N6-methyladenosine modification of a parvovirus-encoded small noncoding RNA facilitates viral DNA replication through recruiting Y-family DNA polymerases.

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.

Human Bocavirus 1 NP1 acts as an ssDNA-binding protein to help AAV2 DNA replication and cooperates with RPA to regulate AAV2 capsid expression.

Adeno-associated virus (AAV) requires co-infection with helper virus for efficient replication. We previously reported that Human Bocavirus 1 (HBoV1) genes, including NP1, NS2, and BocaSR, were critical for AAV2 replication. Here, we first demonstrate the essential roles of the NP1 protein in AAV2 DNA replication and protein expression. We show that NP1 binds to single-strand DNA (ssDNA) at least 30 nucleotides (nt) in length in a sequence-independent manner. Furthermore, NP1 colocalized with the BrdU-labeled AAV2 DNA replication center, and the loss of the ssDNA-binding ability of NP1 by site-directed mutation completely abolished AAV2 DNA replication. We used affinity-tagged NP1 protein to identify host cellular proteins associated with NP1 in cells cotransfected with the HBoV1 helper genes and AAV2 duplex genome. Of the identified proteins, we demonstrate that NP1 directly binds to the DBD-F domain of the RPA70 subunit with a high affinity through the residues 101-121. By reconstituting the heterotrimer protein RPA in vitro using gel filtration, we demonstrate that NP1 physically associates with RPA to form a heterologous complex characterized by typical fast-on/fast-off kinetics. Following a dominant-negative strategy, we found that NP1-RPA complex mainly plays a role in expressing AAV2 capsid protein by enhancing the transcriptional activity of the p40 promoter. Our study revealed a novel mechanism by which HBoV1 NP1 protein supports AAV2 DNA replication and capsid protein expression through its ssDNA-binding ability and direct interaction with RPA, respectively.IMPORTANCERecombinant adeno-associated virus (rAAV) vectors have been extensively used in clinical gene therapy strategies. However, a limitation of these gene therapy strategies is the efficient production of the required vectors, as AAV alone is replication-deficient in the host cells. HBoV1 provides the simplest AAV2 helper genes consisting of NP1, NS2, and BocaSR. An important question regarding the helper function of HBoV1 is whether it provides any direct function that supports AAV2 DNA replication and protein expression. Also of interest is how HBoV1 interplays with potential host factors to constitute a permissive environment for AAV2 replication. Our studies revealed that the multifunctional protein NP1 plays important roles in AAV2 DNA replication via its sequence-independent ssDNA-binding ability and in regulating AAV2 capsid protein expression by physically interacting with host protein RPA. Our findings present theoretical guidance for the future application of the HBoV1 helper genes in the rAAV vector production.

The small nonstructural protein NP1 of human bocavirus 1 directly interacts with Ku70 and RPA70 and facilitates viral DNA replication.

Human bocavirus 1 (HBoV1), a member of the genus Bocaparvovirus of the family Parvoviridae, causes acute respiratory tract infections in young children. Well-differentiated pseudostratified human airway epithelium cultured at an air-liquid interface (HAE-ALI) is an ideal in vitro culture model to study HBoV1 infection. Unique to other parvoviruses, bocaparvoviruses express a small nonstructured protein NP1 of ~25 kDa from an open reading frame (ORF) in the center of the viral genome. NP1 plays an important role in viral DNA replication and pre-mRNA processing. In this study, we performed an affinity purification assay to identify HBoV1 NP1-inteacting proteins. We identified that Ku70 and RPA70 directly interact with the NP1 at a high binding affinity, characterized with an equilibrium dissociation constant (KD) of 95 nM and 122 nM, respectively. Furthermore, we mapped the key NP1-interacting domains of Ku70 at aa266-439 and of RPA70 at aa181-422. Following a dominant negative strategy, we revealed that the interactions of Ku70 and RPA70 with NP1 play a significant role in HBoV1 DNA replication not only in an in vitro viral DNA replication assay but also in HBoV1-infected HAE-ALI cultures. Collectively, our study revealed a novel mechanism by which HBoV1 NP1 enhances viral DNA replication through its direct interactions with Ku70 and RPA70.

Genotypic diversity and long-term impact of human bocavirus on diarrheal disease: Insights from historical fecal samples in Brazil.

