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.

The performances of two commercial human adenoviruses (HAdVs) typing kits were evaluated in pediatric patients with acute respiratory tract infections (ARIs). Respiratory specimens were collected from March to December 2023 for HAdV screening by a multiple real-time quantitative polymerase chain reaction kit (M-qPCR). Using sequence-based HAdV typing as the reference method, the performances of one capillary electrophoresis-based multiplex PCR (CEMP) typing kit (HEALTH) for 12 types of HAdVs and one qPCR typing kit (Uninovo) for 7 types of HAdVs were evaluated. Among 267 specimens positive for HAdVs determined by M-qPCR, there were 215 (80.5%, 215/267) in sequence-based HAdV typing, 216 (80.9%, 216/267) in HAdV-CEMP (HEALTH) and 125 (46.8%, 125/267) in HAdV-qPCR (Uninovo) positive for HAdVs. Though both kits showed strong agreements in HAdV-B3 typing with Kappa value > 0.95, the emerging recombinant P7H3F3 was misidentified as B3 or negative by two kits. Most specimens with high cycle threshold (Ct) value identified as HAdV-C2 and HAdV-C5 by HAdV-CEMP (HEALTH) showed negative typing results in sequence-based HAdV typing. Meanwhile, the recombinants of HAdV-C89 [Px2/H2/F2], HAdV-C108 [Px1ps1/H2/F2], Px1ps3H5F5, and Px2H5F5 identified by sequence-based HAdV typing were misidentified as HAdV-C2 and HAdV-C5 by HAdV-CEMP (HEALTH). In conclusion, the HAdV-CEMP (HEALTH) offered a broad spectrum of HAdVs species and high sensitivity in the detection of species C, and the HAdV-qPCR (Uninovo) provided reliable results for the detection of HAdV-B3. Both of them cannot identify recombinant events.

Objective:LLC-MK2/TMPRSS2 cells constitutively express TMPRSS2, eliminating the requirement for additional trypsin during HPIV3 culture. The efficiency of LLC-MK2/TMPRSS2 for isolating HPIV3 from respiratory specimens was evaluated in comparison with Madin-Darby Canine Kidney (MDCK). Methods:HPIV3-positive respiratory specimens from children with acute respiratory infections (February-June 2025) were inoculated into LLC-MK2/TMPRSS2 and MDCK. The cytopathic effect (CPE) was monitored microscopically, and the proportion of positive cells was evaluated using direct immunofluorescence assay (DFA). Viral infection dynamics were assessed using the cycle threshold (Ct) values obtained by qPCR. Results:Among 50 specimens, 35 strains (35/50, 70%) were successfully isolated using LLC-MK2/TMPRSS2, while 14 strains were isolated using MDCK (14/50, 28%). More pronounced CPE and a higher number of virus-infected positive cells were shown in LLC-MK2/TMPRSS2 compared to that in MDCK ( P < 0.001 and P = 0.001, respectively). Among specimens with an initial Ct < 27, the isolation rate of LLC-MK2/TMPRSS2 was higher and the Ct values were lower (< 27) (82.6%, 19/23). Among specimens with an initial Ct of 23 ≤ Ct < 27, the number of specimens with a supernatant Ct ≥ 27 (63.6%, 7/11) was significantly less than that in MDCK ( P = 0.003). Conclusion:LLC-MK2/TMPRSS2 exhibits superior adaptability and replication efficiency in the isolation of HPIV3 from respiratory specimens.

