Human Bocavirus (HBoV) is an emerging pathogen linked to respiratory and gastrointestinal infections in children. While its role in respiratory diseases is established, its association with liver dysfunction remains unclear. This study presents six paediatric cases of elevated liver enzymes or acute liver failure during HBoV infection, including severe outcomes such as liver transplantation and one fatality. Frequent co-infections with other pathogens were noted, complicating the clinical course. Although direct evidence of HBoV's role in liver involvement is lacking, its potential contribution warrants further investigation to guide clinical management.

The aim of this study was to investigate the clinical characteristics of severe pneumonia caused by human bocavirus (HBoV) infection to explore the associated risk factors.

We conducted a retrospective review of data from children hospitalized with HBoV pneumonia. Based on the severity of pneumonia, patients were categorized into severe pneumonia and non-severe pneumonia groups. Clinical manifestations, laboratory examination results, chest imaging and pathogens were analyzed. Logistic regression was employed to identify the risk factors for severe HBoV pneumonia.

A total of 334 patients were admitted, with 44 (13.17%) patients diagnosed with severe pneumonia and 290 (86.83%) with non-severe pneumonia. There were no significant differences in age distribution, presence of fever, lung moist rales, pleural effusion and reduced breath sounds between the two groups (all P > 0.05). 57.19% of the HBoV-positive children co-infected with other pathogens and HRV was the most common co-infected pathogens with HBoV. No significant differences were observed in the rate of co-infection between the two groups (χ2 = 0.50, p = 0.48). The univariate analysis revealed significant differences between the severe pneumonia group and the non-severe group in terms of gender distribution, presence of underlying chronic diseases, wheezing, premature delivery, lung wheezing rales, pneumothorax, bronchoscopy procedures, length of hospital stay, duration of symptoms prior to admission, neutrophil count, CRP levels, CKMB levels, IgA levels, CD3+(%), CD3+CD4+(%), CD3+CD8+%, and CD3-CD19+% (all P < 0.05). Multivariate logistic regression analysis identified female gender, wheezing and neutrophil count were independent risk factors and the ratio of CD3+CD4+ cells was protective factor for severe HBoV pneumonia. The cut-off values of neutrophil count and the ratio of CD3+CD4+ cells were 6.81 × 109/L and 32.48 respectively.

Our study indicated that female gender, wheezing and neutrophil count greater than 6.81 × 109/L were independent risk factors and the ratio of CD3+CD4+ cells greater than 32.48 was protective factor for severe HBoV pneumonia.

Human bocaviruses (HBoVs) can cause respiratory illness in young children. Although the first HBoV infection in India was reported in 2010, very little information is available about its prevalence, clinical features, or geographic distribution in this country. This study was conducted using 136 respiratory samples from paediatric patients in a tertiary care hospital in Kerala, 21 of which tested positive for HBoV1 and were further characterized through VP1/VP2 gene sequencing. We found that different strains of HBoV1 are co-circulating in the region and that HBoV1 can be detected in children with severe acute respiratory infections, either alone or coinfections with other pathogens, without any significant differences in their clinical characteristics.

Although human bocavirus (HBoV) is primarily linked to respiratory tract infections, its exact role as a respiratory pathogen remains unclear. This study aims to investigate HBoV detection rates, as well as clinical, laboratory, microbiological, and radiological characteristics, length of stay in the emergency department (ED), rate of hospitalization, and severity of illness in cases where HBoV is detected in respiratory secretions.

We conducted a retrospective analysis of all consecutive patients under 18 years who visited a large-volume tertiary pediatric ED from January to December 2023 and tested positive for HBoV in their respiratory viral panel (RVP).

Among the 14,315 patients who underwent RVP testing during the study period, 591 (4%) tested positive for HBoV. After excluding those with incomplete data, 528 patients (57% male) were included in the analyses. The median age was 2.8 [1.2-4.9] years. The most common symptoms were cough (67%), fever (58%), runny nose/nasal congestion/sore throat (34%), and respiratory distress (24%). Thirty percent of the patients had a history of antibiotic use before admission. Thirteen percent of the patients had at least one chronic illness. Co-infection with HBoV occurred in 37% of the patients, with respiratory syncytial virus (RSV) being the most frequently co-detected virus (45%). Lymphopenia was documented in 12% of patients, and 36% had elevated C-reactive protein levels (median 21 [12-38] g/dl). Abnormal chest X-rays were noted in 85% of patients. The management approach included outpatient care for more than half of the patients (69%). Clinical severity was classified as high in 11% of patients (n = 60), necessitating ICU admission.

Although typically mild, HBoV infections can escalate to severe respiratory illnesses, requiring respiratory support and intensive care.

In this study, we present four cases of Human Bocavirus (HBoV) infection in children aged between 1 month and 4 years. Among these cases, two siblings were hospitalized with similar symptoms. Among the four pediatric cases of patients with HBoV infection, three were associated with acute respiratory failure and spontaneous pneumothorax, and two of these presented with subcutaneous emphysema. The presented patients were young children, aged between 1 month and 4 years, two of whom were siblings, suggesting a possible intrafamilial transmission of HBoV1 infection. These cases highlight the importance of considering HBoV as a differential diagnosis in pediatric patients with respiratory and gastrointestinal symptoms. Early recognition and appropriate medical care are important in treating HBoV infection in young children.

Human bocavirus 1 (HBoV1) is a human parvovirus that causes lower respiratory tract infections in young children. It contains a single-stranded (ss) DNA genome of ~5.5 kb that encodes a small noncoding RNA of 140 nucleotides known as bocavirus-encoded small RNA (BocaSR), in addition to viral proteins. Here, we determined the secondary structure of BocaSR in vivo by using DMS-MaPseq. Our findings reveal that BocaSR undergoes N6-methyladenosine (m6A) modification at multiple sites, which is critical for viral DNA replication in both dividing HEK293 cells and nondividing cells of the human airway epithelium. Mechanistically, we found that m6A-modified BocaSR serves as a mediator for recruiting Y-family DNA repair DNA polymerase (Pol) η and Pol κ likely through a direct interaction between BocaSR and the viral DNA replication origin at the right terminus of the viral genome. Thus, this report represents direct involvement of a viral small noncoding RNA in viral DNA replication through m6A modification.