This study aimed to investigate the frequency and genotypic diversity of human bocavirus (HBoV) in historical fecal samples collected before 2005 in Brazil and understand its natural history in patients with diarrhea. Between 1998 and 2005, 3347 samples were tested for HBoV by RT-PCR, with a detection rate of 5.8% (195/3347). Coinfection with norovirus (NoV) and human adenovirus (HAdV) was found in 34.9% (68/195), indicating HBoV's potential role as a causative agent of diarrheal disease. The detection rate varied over the years (p < 0.05), suggesting natural oscillatory fluctuations. HBoV was more prevalent in fall and winter, with higher positivity in children ≤5 years (p < 0.05), reinforcing that HBoV is an important pathogen in childhood diarrhea. Genotyping (32.8%; 64/195) revealed the circulation of HBoV-1 (79.7%, 51/64), HBoV-3 (12.5%, 8/64), HBoV-2 (6.2%, 4/64), and the rare HBoV-4 (1.6%, 1/64). Difference in HBoV-1 and HBoV-2/-3 mono-infections prevalence (p < 0.05), suggests a potential role of HBoV-1 in the pathogenicity of diarrheal disease. The study highlights HBoV's lasting impact on viral gastroenteritis in Brazil and emphasizes its genotypic diversity. Recommending screening for HBoV in public health laboratories is crucial for understanding its role in gastrointestinal diseases. The data also contribute to understanding the molecular characterization of enteric viruses in historical fecal samples.

Sequencing and characterization of human bocavirus genomes from patients diagnosed in Southern France between 2017 and 2022.

The diversity and evolution of the genomes of human bocavirus (HBoV), which causes respiratory diseases, have been scarcely studied. Here, we aimed to obtain and characterize HBoV genomes from patients's nasopharyngeal samples collected between 2017 and 2022 period (5 years and 7 months). Next-generation sequencing (NGS) used Illumina technology after having implemented using GEMI an in-house multiplex PCR amplification strategy. Genomes were assembled and analyzed with CLC Genomics, Mafft, BioEdit, MeV, Nextclade, MEGA, and iTol. A total of 213 genomes were obtained. Phylogeny classified them all as of Bocavirus 1 (HBoV1) species. Five HBoV1 genotypic clusters determined by hierarchical clustering analysis of 27 variable genome positions were scattered over the study period although with differences in yearly prevalence. A total of 167 amino acid substitutions were detected. Besides, coinfection was observed for 52% of the samples, rhinoviruses then adenoviruses (HAdVs) being the most common viruses. Principal component analysis showed that HBoV1 genotypic cluster α tended to be correlated with HAdV co-infection. Subsequent HAdV typing for HBoV1-positive samples and negative controls demonstrated that HAdVC species predominated but HAdVB was that significantly HBoV1-associated. Overall, we described here the first HBoV1 genomes sequenced for France. HBoV1 and HAdVB association deserves further investigation.

Human bocavirus respiratory infection: Tracing the path from viral replication and virus-cell interactions to diagnostic methods.

Human bocaviruses were first described between 2005 and 2010, identified in respiratory and enteric tract samples of children. Screening studies have shown worldwide distribution. Based on phylogenetic analysis, they were classified into four genotypes (HBoV1-4). From a clinical perspective, human bocavirus 1 (HBoV1) is considered the most relevant, since it can cause upper and lower acute respiratory tract infection, mainly in infants, including common cold, bronchiolitis, and pneumonia, as well as wheezing in susceptible patients. However, the specific processes leading to structural, biochemical, and functional changes resulting in the different clinical presentations have not been elucidated yet. This review surveys the interactions between the virus and target cells that can potentially explain disease-causing mechanisms. It also summarises the clinical phenotype of cases, stressing the role of HBoV1 as an aetiological agent of lower acute respiratory infection in infants, together with laboratory tests for detection and diagnosis. By exploring the current knowledge on the epidemiology of HBoV1, insights into the complex scenario of paediatric respiratory infections are presented, as well as the potential effects that changes in the circulation can have on the dynamics of respiratory agents, spotlighting the benefits of comprehensively increase insights into incidence, interrelationships with co-circulating agents and potential control of HBoV1.

Human bocavirus-1 infections in Australian children aged < 2 years: a birth cohort study.

