBoV) 1-4. To reveal their features and functions, HBoV DNA was screened among respiratory specimens from pediatric patients with an acute respiratory infection (ARI) between April 2020 and December 2022, followed by HBoV genotyping. Head-to-tail sequences were detected using nested PCR, TA cloning, and Sanger sequencing, and these findings were confirmed by mNGS and amplicon sequencing. The secondary structure was predicted using the Mfold web server. The results indicated that head-to-tail sequences were detected in 42 specimens through TA cloning from 351 specimens positive for HBoV1 DNA, yielding 92 sequences into 32 types and 2 categories. Additionally, head-to-tail sequences were detected in 16 specimens by amplicon sequencing, yielding 60 sequences categorized into 23 types. The 374nt type, detected in 13 specimens, contains variants 374a and 374b, which differ in the unpaired loop regions of the palindrome or complementary reverse sequences, implying a switch of template chains during the replication process. The mNGS results in three specimens confirmed the presence of circular genome in copies below 1%. In conclusion, head-to-tail sequences of HBoV1 were common in children with ARI and were highly diverse in length and sequences. The variants may be generated by the switch of the template chain in the rolling-circle replication model.

Human bocavirus (HBoVs) is an emerging virus globally, and its prevalence, diversity, and evolution in children with acute gastroenteritis require further study. Fecal specimens collected from outpatients with acute gastroenteritis in Beijing, China, from April 2014 to December 2023, were tested for HBoVs by PCR targeting the NS1 gene. Genotyping was based on the NP1/VP1 boundary region, and nearly full-length HBoV2 sequences were analyzed phylogenetically. A total of 79 HBoVs-positive specimens (2.5%, 79/3,116, 95% CI: 2.0%–3.1%) were detected, with HBoV2 (65.8%, 52/79, 95% CI: 55.4%–76.3%) as the dominant genotype, followed by HBoV1 (25.3%, 20/79, 95% CI: 15.7%–34.9%) and HBoV3 (8.9%, 7/79, 95% CI: 2.6%–15.1%). HBoVs were distributed throughout the year, with higher positive rates observed from August to December 2018–2021. They predominantly infected children younger than 5 years, especially those aged 6–24 months (3.3%, 52/1,591, 95% CI: 2.4%–4.1%). HBoV2C was the prevalent sub-genotype in Beijing. Novel recombination events were detected between HBoV2 sub-genotypes, with breakpoints in the NS1 and VP3 gene regions. Evolutionary analysis estimated the time to the most recent common ancestor (tMRCA) for HBoV2 dating back to 1845, with a mean nucleotide substitution rate of 1.4 × 10 ⁻⁴ substitutions/site/year, and that HBoV2A evolved faster than HBoV2C. The evolutionary rates decreased in the following order: NP1, VP3, VP1, and NS1. A purifying selection was observed on HBoV2 genes, with one positively selected site in the NS1 gene. In conclusion, HBoV2 was the dominant genotype in children with acute gastroenteritis in Beijing, China, with higher susceptibility in those under 2 years old. Novel recombination events between HBoV2 sub-genotypes occurred frequently. IMPORTANCE: Acute gastroenteritis remains a leading cause of morbidity and mortality in children, with viral infections being the primary causative agents. In this study, we investigated the prevalence of human bocavirus (HBoVs) in children with acute gastroenteritis in Beijing from 2014 to 2023, identifying HBoV2C as the predominant sub-genotype. Additionally, this study reported the first estimate of the evolutionary rate for global HBoV2 (1.4 × 10 ⁻⁴ substitutions/site/year) and identified novel intra-genotype recombination events in HBoV2. The results not only filled a gap in the evolutionary studies of global HBoV2 but also offered valuable data for the development of effective surveillance and prevention strategies for controlling acute gastroenteritis in children.

Human bocavirus 1 (HBoV1) causes acute respiratory infections (ARIs) in children, but its diagnosis is complicated by prolonged viral shedding. There are indications that the detection of a circular genome in a clinical specimens may be associated with acute infection. Respiratory specimens collected from pediatric patients with ARIs during January 2021 to July 2024 were screened by a duplex qPCR, which was developed to distinguish circular genome from total viral genomes and evaluated by nested PCR and antigen test. Clinical data were collected from patients with single HBoV1 infection to reveal the association of circular genome with ARIs and the severity of pneumonia. Among 520 specimens positive for HBoV1 DNA, 206 (39.61 %) were positive for circular genomes as determined by duplex qPCR, with the median load of total genomes 10^10.08 (IQR 10^9.26, 10^10.62) copies/mL significantly higher than 10^7.81 (IQR 10^6.88, 10^8.60) copies/mL in the circular genome negative group (p < 0.0001). In the antigen-positive group, the positive rate for circular genomes was 78.57 % (44/56), significantly higher than 34.29 % (108/315) observed in the antigen-negative group. Among patients single positive for HBoV1, the circular genome-positive group (n = 106) showed more severe clinical manifestations and required more intensive treatment. Logistic regression analysis identified the circular genome as a strong independent risk factor for severe pneumonia (OR = 6.38, AUC = 0.82). Circular genome of HBoV1 associated with high load of viral DNA, positive antigen and severe pneumonia in children may serve as a biomarker for acute HBoV1 infection and severe pneumonia.

Human Bocavirus 1 (HBoV1) is one major pathogen that has been associated with wheezing illnesses. However, there is still a lack of effective clinical predictive indicators for wheezing illnesses in children infected with HBoV1. A retrospective cohort study was conducted among pediatric patients with single-HBoV1 infection from September 2016 to August 2023. Then, univariate logistic regression was used to screen potential predictors for wheezing illness, and Least Absolute Shrinkage and Selection Operator (LASSO) regression was applied to minimize overfitting and select key predictors. Finally, a multivariate logistic regression model was constructed in a training dataset comprising 80% of patients and validated in an independent test dataset comprising 20% of patients. Then, its performance was evaluated using the Area Under the Curve (AUC). A total of 330 pediatric patients were enrolled, including 228 in the wheezing-illness group and 102 in the non-wheezing group. Three independent predictors, including abnormal NK cell percentage (OR = 1.101, 95 %CI 1.03–1.27), preterm birth (OR = 1.65, 95 %CI 1.49–1.82) and personal history of allergy (OR = 1.25, 95 %CI 1.11–1.41), were identified. The model achieved AUCs of 0.904 and 0.876 in the training and test sets, respectively. Using a Youden-derived threshold (0.382), the high-risk group in the test set had an observed wheezing rate of 85.3%, compared with 18.7% in the low-risk group (p < 0.001). Calibration was satisfactory (Hosmer–Lemeshow p = 0.324 and 0.576). A validated predictive model incorporating abnormal NK cell percentage, preterm birth and personal history of allergy accurately stratifies the risk of wheezing illness after HBoV1 infection in children, facilitating early clinical intervention.