Wheezing children infected with rhinovirus (RV) have a markedly increased risk of subsequently developing recurrencies and asthma. No previous studies have assessed the association between cytokine response and the severity of acute illness in the first wheezing episode in children infected with RV. Forty-seven children treated both as inpatients and as outpatients infected with RV only, aged 3-23 months, with severe first wheezing episodes were recruited. During acute illness, peripheral blood mononuclear cells (PBMCs) were isolated and stimulated with anti-CD3/anti-CD28 in vitro. A multiplex ELISA was used to quantitatively identify 56 different cytokines. The mean age of the children was 17 months, 74% were males, 79% were hospitalized, and 33% were sensitized. In adjusted analyses, the inpatient group was characterized by decreased expressions of interferon gamma (IFN-γ), interleukin 10 (IL-10), macrophage inflammatory protein 1 alpha (MIP-1α), RANTES (CCL5), and tumor necrosis factor-alpha (TNF-α) and an increased expression of ENA-78 (CXCL5) compared to the outpatient group. The cytokine response profiles from the PBMCs were different between the inpatient and outpatient groups. Our results support that firmly controlled interplay between pro-inflammatory and anti-inflammatory responses are required during acute viral infection to absolve the initial infection leading, to less severe illness.

Respiratory tract infections due to a variety of viruses continue to threaten the human population worldwide, particularly in developing countries. Among the responsible viruses, Human Bocavirus (HBoV), a novel discovered virus, causes respiratory tract and gastroenteritis disorders in young children. In Saudi Arabia, data regarding virus molecular epidemiology and evolution and its implication in respiratory tract infection are scarce. In the current study, genetic diversity and circulation pattern of HBoV-1 among hospitalized children due to acute respiratory tract infection (ARTI) during two consecutive years were charted. We found that 3.44% (2014/2015) and 11.25% (2015/2016) of children hospitalized due to ARTI were infected by HBoV-1. We have shown that HBoV was detected year-round without a marked seasonal peak. HBoV-1 also was co-detected with one or multiple other respiratory viruses. The multisequence analysis showed high sequence identity (∼99%) (few point mutation sites) between strains of each genotype and high sequence variation (∼79%) between HBoV-1 and the other 3 genotypes. Phylogenetic analysis showed the clustering of the study's isolates in the HBoV-1 subclade. Our data reveal that genetically conserved HBoV-1 was circulating among admitted children during the course of the study. Further epidemiological and molecular characterization of multiple HBoV-1 strains for different years and from all regions of Saudi Arabia are required to understand and monitor the virus evolution.

To elaborate the utility of multiplex quantitative polymerase chain reaction (multiplex qPCR) for the accurate diagnosis of severe respiratory tract infections (RTIs) in hospitalized children.

In two separate periods during 2022, 76 respiratory specimens (combined throat/nasopharyngeal swabs) were submitted for multiplex qPCR regarding 26 respiratory pathogens. The specimens were obtained from children with severe RTIs hospitalized in the Institute for Respiratory Diseases in Children, Skopje.

Multiplex qPCR detected at least one respiratory pathogen in all examined specimens (76/76), with 83% (63/76) rate of co-infections. Considering that positive results are only the ones with Ct value below 28, the rates of detected pathogens and co-infections decrease to 75% and 22%, respectively. The most commonly detected pathogens during the spring period were Parainfluenza type 3 (PIV3) followed by Adenovirus (AdV) and Respiratory syncytial virus type B (RSVB) with frequency rate of 23%, 19% and 19%, respectively. During the autumn period, the most common were RSVB and Streptococcus pneumoniae with frequency rate of 31% and 17%, respectively.

Multiplex qPCR is a powerful tool for diagnosing RTIs. Semi-quantification of the viral load by reporting Ct values added higher level of evidence for accurate diagnosis. Seasonal detection of the examined viruses was notable with higher prevalence of PIV3 in spring and RSVB in autumn period.

Wheezing is the cardinal symptom of asthma; its presence early in life, mostly caused by viral infections, is a major risk factor for the establishment of persistent or recurrent disease. Early-life wheezing and asthma exacerbations are triggered by common respiratory viruses, mainly rhinoviruses (RV), and to a lesser extent, respiratory syncytial virus, parainfluenza, human metapneumovirus, coronaviruses, adenoviruses, influenza, and bocavirus. The excess presence of bacteria, several of which are part of the microbiome, has also been identified in association with wheezing and acute asthma exacerbations, including haemophilus influenza, streptococcus pneumoniae, moraxella catarrhalis, mycoplasma pneumoniae, and chlamydophila pneumonia. While it is not clear when asthma starts, its characteristics develop over time. Airway remodeling already appears between the ages of 1 and 3 years of age even prior to the presence of atopic inflammation or an asthma diagnosis. The role of genetic defect or variations hampering the airway epithelium in response to environmental stimuli and severe disease morbidity are now considered as major determinants for early structural changes. Repeated viral infections can induce and perpetuate airway hyperresponsiveness. Allergic sensitization, that often precedes infection-induced wheezing, shifts inflammation toward type-2, while common respiratory infections themselves promote type-2 inflammation. Nevertheless, most children who wheeze with viral infections during infancy and during preschool years do not develop persistent asthma. Multiple factors, including illness severity, viral etiology, allergic sensitization, and the exposome, are associated with disease persistence. Here, we summarize current knowledge and developments in infection epidemiology of asthma in children, describing the known impact of each individual agent and mechanisms of transition from recurrent wheeze to asthma.

Human Bocaviruses (HBoV) can cause acute respiratory tract infections. High coinfection rates cloud its pathogenicity. This study sought to describe the clinical features of HBoV1 disease in children and adults with Influenza-like illness (ILI), exploring associations between viral load, clinical features, and seasonality.