Background: Four genotypes of human bocaviruses (HBoVs) have been identified, with only HBoV1 being detected in respiratory specimens, and with HBoV2 being the predominant human bocavirus in fecal specimens, which implies different tissue tropisms for HBoV1 and HBoV2. It is vital to determine the factors that influence the tissue tropisms. Methods: The major capsid proteins VP3 of HBoV1 and HBoV2 were expressed in eukaryotic cells. Then co-immunoprecipitation (Co-IP) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) (IP-MS) was employed, along with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, to screen host proteins interacting with VP3 of different genotypes. Subsequently, in vitro pull-down assays were conducted to verify the direct virus-host interaction proteins with VP3. Furthermore, molecular docking was performed to predict the interaction interfaces between viral and host proteins. Results: Through IP-MS and enrichment analyses, 50 host proteins that displayed ≥10-fold differential binding affinities between HBoV1 VP3 and HBoV2 VP3 were identified. Among these, seven were considered as high-confidence candidate interactors. Notably, SPLUNC1 and VAMP8 showed predominant expression in respiratory and intestinal tissues, respectively. Subsequent in vitro pull-down assays confirmed that SPLUNC1 specifically bound to HBoV1 VP3, whereas VAMP8 specifically interacted with HBoV2 VP3. Molecular docking analysis further revealed that the binding between SPLUNC1 with HBoV1 VP3, as well as VAMP8 with HBoV2 VP3, was stabilized by extensive hydrophobic interfaces along with specific hydrogen bonds. Conclusions: The specific interactions of host proteins SPLUNC1 with HBoV1 VP3 and VAMP8 with HBoV2 VP3, respectively, provided fundamental evidence that the distinct tissue tropisms of HBoVs may be governed by specific host factors.

Enteroviruses (EVs), which include numerous types, are among the most common pathogens causing infections in children. EVs can cause diverse diseases, including herpangina, hand-foot-and-mouth disease, myocarditis, pneumonia, meningitis, encephalitis, and acute flaccid paralysis. Severe cases may result in pediatric mortality, posing substantial threats to child health and public health. The diversity of EV types combined with rapid viral evolution and antigenic variation presents formidable challenges for EV prevention and control, with dominant genotypes being continuously replaced by emerging variants and novel subtypes. EV invades host cells through specific receptors and hijacks cellular machinery for self-replication, engaging in complex interactions with the host while evolving multiple mechanisms to evade host immune responses. Currently, no specific anti-EV agents are available, and treatment remains primarily supportive and symptomatic. Effective prevention and control strategies for EV are limited, and vaccines are currently available only against selected EV serotypes. This review systematically examines EV infections from multiple perspectives, including epidemiology, virology, viral entry mechanisms, virus-host interactions, clinical diagnosis and management, prevention and control measures, with the aim of providing insights and references for research on EV pathogenesis, precision clinical diagnosis and treatment, and the development of antiviral therapies and vaccines.

The increased circulation of enteroviruses (EVs) from July to December 2024 necessitated a retrospective analysis of hospitalized children with acute respiratory infections in Beijing, China, during 2017-2024. Among the 155 cases confirmed as EVs (0.67%, 155/23,245), with peaks in a triennial pattern and predominance between July and October, 114 were EV-D68, with 82 (71.93%) detected between July and October 2024. Among the 79 VP1 gene sequences of EV-D68 classified into subclades B3 (50.63%, 40/79) and D1 (49.37%, 39/79) through phylogenetic analysis, 31 (77.50%, 31/40) were B3 from 2024, and 34 (87.18%, 34/39) were D1 from 2024. More patients infected with B3 required oxygen supplementation than those infected with D1 (p = 0.042). Therefore, an epidemic of EV-D68 with the co-circulation of B3 and D1 was observed in 2024, with more severe disease caused by B3 than by D1, which underscores the urgent need for surveillance of EV-D68.

Coxsackievirus A4 (CVA4) is an enterovirus associated with diverse clinical syndromes, yet its global evolution and transmission dynamics remain poorly defined. This study aimed to characterize the molecular epidemiology, evolutionary history, and phylodynamic patterns of CVA4 at both local and global scales. In 2024, 34 CVA4 infections were identified during a localized herpangina outbreak in Beijing, primarily affecting children under 5 years of age, with a pronounced peak in July. High detection and isolation rates, together with temporal clustering in mid-2024, indicate a genuine short-term expansion rather than sporadic detection. Phylogenetic analysis of global VP1 sequences resolved six genotypes (A-F), with Beijing strains clustering within genotype E and subgenotype F2, reflecting ongoing worldwide circulation. Bayesian phylodynamic analysis estimated a VP1 evolutionary rate of 1.626 × 10-3 substitutions per site per year (95% highest probability density [HPD], 1.428-1.827 × 10-3) and dated the CVA4 time to the most recent common ancestor back to March 1917 (95% HPD, June 1908-April 1926). Demographic reconstruction revealed fluctuating genetic diversity, with notable expansions in 2013 and 2016, as well as a modest increase in 2023 preceding the outbreak. Phylogeographic analyses identified France and China as key seeding regions. Recombination in the P2 and P3 regions, including evidence of CVA2-related donors, highlights recombination as a major contributor to CVA4 diversity. These findings improve our understanding of CVA4 evolution and transmission and underscore the need for enhanced genomic surveillance.