To determine human bocavirus-1 (HBoV1) infection characteristics in young Australian children. Data were from the Observational Research in Childhood Infectious Diseases (ORChID) study, a Brisbane, Australia-based birth cohort of healthy, term, newborns followed prospectively for 2 years. Parents recorded daily symptoms, maintained an illness-burden diary, and collected weekly nasal swabs, which were tested for 17 respiratory viruses, including HBoV1, by real-time polymerase chain reaction (PCR) assays. Main outcomes measured were infection incidence, risk factors, symptoms, and healthcare use. One hundred fifty-eight children in the ORChID cohort provided 11,126 weekly swabs, of which 157 swabs were HBoV1 positive involving 107 incident episodes. Co-detections were observed in 65/157 (41.4%) HBoV1-positive swabs (or 41/107 [38.3%] infection episodes), principally with rhinovirus. Shedding duration was 1 week in 64.5% of episodes. The incidence of HBoV1 infections in the first 2 years of life was 0.58 episodes per child-year (95% confidence interval [CI] 0.47-0.71), including 0.38 episodes per child-year (95% CI 0.30-0.49) associated with respiratory symptoms. Recurrent episodes occurred in 18/87 (20.7%) children following their primary infection. In the first 2 years of life, incidence of HBoV1 episodes increased with age, during winter and with childcare attendance. Overall, 64.2% of HBoV1 episodes were symptomatic, with 26.4% having healthcare contact. Viral load estimates were higher when children were symptomatic than when asymptomatic (mean difference = 3.4; 95% CI 1.0-5.7 PCR cycle threshold units). After age 6 months, HBoV1 is detected frequently in the first 2 years of life, especially during winter. Symptoms are usually mild and associated with higher viral loads.

Molecular characterization and clinical impact of human bocavirus at a tertiary hospital in Barcelona (Catalonia, Spain) during the 2014-2017 seasons.

PURPOSE: The aim was to describe the prevalence, molecular epidemiology and clinical manifestations of human bocavirus (HBoV) in patients attended at a tertiary hospital in Barcelona, Spain. METHODS: From October 2014 to May 2017, respiratory specimens from paediatric patients were collected for respiratory viruses' laboratory-confirmation. Phylogenetic analyses from partial VP1 sequences were performed from all HBoV laboratory-confirmed specimens. Clinical features were retrospectively studied. RESULTS: 178/10271 cases were HBoV laboratory-confirmed. The median age was 1.53 (IQR 1.0-2.3). Co-detection was highly reported (136; 76%). All viruses belonged into HBoV1 genotype but one into HBoV2. Non-reported mutations were observed and two sites were suggestive to be under negative selection. 61% (109/178) cases had lower RTI (LRTI), of whom 84 had co-detections (77%) and 76 had comorbidities (70%). LRTI was the cause of hospitalization in 85 out of 109 cases (78%), and no differences were found regarding severity factors during hospitalization between co- and single-detections, except for median length of respiratory support, which was longer in cases with co-detections. CONCLUSIONS: Close monitoring of predominant HBoV1 showed a high similarity between viruses. The presence of comorbidities might explain the high prevalence of LRTI. Symptomatology in HBoV single-detected cases suggest that HBoV is a true pathogen.

Human bocavirus 1 epidemiology in children in relation to virus load and codetection.

AIM: Human bocavirus 1 (HBoV1) has been associated with respiratory tract infections in children. We aimed at retrospectively describing patient characteristics, seasonality, pre-existing medical conditions, codetections, clinical manifestations and complications of HBoV1 infection in relation to viral load in the child population in Stockholm, with the overarching aim of elucidating the clinical significance of HBoV1. METHODS: We included all hospitalised children 0-17 years testing positive for HBoV1 by real-time polymerase chain reaction on nasopharyngeal aspirates 1 July 2008-30 June 2019. Patients with HBoV1 single detection, high viral load expressed as an HBoV1-DNA cycle threshold (Ct) < 25, or both, were separately analysed. We retrieved information on pre-existing conditions and clinical course from the medical records. RESULTS: We found 768 episodes in 727 children, 496 (64.6%) male and 441 (60.7%) previously healthy. The median age was 17.6 months. Most (476/768, 62.0%) episodes occurred during December-March. HBoV1 was in 549 episodes (71.5%) codetected with other viruses. Ct < 25 was independently associated with young age, single detection of HBoV1 and presentation early in the epidemic season. We saw few differences in clinical manifestations between the subgroups. CONCLUSION: Our findings are consistent with primary HBoV1 infection causing mild-to-severe respiratory tract manifestations in young children.

Hairpin Transfer-Independent Parvovirus DNA Replication Produces Infectious Virus.