Patients who tested positive for HBoV1 by polymerase chain reaction, enrolled from April 2010 to March 2014 in the ILI002 prospective observational cohort study were included in this cross-sectional nested study. Participants were included in ILI002 if they presented with signs and/or symptoms suggestive of influenza-like illness. Samples were tested for viral load, and NP1 and VP1/VP2 phylogenetic analyses, except for the samples lacking suitable and viable clinical material for genotyping.

We identified HBoV1 in 157 (2.8%) of participants. Prevalence was 4.5% in children and 1.8% in adults. Single HBoV1 detection occurred in 41.1% and 46.3% of children and adults, respectively. Children commonly experienced fever (83.3%), cough with sputum (74.4%), and shortness of breath (72.2%). In the multivariate analysis of children, significant positive associations were detected between viral loads and age (0.20 [95% CI: 0.07, 0.33]), and the presence of fever (2.64 [95% CI: 1.35, 3.94]), nasal congestion (1.03 [95% CI: 0.07, 1.99]), dry cough (1.32 [95% CI: 0.42, 2.22]), chest congestion (1.57 [95% CI: 0.33, 2.80]), red eyes (1.25 [95% CI: 0.35, 2.14]), cough with sputum (1.79 [95% CI: 0.80, 2.78]), and other signs and symptoms such as chills, dizziness, and diaphoresis (1.73 [95% CI: 0.19, 3.27]). In contrast, significant negative associations were found between viral loads and percent neutrophils on the blood count (-0.04 [95% CI: -0.06, -0.02]), fatigue (-1.60 [95% CI: -2.46, -0.74]) and the presence of other symptoms or signs, including adenopathy and rash (-1.26 [95% CI: -2.31, -0.21]). Adults commonly experienced sore throat (73.1%), fatigue (77.4%), and headache (73.1%). In the multivariate analysis of adults, significant positive associations were detected between viral load and body mass index (0.13 [95% CI: 0.04, 0.21]), and the presence of confusion (1.54 [95% CI: 0.55, 2.53]), and sore throat (1.03 [95% CI: 0.20, 1.85]), and significant negative associations were detected between viral load and chest congestion (-1.16 [95% CI: -2.07, -0.24]). HBoV1 was detected throughout the year irrespective of season, temperature, and humidity.

This study demonstrated the importance of detecting HBoV1 in patients with influenza-like illness either as single infection or co-infection, in both adults and children, and improves the characterization of HBoV1 seasonality, clinical features, and viral load. Phylogenetic analyses show a high conservation.

The Mexican Emerging Infectious Diseases Clinical Research Network (LaRed), CONACYT (Fondo Sectorial SSA/IMSS/ISSSTE, Projects No. 71260 and No. 127088), Fondos federales no. HIM/2015/006, NIAID, NIH through a contract with Westat, Inc. (HHSN2722009000031, HHSN27200002), NCI, NIH (75N91019D00024, 75N91019F00130). Additional information at the end of the manuscript.

Rhinovirus (RV)-induced first wheezing episodes in children are associated with a markedly increased risk of asthma. Previous studies have suggested that human bocavirus 1 (HBoV1) may modify RV-induced immune responses in young children. We investigated cytokine profiles of sole RV- and dual RV-HBoV1-induced first wheezing episodes, and their association with severity and prognosis.

Fifty-two children infected with only RV and nine children infected with dual RV-HBoV1, aged 3-23 months, with severe first wheezing episodes were recruited. At acute illness and 2 weeks later, peripheral blood mononuclear cells were isolated, and stimulated with anti-CD3/anti-CD28 in vitro. Multiplex ELISA was used to quantitatively identify 56 different cytokines at both study points. Patients were prospectively followed for 4 years.

The mean age of the children was 14.3 months, and 30% were sensitized. During the acute illness, the adjusted analyses revealed a decrease in the expression of IL-1b, MIP-1b, Regulated upon activation, normal T cell expressed and presumably secreted (CCL5), TNF-a, TARC, and ENA-78 in the RV-HBoV1 group compared with the RV group. In the convalescence phase, the RV-HBoV1 group was characterized by decreased expression of Fractalkine, MCP-3, and IL-8 compared to the RV group. Furthermore, the hospitalization time was associated with the virus group and cytokine response (interaction p < 0.05), signifying that increased levels of epidermal growth factor and MIP-1b were related with a shorter duration of hospitalization in the RV-HBoV1 coinfection group but not in the RV group.

Different cytokine response profiles were detected between the RV and the RV-HBoV1 groups. Our results show the idea that RV-induced immune responses may be suppressed by HBoV1.

To determine the epidemiological status of influenza and understand the distribution of common respiratory viruses in adult patients with influenza-like illness (ILI) cases in Taiyuan City, Shanxi Province, China, epidemiological data between 2018 and 2019 were retrieved from the China Influenza Surveillance Information System, and two sentinel ILI surveillance hospitals were selected for sample collection. All specimens were screened for influenza virus (IFV) and the other 14 common respiratory viruses using real-time polymerase chain reaction. The results of the 2-year ILI surveillance showed that 26,205 (1.37%) of the 1,907,869 outpatients and emergency patients presented with ILI, with an average annual incidence of 297.75 per 100,000 individuals, and ILI cases were predominant in children <15 years (21,348 patients, 81.47%). Of the 2713 specimens collected from adult patients with ILI, the overall detection rate of respiratory viruses was 20.13%, with IFV being the most frequently detected (11.79%) and at a relatively lower rate than other respiratory viruses. Further subtype analysis indicated an alternating or mixed prevalence of H1N1 (2009), H3N2, Victoria, and Yamagata subtypes. This study provides a baseline epidemiological characterization of ILI and highlights the need for a nationwide detection and surveillance system for multiple respiratory pathogens.

Numerous delivery strategies, primarily novel nucleic acid delivery carriers, have been developed and explored to enable therapeutically relevant lung gene therapy. However, its clinical translation is yet to be achieved despite over 30 years of efforts, which is attributed to the inability to overcome a series of biological barriers that hamper efficient nucleic acid transfer to target cells in the lung.

This review is initiated with the fundamentals of nucleic acid therapy and a brief overview of previous and ongoing efforts on clinical translation of lung gene therapy. We then walk through the nature of biological barriers encountered by nucleic acid carriers administered via respiratory and/or systemic routes. Finally, we introduce advanced strategies developed to overcome those barriers to achieve therapeutically relevant nucleic acid delivery efficiency in the lung.