Background/Objectives: The aim of this study was to clarify the antimicrobial susceptibility and distribution characteristics of Mycoplasma pneumoniae (M. pneumoniae, MP) collected from children in Beijing, China, from 2017 to 2025. Methods: A total of 197 MP isolates were analyzed. Mutations in macrolide-resistant loci of MP strains were detected via real-time fluorescent quantitative polymerase chain reactions. We used the broth microdilution method to determine the minimum inhibitory concentrations (MICs) of erythromycin, azithromycin, tetracycline, levofloxacin, and moxifloxacin against these isolates. The distribution characteristics of MIC values were further analyzed according to the isolates’ collection year, epidemic phase (low epidemic phase, epidemic initiation phase, ultra-low epidemic phase, outbreak phase, and epidemic recovery phase), and the corresponding patient age group (<3 years, 3–6 years, and ≥6 years). Results: All 197 isolates were found to be resistant to erythromycin and azithromycin, with a resistance rate of 100%. In contrast, the strains remained susceptible to tetracycline, levofloxacin and moxifloxacin. The highest resistance rate was 100% for macrolides. The MIC90 values were 1024 μg/mL for erythromycin, 256 μg/mL for azithromycin, 0.5 μg/mL for tetracycline, 1 μg/mL for levofloxacin, and 0.125 μg/mL for moxifloxacin, respectively. Distinct differences in MIC distributions of erythromycin and azithromycin were observed across collection years, epidemic phases, and age groups. Conclusions: The resistance of MP to macrolides in children is closely associated with the epidemic intensity and age of the patient. Erythromycin is no longer suitable as an empirical therapy for MP infections during epidemic periods, whereas azithromycin can be cautiously administered in young children according to age stratification and MIC detection results. Meanwhile, it is imperative to strengthen the prevention and control of cluster MP infections during epidemic phases to reduce the transmission of drug-resistant MP strains.

It has been reported that chronic infection of human norovirus (HuNoV) may potentially serve as a reservoir for viral variants with the possibility to evade population immunity or alter the binding sites of HBGA receptors. In this study, a child diagnosed with Burkitt lymphoma and positive for HuNoV determined by real-time PCR (qPCR) firstly in 15 August 2016, was followed up until 20 March 2018, and 26 fecal specimens and one vomitus were collected to trace the evolutionary characteristics of HuNoV by phylogenetic analysis, meta-genomics next-generation sequencing (mNGS), and temporal evolutionary analysis of VP1 among 23 specimens positive for HuNoV. There were 15 specimens with partial RdRp gene sequences forming an independent cluster with sequences of GII.P31, 14 with the region C sequences and 11 with P domain sequences of VP1 gene clustered together with HuNoV GII.4 Sydney_2012. All these sequences showed that mutations accumulated nearly in a time order, and more mutations were shown in the key epitopes A-E or near the binding sites for HBGA in subdomain P2 with higher evolutionary rates. Analysis of NGS data identified intra-host viral quasi-species, and two genome sequences of the same length from mNGS were assembled from N705, with mutations located in the region of subdomain P2 (1171 nt-1202 nt) which led to five amino acid mutations. In conclusion, the accumulated mutations of HuNoV, especially in subdomain P2, were explored in a child with Burkitt lymphoma, and the sequencing of HuNoV from immunocompromised individuals was proven critical for monitoring intra-host quasi-species evolution and potential variant emergence, providing basic data for clinical infection control.