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 293 (HEK293) 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 to 20 times lower than that of 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-nucleotide (nt) sequence (nt 5403 to 5420) 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 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 by 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 of viral DNA replication and may have implications in the development of parvovirus-based viral vectors with alternative properties.

Evaluation of human bocavirus as well as other well-known viral etiologic agents by PCR in the cerebrospinal fluid of children with clinical impression of viral meningoencephalitis referred to the Children's Medical Center in Tehran, Iran, from 2019 to 2020.

Viral meningoencephalitis is one of the most important diseases that most commonly affect children. In many cases of viral meningoencephalitis, the underlying cause of the disease is not identified, raising the possibility of a variety of pathogens that are not routinely tested. Bocaviruses belong to a newly identified class of viruses that have been reported in some studies to be associated with viral encephalitis. In the present study, we investigated the prevalence of bocaviruses and other viruses in the patients suspected of having viral encephalitis and their associations with various demographic and clinical variables. Two hundred patients with suspected viral meningoencephalitis referred to Children's Medical Center were studied from 2019 to 2020. Age, sex, length of hospitalization, and course of the disease were gathered. Cerebrospinal fluid (CSF) samples were taken from the patients and subjected to biochemical examinations and PCR to identify the underlying cause. Bocaviruses were detected in none of the DNA samples extracted from the CSF specimens. The most identified organisms were mumps and enteroviruses. In 92% of cases, the underlying cause was not identified. PCR-based identification of the underlying causes of viral meningoencephalitis in CSF specimens was not successful in most cases. Bocavirus was not found in any of the collected CSF samples. Further studies are required for drawing more accurate conclusions.

Human bocavirus detection and quantification in fecal and serum specimens from recipients of allogeneic hematopoietic stem cell transplantation: A longitudinal study.

OBJECTIVES: The aim of this study was to evaluate the occurrence of human bocavirus (HBoV) and to determine viral loads in samples of patients admitted for allogeneic hematopoietic stem cell transplantation (allo-HSCT). METHODS: Fecal and serum samples were collected from 19 patients, during a 24-month period. Samples were screened by quantitative polymerase chain reaction TaqMan assay, with specific probe and primers targeting the NP1 gene of all HBoVs genotypes (HBoV-1 to - 4), and viral loads were determined using serial dilutions of a recombinant plasmid. RESULTS: HBoV DNA was detected in 42.1% (8 of 19) of the patients in at least one type of sample (feces and/or serum) during the study period, with 75% (6 of 8) of the patients being positive in both types of sample. Viral shedding in feces had a median of 26 days (range, 5 to 121) and viremia was detected in 87.5% (7 of 8) of the patients. The HBoV loads in fecal samples were higher than in sera and, in most cases, HBoV was detected earlier in fecal than in sera samples. In six HBoV-positive patients (6 of 8) diarrhea was observed concomitantly to viral detection in fecal samples. CONCLUSIONS: A high frequency and loads of HBoV in allo-HSCT recipients was observed, especially in fecal samples. Positivity in fecal samples was an early predictor of HBoV presence.

Human bocavirus 2 detected in Rattus norvegicus feces in China.

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.

Severe Human Bocavirus-Associated Pneumonia in Adults at a Referral Hospital, Seoul, South Korea.

We report a case series of severe human bocavirus-associated pneumonia in adults in Seoul, South Korea. The virus accounted for 0.5% of all severe pneumonia cases. Structural lung disease and hematologic malignancy were common underlying diseases. Overall death rate was 54.5%. Higher death rates were associated with co-infection (83.3%) and immunocompromise (80.0%).

Cellular Cleavage and Polyadenylation Specificity Factor 6 (CPSF6) Mediates Nuclear Import of Human Bocavirus 1 NP1 Protein and Modulates Viral Capsid Protein Expression.