We are now stepping close to the clinical translation of lung gene therapy, thanks to the discovery of novel delivery strategies that overcome biological barriers via comprehensive preclinical studies. However, preclinical findings should be cautiously interpreted and validated to ultimately realize meaningful therapeutic outcomes with newly developed delivery strategies in humans. In particular, individual strategies should be selected, tailored, and implemented in a manner directly relevant to specific therapeutic applications and goals.

Human bocavirus (HBoV) is an important respiratory pathogen, especially in children, but it is often found in co-detection with other respiratory viruses, which makes the diagnostic approach challenging. We compared multiplex PCR and quantitative PCR for HBoV with multiplex tandem PCR (MT-PCR) in 55 cases of co-detection of HBoV and other respiratory viruses. In addition, we investigated whether there is a connection between the severity of the disease, measured by the localization of the infection, and amount of virus detected in the respiratory secretions. No statistically significant difference was found, but children with large amount of HBoV and other respiratory virus had a longer stay in hospital.

Respiratory tract infections (RTIs) in infants are often caused by viruses. Although respiratory syncytial virus (RSV), influenza virus and human metapneumovirus (hMPV) can be considered the most pathogenic viruses in children, rhinovirus (RV) is often found in asymptomatic infants as well. Little is known about the health consequences of viral presence, especially early in life. We aimed to examine the dynamics of (a)symptomatic viral presence and relate early viral detection to susceptibility to RTIs in infants.

In a prospective birth cohort of 117 infants, we tested 1304 nasopharyngeal samples obtained from 11 consecutive regular sampling moments, and during acute RTIs across the first year of life for 17 respiratory viruses by quantitative PCR. Associations between viral presence, viral (sub)type, viral load, viral co-detection and symptoms were tested by generalized estimating equation (GEE) models.

RV was the most detected virus. RV was negatively associated [GEE: adjusted odds ratio (aOR) 0.41 (95% CI 0.18-0.92)], and hMPV, RSV, parainfluenza 2 and 4 and human coronavirus HKU1 were positively associated with an acute RTI. Asymptomatic RV in early life was, however, associated with increased susceptibility to and recurrence of RTIs later in the first year of life (Kaplan-Meier survival analysis: P = 0.022).

Respiratory viruses, including the seasonal human coronaviruses, are often detected in infants, and are often asymptomatic. Early life RV presence is, though negatively associated with an acute RTI, associated with future susceptibility to and recurrence of RTIs. Further studies on potential ecologic or immunologic mechanisms are needed to understand these observations.

Acute lower respiratory infection (ALRI) remains the leading cause of death in children worldwide, and viruses have been the major cause of ALRI. In Myanmar, ALRI is associated with high morbidity and mortality in children, and detailed information on ALRI is currently lacking.

This prospective study investigated the viral aetiologies, clinical manifestations, and outcomes of ALRI in hospitalised children aged 1 month to 12 years at the Yankin Children Hospital, Yangon, Myanmar from May 2017 to April 2019. The sample size was set to 300 patients for each year. Two nasopharyngeal swabs were obtained for the patients with suspected viral ALRI; one for rapid tests for influenza and respiratory syncytial virus (RSV), and the other for real-time PCR for the 16 ALRI-causing viruses. Pneumococcal colonization rates were also investigated using real-time PCR. Clinical information was extracted from the medical records, and enrolled patients were categorised by age and severity for comparison.

Among the 5463 patients admitted with a diagnosis of ALRI, 570 (10.4%) were enrolled in this study. The median age of the patients was 8 months (interquartile range, 4-15 months). The most common symptoms were cough (93%) and difficulty in breathing (73%), while the most common signs of ALRI were tachypnoea (78%) and chest indrawing (67%). A total of 16 viruses were detected in 502 of 570 patients' samples (88%), with RSV B (36%) and rhinovirus (28%) being the most commonly detected. Multiple viruses were detected in 221 of 570 samples (37%) collected from 570 patients. Severe ALRI was diagnosed in 107 of 570 patients (19%), and RSV B and human rhinovirus were commonly detected. The mortality rate was 5%; influenza virus A (29%) and RSV B (21%) were commonly detected, and stunting and lack of immunization were frequently observed in such cases. Additionally, 45% (259/570) of the patients had pneumococcal colonization.

Viral ALRI in hospitalised children with a median of 8 months has significant morbidity and mortality rates in Myanmar. RSV and rhinovirus were the most commonly detected from nasopharyngeal swabs, while influenza virus and RSV were the most frequently associated with fatal cases.

Human bocavirus 1 (HBoV1), an autonomous human parvovirus, causes acute respiratory tract infections in young children. HBoV1 infects well-differentiated (polarized) human airway epithelium cultured at an air-liquid interface (HAE-ALI). HBoV1 expresses a large nonstructural protein, NS1, that is essential for viral DNA replication. HBoV1 infection of polarized human airway epithelial cells induces a DNA damage response (DDR) that is critical to viral DNA replication involving DNA repair with error-free Y-family DNA polymerases. HBoV1 NS1 or the isoform NS1-70 per se induces a DDR. In this study, using the second-generation proximity-dependent biotin identification (BioID2) approach, we identified that Ku70 is associated with the NS1-BioID2 pulldown complex through a direct interaction with NS1. Biolayer interferometry (BLI) assay determined a high binding affinity of NS1 with Ku70, which has an equilibrium dissociation constant (KD) value of 0.16 μM and processes the strongest interaction at the C-terminal domain. The association of Ku70 with NS1 was also revealed during HBoV1 infection of HAE-ALI. Knockdown of Ku70 and overexpression of the C-terminal domain of Ku70 significantly decreased HBoV1 replication in HAE-ALI. Thus, our study provides, for the first time, a direct interaction of parvovirus large nonstructural protein NS1 with Ku70. IMPORTANCE Parvovirus infection induces a DNA damage response (DDR) that plays a pivotal role in viral DNA replication. The DDR includes activation of ATM (ataxia telangiectasia mutated), ATR (ATM- and RAD3-related), and DNA-PKcs (DNA-dependent protein kinase catalytic subunit). The large nonstructural protein (NS1) often plays a role in the induction of DDR; however, how the DDR is induced during parvovirus infection or simply by the NS1 is not well studied. Activation of DNA-PKcs has been shown as one of the key DDR pathways in DNA replication of HBoV1. We identified that HBoV1 NS1 directly interacts with Ku70, but not Ku80, of the Ku70/Ku80 heterodimer at high affinity. This interaction is also important for HBoV1 replication in HAE-ALI. We propose that the interaction of NS1 with Ku70 recruits the Ku70/Ku80 complex to the viral DNA replication center, which activates DNA-PKcs and facilitates viral DNA replication.