Background 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. Methods 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. Results 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 1010.08 (IQR 109.26, 1010.62) copies/mL significantly higher than 107.81 (IQR 106.88, 108.60) 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). Conclusion 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 adenovirus (HAdV)-114 (P7H3F3) is a newly identified recombinant genotype, while recent studies suggest that it likely represents a previously circulating HAdV-3 strain. A total of 413 strains previously identified as HAdV-3 from 20 Chinese provinces during 2000-2024, along with 125 GenBank-derived HAdV-3 strains (1988-2023), underwent genotype reconfirmation and genetic evolutionary analysis using the penton base, hexon, and fiber genes. Furthermore, 97 representative strains were selected for whole-genome analysis to determine their genetic recombination patterns. Of the 538 analyzed strains, 537 were identified as HAdV-114 using three genes, with only one 2010 U.S strain classified as HAdV-3. Phylogenetic analysis revealed three HAdV-114 clades (Clades 1-3): Clade 1 (1988-2019) displayed sporadic circulation, Clade 2 (2023-2024) appeared geographically restricted to three Chinese provinces, and Clade 3 (2000-2024) likely represented the globally dominant strain. Genetic evolutionary analyses indicated that, although HAdV-114 originated from recombination between HAdV-7 and HAdV-3, its three Clades diverged from the parental trajectories, with Clades 1 and 3 sharing a common evolutionary origin and Clade 2 forming an independent terminal cluster. Genomic recombination analysis identified two recombination patterns across the three clades. All clades shared a 3' terminal genomic region (nt 18,794-end) derived from HAdV-3, whereas their 5' ends comprised gene fragments recombined from unknown sources and epidemic HAdV-7 strains, with varied breakpoints. HAdV-114, historically identified as HAdV-3, is the predominant genotype associated with HAdV-related respiratory infections. Therefore, enhanced surveillance of recombinant HAdV strains is imperative for effective disease control.

Introduction:Human respiratory syncytial virus subgroup A (HRSV-A) lineage A.D is characterized by a unique 72-nucleotide duplication in the second hypervariable region of the G-gene (G-HVR2) and has become the predominant global circulating strain since 2010. Molecular evolutionary features of this region should be monitored. Methods:A global HRSV-A G-HVR2 sequence dataset (2010-2024) was assembled that included 144 Chinese sequences and the molecular characteristics and evolutionary patterns in lineage A.D were analyzed. Results:Molecular clock analysis based on G-HVR2 estimated that HRSV-A lineage A.D originated in 2006, with subsequent diversification into four distinct lineages. A.D.4 and A.D.5 were grouped as A.D.4/A.D.5. Lineages circulate globally with temporal fluctuations and sequential dominance shifts. The evolutionary rate of A.D was high, with the highest rate observed in A.D.4/A.D.5. Amino acid analysis indicated five shared, lineage-specific mutations in A.D.1 and A.D.4/A.D.5 that coincided with high-frequency mutation sites. Nine positively-selected sites were predicted across all lineages, three of which resided within the 72-nucleotide duplication. A.D.1 and A.D.4/A.D.5 lost conserved N- and O-glycosylation sites. A 72-nucleotide duplication in lineage A.D introduced two extra β-strands and two unique α-helical motifs relative to the prototype strain. Conclusion:Adaptive evolution in the G-HVR2 region of HRSV-A lineage A.D was evident, likely facilitating the rapid transmission of this lineage. Sustained monitoring of lineage-specific evolution in this region is critical for targeted prevention and control of HRSV infections.

Stabilizing the RSV F protein in its prefusion conformation is crucial for effective vaccine development but has remained a significant challenge. Traditional stabilization methods, such as disulfide bonds and cavity-filling mutations, have been labor-intensive and have often resulted in suboptimal expression levels. Here, we report the design of an RSV prefusion F (preF) antigen using a proline-scanning strategy, incorporating seven proline substitutions to achieve stabilization. The resulting variant, preF7P, is structurally and biochemically validated to maintain the correct prefusion state. PreF7P demonstrates superior immunogenicity with a 1.8-fold increase in neutralizing antibody titers when compared to DS-cav2, and provides protection from clinical disease against both RSV A and B strains in female murine and female cotton rat models. In clinical development, preF7P exhibits high expression levels (~10 g/L) in clinical-grade CHO cells. The clinical-grade vaccine elicits robust immunogenic responses across female mice, female SD rats, and both male and female cynomolgus macaques, significantly boosting RSV pre-infection neutralizing antibody titers, and providing sustained protection for at least six months in female mice. This proline-scanning strategy offers a streamlined approach for stabilizing class I fusion proteins, potentially accelerating the development of vaccines for other pathogens.