Human bocavirus 1 (HBoV1), which belongs to the genus Bocaparvovirus of the Parvoviridae family, causes acute respiratory tract infections in young children. In vitro, HBoV1 infects polarized primary human airway epithelium (HAE) cultured at an air-liquid interface (HAE-ALI). HBoV1 encodes a small nonstructural protein, nuclear protein 1 (NP1), that plays an essential role in the maturation of capsid protein (VP)-encoding mRNAs and viral DNA replication. In this study, we determined the broad interactome of NP1 using the proximity-dependent biotin identification (BioID) assay combined with mass spectrometry (MS). We confirmed that two host mRNA processing factors, DEAH-box helicase 15 (DHX15) and cleavage and polyadenylation specificity factor 6 (CPSF6; also known as CFIm68), a subunit of the cleavage factor Im complex (CFIm), interact with HBoV1 NP1 independently of any DNA or mRNAs. Knockdown of CPSF6 significantly decreased the expression of capsid protein but not that of DHX15. We further demonstrated that NP1 directly interacts with CPSF6 in vitro and colocalizes within the virus replication centers. Importantly, we revealed a novel role of CPSF6 in the nuclear import of NP1, in addition to the critical role of CPSF6 in NP1-facilitated maturation of VP-encoding mRNAs. Thus, our study suggests that CPSF6 interacts with NP1 to escort NP1 imported into the nucleus for its function in the modulation of viral mRNA processing and viral DNA replication.IMPORTANCE Human bocavirus 1 (HBoV1) is one of the significant pathogens causing acute respiratory tract infections in young children worldwide. HBoV1 encodes a small nonstructural protein (NP1) that plays an important role in the maturation of viral mRNAs encoding capsid proteins as well as in viral DNA replication. Here, we identified a critical host factor, CPSF6, that directly interacts with NP1, mediates the nuclear import of NP1, and plays a role in the maturation of capsid protein-encoding mRNAs in the nucleus. The identification of the direct interaction between viral NP1 and host CPSF6 provides new insights into the mechanism by which a viral small nonstructural protein facilitates the multiple regulation of viral gene expression and replication and reveals a novel target for potent antiviral drug development.

Predominance of Human Bocavirus Genotypes 1 and 2 in Oysters in Thailand.

Human bocavirus (HBoV) has been recognized as an important pathogen that causes respiratory infection and acute gastroenteritis in young children worldwide. HBoV is most likely transmitted by the respiratory route and by fecal-oral transmission. Recently, HBoV has been detected in several types of environmental water and in bivalve shellfish. However, study of the existence of HBoV in oysters is still undocumented in Thailand. In this study, 144 oyster samples collected from different markets in Chiang Mai, Thailand, in 2017 and 2018 were investigated for the presence of HBoV by nested PCR and sequencing. HBoV was detected in 11 out of 144 samples (7.6%). Nine HBoV-positive samples (81.8%) were identified as genotype 1 (HBoV1) and two (18.2%) as HBoV2. A monthly investigation of HBoV in oyster samples from July 2017 to June 2018 showed that HBoV was sporadically detected in particular months spanning the rainy and colder season, with a peak in January. This study demonstrates the presence and genotype diversity of HBoV in oyster samples in Thailand. The findings contribute to evaluating the risk of foodborne transmission of HBoV and to monitoring outbreaks of HBoV in Thailand and in other countries. IMPORTANCE Human bocavirus is recognized as an important cause of respiratory infection and of acute gastroenteritis in children worldwide. Human bocavirus has been widely detected in many clinical specimens, as well as in several types of environmental samples. Most previous studies describe the incidence of bocavirus infection in humans, whereas few data are available for the occurrence of human bocavirus in food materials, particularly that in bivalve shellfish. Our findings provide evidence for the existence and prevalence of human bocavirus in oysters, suggesting that further monitoring of the potential risk of food- and waterborne transmission of this virus to humans should be undertaken.

Detection of respiratory viruses in primary cholesteatoma tissues.

Cholesteatomas are frequent middle ear benign tumors of unknown etiology. Infectious agents have been considered as possible contributing factors in the pathogenesis of cholesteatomas. Aiming to investigate the presence of respiratory viruses in primary cholesteatoma tissues, 26 formalin-fixed paraffin-embedded primary cholesteatoma tissues obtained from patients seen at the of the Clinical Hospital of the University of São Paulo School of Medicine, in Ribeirão Preto, Brazil were tested by real-time polymerase chain reaction (PCR). Considering the PCR results, 35% of the tissues were positive for human rhinovirus (HRV), 15.3% for human enterovirus (EV), 3.8% for human metapneumovirus (HMPV), and 3.8% for human bocavirus (HBoV). Serial immunohistochemistry for virus antigens and cell surface markers evidenced that the viruses were associated with fibroblasts, dendritic cells, macrophages, B lymphocytes, CD4+ , and CD8+ T lymphocytes. These findings indicate for the first time the presence of active respiratory virus infection in primary cholesteatoma tissues, suggesting that persisting virus infection in the middle could play a role in the pathogenesis and evolution of cholesteatomas.