Human bocavirus 1 (HBoV1) belongs to the family Parvoviridae and it is acknowledged that HBoV1 is a respiratory pathogen. We report the case of a 13-month-old boy who presented with a cough, shortness of breath, and wheezing, and who eventually died of severe pneumonia and acute respiratory distress syndrome (ARDS). Metagenomics next-generation sequencing (mNGS) showed that HBoV1 was the only detected pathogen. The nasopharyngeal aspirate viral load was 2.08 × 10¹⁰ copies/ml and the serum viral load was 2.37 × 10⁵ copies/ml. The child was still oxygen deficient under mechanical ventilation. Chest imaging suggested diffuse lesions in both lungs, an injury caused by ARDS. In this case, the clinical symptoms and signs of the child, the high viral load, viremia, and the detection of mNGS in the tracheal aspirate all supported that HBoV1 could cause severe acute respiratory tract infection in children without other pathogen infections.

Нuman bocaviruses (hBoVs) are often associated with acute respiratory infections (ARIs). Information on the distribution and molecular epidemiology of hBoVs in Bulgaria is currently limited. The objectives of this study were to investigate the prevalence and genetic characteristics of hBoVs detected in patients with ARIs in Bulgaria. From October 2016 to September 2019, nasopharyngeal/oropharyngeal swabs were prospectively collected from 1842 patients of all ages and tested for 12 common respiratory viruses using a real-time RT-PCR. Phylogenetic and amino acid analyses of the hBoV VP1/VP2 gene/protein were performed. HBoV was identified in 98 (5.3%) patients and was the 6^th most prevalent virus after respiratory-syncytial virus (20.4%), influenza A(H1N1)pdm09 (11.1%), A(H3N2) (10.5%), rhinoviruses (9.9%), and adenoviruses (6.8%). Coinfections with other respiratory viruses were detected in 51% of the hBoV-positive patients. Significant differences in the prevalence of hBoVs were found during the different study periods and in patients of different age groups. The detection rate of hBoV was the highest in patients aged 0-4 years (6.9%). In this age group, hBoV was the only identified virus in 9.7%, 5.8%, and 1.1% of the children diagnosed with laryngotracheitis, bronchiolitis, and pneumonia, respectively. Among patients aged ≥5 years, hBoV was detected as a single agent in 2.2% of cases of pneumonia. Phylogenetic analysis showed that all Bulgarian hBoV strains belonged to the hBoV1 genotype. A few amino acid substitutions were identified compared to the St1 prototype strain. This first study amongst an all-age population in Bulgaria showed a significant rate of hBoV detection in some serious respiratory illnesses in early childhood, year-to-year changes in the hBoV prevalence, and low genetic variability in the circulating strains.

To evaluate the pathogenicity of a broad range of 11 possible gastroenteritis viruses, by means of statistical relationships with cases vs. controls, or Ct-values, in order to establish the most appropriate diagnostic panel for our general practitioner (GP) patients in the Netherlands (2010-2012).

Archived stool samples from 1340 cases and 1100 controls were retested using internally controlled multiplex real-time PCRs for putative pathogenic gastroenteritis viruses: adenovirus, astrovirus, bocavirus, enterovirus, norovirus GI and GII, human parechovirus, rotavirus, salivirus, sapovirus, and torovirus.

The prevalence of any virus in symptomatic cases and asymptomatic controls was 16.6% (223/1340) and 10.2% (112/1100), respectively. Prevalence of astrovirus (adjusted odds ratio (aOR) 10.37; 95% confidence interval (CI) 1.34-80.06) and norovirus GII (aOR 3.10; CI 1.62-5.92) was significantly higher in cases versus controls. Rotavirus was encountered only in cases. We did not find torovirus and there was no statistically significant relationship with cases for salivirus (aOR 1,67; (CI) 0.43-6.54)), adenovirus non-group F (aOR 1.20; CI 0.75-1.91), bocavirus (aOR 0.85; CI 0.05-13.64), enterovirus (aOR 0.83; CI 0.50-1.37), human parechovirus (aOR 1.61; CI 0.54-4.77) and sapovirus (aOR 1.15; CI 0.67-1.98). Though adenovirus group F (aOR 6.37; CI 0.80-50.92) and norovirus GI (aOR 2.22, CI: 0.79-6.23) are known enteropathogenic viruses and were more prevalent in cases than in controls, this did not reach significance in this study. The Ct value did not discriminate between carriage and disease in PCR-positive subjects.

In our population, diagnostic gastroenteritis tests should screen for adenovirus group F, astrovirus, noroviruses GI and GII, and rotavirus. Case-control studies as ours are lacking and should also be carried out in populations from other epidemiological backgrounds.

Bocaviruses are typical zoonotic pathogens with a wide range of hosts. Here, we report the detection of human bocavirus (HBoV) in Rattus norvegicus captured in China and the results of sequencing and phylogenetic analysis based on the partial VP1 region and the entire viral genome. A total of 357 fecal samples from rats were collected in 2015-2017 and analyzed for HBoV using PCR. The detection rate of HBoV was 0.84% (3/357). Phylogenetic analysis revealed that this virus is genetically closely related to HBoV-2. R. norvegicus may be a carrier of HBoV, and its impact on public health merits attention.