Introduction:Recent sentinel surveillance has revealed a rising prevalence of human adenovirus type 21 (HAdV-21) among HAdV infections in China. This study aimed to elucidate the molecular features of currently circulating HAdV-21 strains in China. Methods:Whole-genome sequencing (WGS) was performed on 23 HAdV-21 strains isolated from acute respiratory infection cases, 56.5% involving lower respiratory tract infections, across 7 Chinese sentinel surveillance provincial-level administrative divisions (PLADs) (2023-2024). These sequences, along with 50 previously reported HAdV-21 genomes from 6 countries (1956-2019), were integrated into a WGS dataset for comprehensive phylogenetic, genetic variation, and recombination analyses. Results:WGS categorized the HAdV-21 strains into 3 subtypes: HAdV-21a, HAdV-21b, and historical HAdV-21p (isolated in the 1950s). HAdV-21a (1956-2024, involving 5 of the 6 countries) and HAdV-21b (2005-2024, involving 3 of the 6 countries) exhibited extensive spatiotemporal distributions. Recent Chinese strains (2023-2024) belonged to HAdV-21a and HAdV-21b (HAdV-21a/b), showing extremely high genetic homology with Chinese 2019 strains (genetic distance: 0.00007) and global strains (distance: <0.00040). Phylogenetic analysis confirmed that HAdV-21a/b shared a common ancestor and maintained a highly conserved genome despite decades of circulation. Sequence variation analysis identified shared and subtype-specific mutations in these two subtypes. Recombination pattern analysis further revealed that HAdV-21a/b acquired an HAdV-3-derived fragment in the E4 region (breakpoint: nt32,843). Conclusions:Recombinant HAdV-21a/b subtypes have co-circulated in China in recent years with remarkable genetic conservation. Enhanced surveillance is essential to quantify associated disease burden and guide targeted prevention and control strategies.

Introduction:Coxsackievirus A12 (CVA12) is a serotype of Enterovirus A. Its evolutionary and molecular characteristics remain poorly understood. Methods:The metagenomic Next-Generation Sequencing (mNGS) strategy were used to investigate the viral diversity. The viral isolation, proliferation assays, phylogenetic relationships and recombination events were analyzed. Results:In this study, nine clinical specimens collected in Beijing, China, during March 2010 to October 2019 were identified as CVA12 positive, among which five were confirmed by mNGS. Then five CVA12 strains were isolated, and the proliferation assays demonstrated the preferential replication of CVA12 in rhabdomyosarcoma (RD) cells, with rapid intracellular replication before being released extracellularly, over Hep-2 cells. Transcriptomic profiling of infected RD cells revealed that the significant up-regulated genes were involved in inflammatory responses and transcriptional regulation (e.g., JUN, FOS), suggesting robust host immune activation. Phylogenetic analysis identified that four strains were clustered into genogroup E, indicating a lineage undergoing active transmission in Beijing, China, the other one into genogroups B. Recombination analysis revealed that strain s7275 exhibited recombination with CVA5 (strain 3,490, GenBank access number OK334538) at the breakpoint position 3,373-6,634, while the others showed recombination with EV-A71 (strain EV71/P1034/2013/China, GenBank access number KP289419) at breakpoint position 3,370-6,645. Discussion:These findings underscored the genetic diversity and recombination dynamics which provided insights into the evolutionary implications of CVA12, and its proliferation features in RD cells of CVA12. Further research is needed to elucidate the functional mechanisms of CVA12 infection and its role for disease.

Objective:Recombination events are common and serve as the primary driving force of diverse human adenovirus (HAdV), particularly in children with acute respiratory tract infections (ARIs). Therefore, continual monitoring of these events is essential for effective viral surveillance and control. Methods:Respiratory specimens were collected from children with ARIs between January 2022 and December 2023. The penton base, hexon, and fiber genes were amplified from HAdV-positive specimens and sequenced to determine the virus type. In cases with inconsistent typing results, genes were cloned into the pGEM-T vector to detect recombination events. Metagenomic next-generation sequencing (mNGS) was performed to characterize the recombinant HAdV genomes. Results:Among 6,771 specimens, 277 (4.09%, 277/6,771) were positvie for HAdV, of which 157 (56.68%, 157/277) were successfully typed, with HAdV-B3 being the dominant type (91.08%, 143/157), and 14 (5.05%, 14/277) exhibited inconsistent typing results, six of which belonged to species B. The penton base genes of these six specimens were classified as HAdV-B7, whereas their hexon and fiber genes were classified as HAdV-B3, resulting in a recombinant genotype designated P7H3F3, which closely resembled HAdV-B114. Additionally, a partial gene encoding L1 52/55 kD was identified, which originated from HAdV-B16. Conclusion:A novel recombinant, P7H3F3, was identified, containing sequences derived from HAdV-B3 and HAdV-B7, which is similar to HAdV-B114, along with additional sequences from HAdV-B16.