Molecular epidemiology of human bocavirus infection in hospitalized children with acute gastroenteritis in South Africa, 2009-2015.

Human bocavirus (HBoV) is known to be associated with a variety of clinical manifestation including acute gastroenteritis (AGE). Despite their global prevalence, no data is available on the epidemiology of HBoV associated with AGE in South Africa (SA). Between April 2009 and April 2015, 3765 stool specimens were collected from children less than 5 years of age hospitalized with diarrhea. Specimens were screened for selected enteric viruses by enzyme immunoassay and quantitative polymerase chain reaction, bacteria by culture and parasites by staining and microscopy. HBoV was detected in 5.63% (212 of 3765) of cases, the majority of which were children ≤2 years (92%, 195 of 212), and were common in the summer and autumn months (60%; 128 of 212). Further investigations of coinfections showed that bacteria (adjusted odds ratio [aOR] = 2.20; 95% confidence interval [CI], 1.41-3.45; P = .001) and sapovirus (aOR = 2.05; 95% CI, 1.08-3.86; P = .027) were significantly associated with HBoV in multivariate analysis. HBoV genotyping was successful in 191 of the 212 samples with HBoV-1 being the most prevalent genotype observed (79.6%; 152 of 191) followed by HBoV-3 (13.6%; 26 of 191), HBoV-2 (5.2%; 10 of 191), and HBoV-4 (1.6%; 3 of 191). The high prevalence of HBoV-1, a virus known to be associated with respiratory infections, and the association between HBoV-positive specimens and already established AGE agents, suggests that HBoV may play a limited role in the observed AGE cases in SA.

No Correlation Between Nasopharyngeal Human Bocavirus 1 Genome Load and mRNA Detection or Serology in Adeno-/Tonsillectomy Patients.

Human bocavirus 1 (HBoV1) can persist in nasopharynx and tonsils. Using HBoV1 serology, reverse-transcription polymerase chain reaction (PCR) for detecting messenger RNA (mRNA) and quantitative PCR for HBoV1 genome load count, we studied to what extent the HBoV1 DNA loads in nasopharynx correlate with acute infection markers. Tonsillar tissue, nasopharyngeal aspirate, and serum were obtained from 188 elective adeno-/tonsillectomy patients. Relatively high loads of HBoV1 DNA were detected in the nasopharynx of 14 (7%) primarily asymptomatic subjects with negative mRNA and/or serodiagnostic results. Quantitative HBoV1 DNA PCR may have lower specificity than HBoV1 mRNA detection for diagnosing symptomatic infection.

The Human Bocavirus 1 NP1 Protein Is a Multifunctional Regulator of Viral RNA Processing.

Human bocavirus 1 (HBoV1) encodes a genus-specific protein, NP1, which regulates viral alternative pre-mRNA processing. Similar to NP1 of the related bocavirus minute virus of canine (MVC), HBoV1 NP1 suppressed cleavage and polyadenylation of RNAs at the viral internal polyadenylation site (pA)p. HBoV1 (pA)p is a complex region. It contains 5 significant cleavage and polyadenylation sites, and NP1 was found to regulate only the three of these sites that are governed by canonical AAUAAA hexamer signals. HBoV1 NP1 also facilitated splicing of the upstream intron adjacent to (pA)p. Alternative polyadenylation and splicing of the upstream intron were independent of each other, functioned efficiently within an isolated transcription unit, and were responsive independent of NP1. Characterization of HBoV1 NP1 generalizes its function within the genus Bocaparvovirus, uncovers important differences, and provides important comparisons with MVC NP1 for mechanistic and evolutionary considerations.IMPORTANCE The Parvovirinae are small nonenveloped icosahedral viruses that are important pathogens in many animal species, including humans. The NP1 protein of human bocavirus 1 (HBoV1), similar to NP1 of the bocavirus minute virus of canine (MVC), regulates viral alternative RNA processing by both suppressing polyadenylation at an internal site, (pA)p, and facilitating splicing of an upstream adjacent intron. These effects allow both extension into the capsid gene and splicing of the viral pre-mRNA that correctly registers the capsid gene open reading frame. Characterization of HBoV1 NP1 generalizes this central mode of parvovirus gene regulation to another member of the bocavirus genus and uncovers both important similarities and differences in function compared to MVC NP1 that will be important for future comparative studies.