Parvoviruses package a linear single-stranded DNA genome with hairpin structures at both ends. It has been thought that terminal hairpin sequences are indispensable for viral DNA replication. Here, we provide evidence that the hairpin-deleted duplex genomes of human bocavirus 1 (HBoV1) replicate in human embryonic kidney (HEK) 293 cells. We propose an alternative model for HBoV1 DNA replication in which the leading strand can initiate strand-displacement without "hairpin-transfer." The transfection of the HBoV1 duplex genomes that retain a minimal replication origin at the right-end (OriR), but with extensive deletions in the right-end hairpin (REH), generated viruses in HEK293 cells at a level 10-20 times lower than the wild-type (WT) duplex genome. Importantly, these viruses that have a genome with various deletions after the OriR, but not the one retaining only the OriR, replicated in polarized human airway epithelia. We discovered that the 18-nt sequence (nt 5,403-5,420) beyond the OriR was sufficient to confer virus replication in polarized human airway epithelia, although its progeny virus production was ∼5 times lower than that of the WT virus. Thus, our study demonstrates that hairpin transfer-independent productive parvovirus DNA replication can occur. Importance Hairpin transfer-independent parvovirus replication was modeled with human bocavirus 1 (HBoV1) duplex genomes whose 5' hairpin structure was ablated by various deletions. In HEK293 cells, these duplex viral genomes with ablated 5'/hairpin sequence replicated efficiently and generated viruses that productively infected polarized human airway epithelium. Thus, for the first time, we reveal a previously unknown phenomenon that the productive parvovirus DNA replication does not depend on the hairpin sequence at REH to initiate "rolling hairpin" DNA replication. Notably, the intermediates of viral DNA replication, as revealed two-dimensional electrophoresis, from transfections of hairpin sequence-deleted duplex genome and full-length genome in HEK293 cells, as well as from virus infection of polarized human airway epithelia are similar. Thus, the establishment of the hairpin transfer-independent parvoviral DNA replication deepens our understanding in viral DNA replication and may have implications in development of parvovirus-based viral vectors with alternative properties.

Human bocaviruses (HBoVs) are recently described as human emergent viruses, especially in young children. In this study, we undertook a systematic review and meta-analysis to estimate their prevalence in Europe. PubMed, Web of Science and Scopus databases were systematically screened for clinical studies, up to October 2020. Study eligibility criteria were primary full-text articles from clinical studies, conducted using valid screening test methods and published in peer-reviewed journals, in English or Spanish and from European countries. The overall pooled prevalence, prevalence by country as well as the prevalence of HBoV as a single or co-pathogen were estimated using a random-effects model. Sub-group and meta-regression analyses explored potential sources of heterogeneity in the data. A total of 35 studies involving 32,656 subjects from 16 European countries met the inclusion criteria. Heterogeneity (I²  = 97.0%, p < .01) was seen among studies; HBoV prevalence varied from 2.0 to 45.69% with a pooled estimate of 9.57% (95%CI 7.66-11.91%). The HBoV prevalence both as a single infection (3.99%; 95%CI 2.99-5.31%) or as co-infection with other viruses (5.06%; 95%CI 3.88-6.58%) was also analysed. On a geographic level, prevalence by country did not show statistical differences, ranging from 3.24% (Greece) to 21.05% (Denmark). An odds ratio analysis was also included in order to evaluate the relevance of the variable 'age' as a risk factor of HBoV infection in children <5 years old. The OR value of 1.77 (95%CI 1.13-2.77; p < .01) indicated that being <5 years old is a risk factor for HBoV infection. This study showed that HBoV has a moderate prevalence among European countries.

Persistent human bocavirus 1 (HBoV1) infection is a common finding in patients suffering from chronic tonsillar disease. However, the associations between HBoV1 infection and specific immune reactions are not completely known. We aimed to compare in vivo expression of T-cell cytokines, transcription factors, and type I/III interferons in human tonsils between HBoV1-positive and -negative tonsillectomy patients.

Tonsil tissue samples, nasopharyngeal aspirate (NPA), and serum samples were obtained from 143 immunocompetent adult and child tonsillectomy patients. HBoV1 and 14 other respiratory viruses were detected in NPAs and tonsil tissues by polymerase chain reaction (PCR). Serology and semi-quantitative PCR were used for diagnosing HBoV1 infections. Expression of 14 cytokines and transcription factors (IFN-α, IFN-β, IFN-γ, IL-10, IL-13, IL-17, IL-28, IL-29, IL-37, TGF-β, FOXP3, GATA3, RORC2, Tbet) was analyzed by quantitative reverse-transcription (RT)-PCR in tonsil tissues.

HBoV1 was detected by PCR in NPA and tonsils from 25 (17%) study patients. Serology results indicated prior nonacute infections in 81% of cases. Tonsillar cytokine responses were affected by HBoV1 infection. The suppression of two transcription factors, RORC2 and FOXP3, was associated with HBoV1 infection (p < 0.05). Furthermore, intratonsillar HBoV1-DNA loads correlated negatively with IFN-λ family cytokines and IL-13.

Our study shows distinctively decreased T-helper17 and T-regulatory type immune responses in local lymphoid tissue in HBoV1-positive tonsillectomy patients. HBoV1 may act as a suppressive immune modulator.

Several viruses target the human respiratory tract, causing different clinical manifestations spanning from mild upper airway involvement to life-threatening acute respiratory distress syndrome (ARDS). As dramatically evident in the ongoing SARS-CoV-2 pandemic, the clinical picture is not always easily predictable due to the combined effect of direct viral and indirect patient-specific immune-mediated damage. In this review, we discuss the main RNA (orthomyxoviruses, paramyxoviruses, and coronaviruses) and DNA (adenoviruses, herpesviruses, and bocaviruses) viruses with respiratory tropism and their mechanisms of direct and indirect cell damage. We analyze the thin line existing between a protective immune response, capable of limiting viral replication, and an unbalanced, dysregulated immune activation often leading to the most severe complication. Our comprehension of the molecular mechanisms involved is increasing and this should pave the way for the development and clinical use of new tailored immune-based antiviral strategies.