The heterogeneity of childhood wheezing illnesses is associated with viral and host factors. Human rhinoviruses (HRV) are the major pathogens in severe wheezing in young children. The single nucleotide polymorphism (SNP) rs6967330 G > A proved to heighten the risk of wheezing. However, the relation between rs6967330 variants of cadherin-related family member 3 (CDHR3) and wheezing induced by human rhinovirus (HRV)-C has not been determined. A total of 11,756 respiratory specimens collected from hospitalized children with acute respiratory infections (ARIs) between September 2017 and March 2023 were screened for enterovirus (EV)/HRVs by the capillary electrophoresis-based multiplex PCR (CEMP) assay, and those positive only for HRVs were amplified and sequenced for HRV and CDHR3 genotyping. The clinical data of the enrolled patients were obtained and analyzed. EV/HRVs (15.2

Human bocavirus (HBoV) is a common respiratory virus among patients with acute respiratory infection (ARI). To investigate the prevalence and genetic characteristics of HBoV, clinical specimens from 13,109 ARI patients were collected through active surveillance from 12 provinces of China during 2012-2021. Extracted nucleic acid was screened and the viral protein 1 (VP1) gene was directly amplified and sequenced in HBoV-positive specimens. 3.51 % of patients were HBoV-positive, with children under 5 years old accounting for 93.48 % of cases. HBoV detection rate increased from 2.35 % in 2012-2019 to 5.38 % in 2020 and 7.68 % in 2021, with a pronounced increase in children aged 2-4 years and in Southern China. The age group with the highest detection rate shifted from infants under 2 years in 2012-2019 to children aged 2-4 years in 2020-2021. The proportion of HBoV co-detections increased significantly in 2020-2021, from 43.98 % to over 60.00 %. All HBoV cases were identified as HBoV-1 with 165 full length sequences of VP1 gene obtained. No temporal or geographic clustering was observed. The VP1 gene evolved at a rate of 7.99 × 10 -5 substitutions/site per year, with ω-value less than 1, indicating that the VP1 protein was under negative selection pressure. Multiple antigen-associated amino acid mutations and positive selection sites were found in the VP1 protein. In conclusion, HBoV1 remains a major cause of pediatric ARI in China, but its epidemic pattern exhibited dynamic shifts during the coronavirus disease 2019 pandemic, while the viral genetic evolution remained relatively stable.

To better understand the epidemiological characteristics of human adenovirus (HAdV) infections in China during and after the coronavirus disease 2019 (COVID-19) pandemic, respiratory specimens were collected from 17,562 enrolled patients with acute respiratory infections (ARIs) in 14 sentinel surveillance provinces during 2020-2023. Eight common respiratory viruses were detected using commercially available nucleic acid detection kits. HAdV-positive cases were statistically analyzed for detection rates, geographic distribution, seasonal patterns, demographic characteristics, and co-infection status. The results of this study showed that the overall HAdV detection rate was 5.09 % (894/17,562) during 2020-2023, with a gradual decrease in the annual detection rate from 6.66 % in 2020 to 3.89 % in 2022 and a rebound in 2023 (5.19 %). The overall HAdV detection rate was significantly higher in the southern region (6.15 %) than in the northern region (4.76 %) (P < 0.001). The median age of patients with HAdV infection was 3 years, with infants aged 0-2 years accounting for the majority (41.39 %). HAdV-positive cases were detected throughout the year, with no clear seasonal pattern, and the HAdV epidemic in China during 2020-2023 may have been driven primarily by the virus infection in the southern region. Co-infections were frequent in HAdV-positive cases (overall rate: 36.01 %), primarily consisting of dual infections (79.28 %), with human rhinovirus and human respiratory syncytial virus being the most common coinfecting pathogens. In conclusion, this study suggested the significant regional and temporal variation in HAdV detection rate in China during 2020-2023, and thus ongoing surveillance should be conducted to elucidate the epidemiological dynamics of HAdV infections.

ABSTRACT 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.IMPORTANCEAcute 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.