Human bocavirus 1 (HBoV1) is a small DNA virus that belongs to the Bocaparvovirus genus of the Parvoviridae family. HBoV1 is a common respiratory pathogen that causes mild to life-threatening acute respiratory tract infections in children and immunocompromised individuals, infecting both the upper and lower respiratory tracts. HBoV1 infection causes death of airway epithelial cells, resulting in airway injury and inflammation. In vitro, HBoV1 only infects well-differentiated (polarized) human airway epithelium cultured at an air-liquid interface (HAE-ALI), but not any dividing human cells. A full-length HBoV1 genome of 5543 nucleotides has been cloned from DNA extracted from a human nasopharyngeal swab into a plasmid called HBoV1 infectious clone pIHBoV1. Transfection of pIHBoV1 replicates efficiently in human embryonic kidney 293 (HEK293) cells and produces virions that are highly infectious. This article describes protocols for production of HBoV1 in HEK293 cells, generation of HAE-ALI cultures, and infection with HBoV1 in HAE-ALI. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Human bocavirus 1 production in HEK293 cells Support Protocol 1: HEK293 cell culture and transfection Support Protocol 2: Quantification of human bocavirus 1 using real-time quantitative PCR Basic Protocol 2: Differentiation of human airway cells at an air-liquid interface Support Protocol 3: Expansion of human airway epithelial cell line CuFi-8 Support Protocol 4: Expansion of human airway basal cells Support Protocol 5: Coating of plastic dishes and permeable membranes of inserts Support Protocol 6: Transepithelial electrical resistance measurement Basic Protocol 3: Human bocavirus 1 infection in human airway epithelium cultured at an air-liquid interface Support Protocol 7: Isolation of infected human airway epithelium cells from inserts Basic Protocol 4: Transduction of airway basal cells with lentiviral vector.

Wheezing is a major problem in children, and respiratory viruses are often believed to be the causative agent. While molecular detection tools enable identification of respiratory viruses in wheezing children, it remains unclear if and how these viruses are associated with wheezing. The objective of this systematic review is to clarify the prevalence of different respiratory viruses in children with wheezing.

We performed an electronic in Pubmed and Global Index Medicus on 01 July 2019 and manual search. We performed search of studies that have detected common respiratory viruses in children ≤18 years with wheezing. We included only studies using polymerase chain reaction (PCR) assays. Study data were extracted and the quality of articles assessed. We conducted sensitivity, subgroup, publication bias, and heterogeneity analyses using a random effects model.

The systematic review included 33 studies. Rhinovirus, with a prevalence of 35.6% (95% CI 24.6-47.3, I2 98.4%), and respiratory syncytial virus, at 31.0% (95% CI 19.9-43.3, I2 96.4%), were the most common viruses detected. The prevalence of other respiratory viruses was as follows: human bocavirus 8.1% (95% CI 5.3-11.3, I2 84.6%), human adenovirus 7.7% (95% CI 2.6-15.0, I2 91.0%), influenza virus6.5% (95% CI 2.2-12.6, I2 92.4%), human metapneumovirus5.8% (95% CI 3.4-8.8, I2 89.0%), enterovirus 4.3% (95% CI 0.1-12.9, I2 96.2%), human parainfluenza virus 3.8% (95% CI 1.5-6.9, I2 79.1%), and human coronavirus 2.2% (95% CI 0.6-4.4, I2 79.4%).

Our results suggest that rhinovirus and respiratory syncytial virus may contribute to the etiology of wheezing in children. While the clinical implications of molecular detection of respiratory viruses remains an interesting question, this study helps to illuminate the potential of role respiratory viruses in pediatric wheezing.

PROSPERO, CRD42018115128.

Viral respiratory tract infections are the leading cause of acute wheezing in children with a significant risk of hospital admission, risk of recurrence and subsequent asthma. Human respiratory syncytial virus (RSV) and human rhinovirus (RV) in childhood wheezing are widely studied; however, accessible PCR assays enabled diagnosis of other pathogens, including bocavirus (hBOV) and metapneumovirus (hMPV).

The aim of the study was to evaluate the prevalence of respiratory viruses in children hospitalized for acute wheezing along with demographic and clinical data.

We enrolled 101 children, n = 50 (49.5%) with wheezy bronchitis, n = 34 (33.7%) with acute bronchiolitis and n = 17 (16.8%) with exacerbation of asthma; (median age 1.41 ± 2.84 years). Multiplex real-time PCR assay was used for virus detection.

One or more viruses were detected in 83.2% subjects: RSV in 44.6%, followed by RV (23.8%), hBOV and hMPV (both 11.9%); other viruses were less frequent (<8%). Viral coinfection was found in 38 (37.6%) of children. ANCOVA analysis revealed significantly higher total IgE concentrations in the hMPV-positive subgroup compared to RSV (34 kU/L vs 12.7 kU/L; P = .009) and RV (13.3 kU/L, P = .022). For both hMPV and hBOV an association with atopic dermatitis (AD) was observed: aOR for hMPV and AD was 5.6 (95%CI: 1.4-22.7; P = .016) and 4.7 for hBOV and AD (95%CI: 1.3-18; P = .024).

Viral detection ratio in wheezy respiratory tract infections in Polish children is high (83.2%), with both hBOV and hMPV at 11.9% The results also suggest possible relationship of hBOV wheezy infection with nonspecific markers of atopy in children.

Acute bronchiolitis is one of the most common lower respiratory tract infections in children with less than 2 years of age. Nowadays, molecular methods provide an opportunity to better understand the etiology of bronchiolitis. Several viral agents including Respiratory syncytial virus (RSV), Rhinovirus, Parainfluenza and Human bocavirus (HBoV) are responsible for acute bronchiolitis. There are growing studies on the prevalence of HBoV in patients with bronchiolitis. The present systematic review and meta-analysis were conducted to determine the pooled prevalence of HBoV in the respiratory samples of children with acute bronchiolitis. A literature search was conducted in the databases of PubMed, Scopus and Web of Science to recruit studies reporting the frequency of HBoV in <2-year-old children with acute bronchiolitis from 2005 to 2019. Only studies that used polymerase chain reaction (PCR)-based methods to detect the virus in nasopharyngeal samples were included. A total of 22 studies assessing 6751 cases were analyzed. According to the meta-analysis based on the random-effects model, the overall prevalence of HBoV in children with <2 years old was obtained 13% (95% CI: 0.09-0.17). Additionally, the rates of single (as the sole organism) and mixed (in combination with other viruses) HBoV infections were 4% and 9%, respectively. This study showed a high rate of HBoV detection in children with acute bronchiolitis. This should be considered as part of a diagnostic test panel for respiratory infections in children with bronchiolitis.

Viral primary infections and reactivations are common complications in patients after solid organ transplantation (SOT) and hematopoietic stem cell transplantation (HSCT) and are associated with high morbidity and mortality. Among these patients, viral infections are frequently associated with viremia. Beyond the usual well-known viruses that are part of the routine clinical management of transplant recipients, numerous other viral signatures or genomes can be identified in the blood of these patients. The identification of novel viral species and variants by metagenomic next-generation sequencing has opened up a new field of investigation and new paradigms. Thus, there is a need to thoroughly describe the state of knowledge in this field with a review of all viral infections that should be scrutinized in high-risk populations. Here, we review the eukaryotic DNA and RNA viruses identified in blood, plasma, or serum samples of pediatric and adult SOT/HSCT recipients and the prevalence of their detection, with a particular focus on recently identified viruses and those for which their potential association with disease remains to be investigated, such as members of the Polyomaviridae, Anelloviridae, Flaviviridae, and Astroviridae families. Current knowledge of the clinical significance of these viral infections with associated viremia among transplant recipients is also discussed. To ensure a comprehensive description in these two populations, individuals described as healthy (mostly blood donors) are considered for comparative purposes. The list of viruses that should be on the clinicians' radar is certainly incomplete and will expand, but the challenge is to identify those of possible clinical significance.

Limited data exist regarding the impact of human bocavirus (BoV) in hematopoietic cell transplant (HCT) recipients.

In a longitudinal surveillance study among allogeneic HCT recipients, pre-HCT and weekly post-HCT nasal washes and symptom surveys were collected through day 100, then at least every 3 months through 1 year post-HCT at the Fred Hutch (2005-2010). Samples were tested by multiplex semi-quantitative PCR for 12 viruses. Plasma samples from BoV+ subjects were analyzed by PCR. Separately, we conducted a retrospective review of HCT recipients with BoV detected in lower respiratory tract specimens.

Among 51 children and 420 adults in the prospective cohort, 21 distinct BoV respiratory tract infections (RTIs) were observed by 1 year post-HCT in 19 patients. Younger age and exposure to children were risk factors for BoV acquisition. Univariable models among patients with BoV RTI showed higher peak viral load in nasal samples (p=0.04) and presence of respiratory copathogens (p=0.03) were associated with presence of respiratory symptoms but BoV plasma detection was not. Only watery eyes and rhinorrhea were associated with BoV RTI in adjusted models. With additional chart review, we identified 6 HCT recipients with BoV detected in lower respiratory tract specimens [incidence rate of 0.4% (9/2509) per sample tested]. Although all cases presented with hypoxemia, 4 had respiratory copathogens or concomitant conditions that contributed to respiratory compromise.

BoV RTI is infrequent in transplant recipients and associated with mild symptoms. Our studies did not demonstrate convincing evidence that BoV is a serious respiratory pathogen.

Introduction. Globally, human bocavirus (HBoV) has been detected in respiratory samples from patients suffering from upper and lower respiratory diseases. In Kuwait, little is known about the epidemiological and clinical characterization of the virus and genetic characterization of the virus as a respiratory pathogen is unknown.Aim. This study aims to explore the molecular epidemiology and clinical features of HBoV isolates in patients with respiratory diseases.Methodology. Retrospectively, between 2018 and 2020, 5941 respiratory samples from patients with respiratory diseases were screened for respiratory viruses using multiplex real-time PCR. Samples that were positive for HBoV were then subjected to NP1 and VP1/PV2 phylogenetic analysis.Results. HBoV was detected in 1.9 % of the patients, with a peak incidence of infection among children <1 year old. Co-infection with other respiratory viruses was observed in 56.8 % of HBoV-positive patients. Fever, cough and respiratory distress were the most common clinical features of HBoV infection. Phylogenetic analysis of the Kuwaiti HBoV isolates revealed that all the isolates were of the HBoV-1 genotype, with slight sequence variations among the isolates.Conclusion. This study illustrated the predominance of the HBoV-1 genotype in patients with respiratory diseases in Kuwait with minimal genetic variability. It also highlighted the clinical features of HBoV-1 infection, verifying its role in respiratory diseases.

Human bocavirus (HBoV) is a newly identified pathogen that can cause upper and lower respiratory infections usually in children; however, its clinical characteristics and significance in respiratory infections in adults have not been well known. Our objective was to evaluate the clinical features of respiratory HBoV infection and to describe the CT findings of HBoV pneumonia in adults.

A total of 185 adult patients diagnosed with HBoV infection at a tertiary referral center between January 2010 and December 2017 were retrospectively evaluated with respect to the clinical characteristics of HBoV infection and its risk factors for pneumonia. Chest CT findings for 34 patients with HBoV pneumonia without co-infection were analyzed and compared between immunocompetent (n = 18) and immunocompromised (n = 16) patients.

HBoV infections were predominantly noted between February and June. Among the 185 patients with HBoV infection, 119 (64.3%) had community-acquired infections and 110 (59.5%) had pneumonia. In multivariable analysis, older age (odds ratio [OR], 1.02; 95% confidence interval [CI], 1.00-1.04; p = 0.045) and nosocomial infection (OR, 2.07; 95% CI, 1.05-4.10; p = 0.037) were associated with HBoV pneumonia. The main CT findings were bilateral consolidation (70.6%) and/or ground-glass opacities (64.7%); centrilobular nodules (14.7%) were found less frequently. The pattern of CT findings were not significantly different between immunocompetent and immunocompromised patients (all, p > 0.05).

HBoV infection can be a potential respiratory tract infection in adults. The most frequent CT findings of HBoV pneumonia were bilateral consolidation and/or ground-glass opacities.