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.

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.

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.

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.

Demographic and clinical characteristics of human bocavirus-1 infection in patients with acute respiratory tract infections during the COVID-19 pandemic in the Central Province of Sri Lanka.

BACKGROUND: Human bocavirus-1 (hBoV-1) was first detected in respiratory specimens in 2005. Due to high co-infection rates and prolonged shedding of the virus, the pathogenic role of hBoV-1 as a primary causative agent of respiratory infections is still under discussion. This study aimed to determine the prevalence of hBoV-1 infection in patients with acute respiratory tract infections (ARTIs) during the COVID-19 pandemic in the Central Province of Sri Lanka. METHODS: A total of 1021 patients (Age 12 days to ≤ 85 years) with ARTI symptoms including fever, cough, cold, sore throat and shortness of breath within first 7 days of the illness were included. The study was carried out at the National Hospital, Kandy, Sri Lanka from January 2021 to October 2022. Respiratory specimens were tested to detect 23 pathogens including hBoV-1 using a real time PCR. Prevalence of hBoV-1 co-infections with other respiratory pathogens and distribution of hBoV-1 infection among different age groups were determined. Moreover, clinical and demographic characteristics of hBoV-1 mono-infection associated ARTI were compared with that of the hBoV-1 co-infections. RESULTS: Respiratory infections were detected in 51.5% (526/1021) of the patients and of these 82.5% were mono- and 17.1% were co-infections. hBoV-1 was detected in 66 patients and this was the most prevalent respiratory virus associated with 40% co-infections. Of the 66 hBoV-1 positive patients, 36 had co-infections and of these 33 had dual and 3 had triple infections. Most of the hBoV-1 co-infections were identified in children aged 2-<5 years. hBoV-1 co-infections were most frequently detected with respiratory syncytial virus (RSV) and Rhino/ Entero viruses (Rh/EnV). No differences were observed in age, gender and clinical presentations in those with hBoV-1 mono- compared to co-infections. Intensive care admissions were less among hBoV-1 mono-infected than hBoV-1 co-infected patients. CONCLUSION: This study shows a prevalence of 12.5% for hBoV-1 infections in patients with ARTI. RSV and Rh/EnV were the most common co-infecting pathogens with hBoV-1. Clinical features of hBoV-1 mono-infections were not different to that of the hBoV-1 co-infections. Interactions between hBoV-1 and other respiratory pathogens need investigation to identify the role of hBoV-1 in clinical severity of co-infections.

Respiratory virus infections after allogeneic stem cell transplantation: Current understanding, knowledge gaps, and recent advances.

Before the COVID-19 pandemic, common community-acquired seasonal respiratory viruses (CARVs) were a significant threat to the health and well-being of allogeneic hematopoietic cell transplant (allo-HCT) recipients, often resulting in severe illness and even death. The pandemic has further highlighted the significant risk that immunosuppressed patients, including allo-HCT recipients, face when infected with SARS-CoV-2. As preventive transmission measures are relaxed and CARVs circulate again among the community, including in allo-HSCT recipients, it is crucial to understand the current state of knowledge, gaps, and recent advances regarding CARV infection in allo-HCT recipients. Urgent research is needed to identify seasonal respiratory viruses as potential drivers for future pandemics.

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.

Best of most possible worlds: Hybrid gene therapy vectors based on parvoviruses and heterologous viruses.

Parvoviruses and especially the adeno-associated virus (AAV) species provide an exciting and versatile platform for the rational design or molecular evolution of human gene-therapy vectors, documented by literature from over half a century, hundreds of clinical trials, and the recent commercialization of multiple AAV gene therapeutics. For the last three decades, the power of these vectors has been further potentiated through various types of hybrid vectors created by intra- or inter-genus juxtaposition of viral DNA and protein cis elements or by synergistic complementation of parvoviral features with those of heterologous, prokaryotic, or eukaryotic viruses. Here, we provide an overview of the history and promise of this rapidly expanding field of hybrid parvoviral gene-therapy vectors, starting with early generations of chimeric particles composed of a recombinant AAV genome encapsidated in shells of synthetic AAVs or of adeno-, herpes-, baculo-, or protoparvoviruses. We then dedicate our attention to two newer, highly promising types of hybrid vectors created via (1) pseudotyping of AAV genomes with bocaviral serotypes and capsid mutants or (2) packaging of AAV DNA into, or tethering of entire vector particles to, bacteriophages. Finally, we conclude with an outlook summarizing critical requirements and improvements toward clinical translation of these original concepts.

Human bocavirus and human metapneumovirus in children with lower respiratory tract infections: Effects on clinical, microbiological features and disease severity .

BACKGROUND: We aimed to compare the clinical findings of human bocavirus (HBoV) and human metapneumovirus (HMPV) infections, and to analyze the effects of coinfections on clinical features and disease severity in children with HBoV and HMPV infections. METHODS: Data were collected from 125 children with lower respiratory tract infections due to HBoV or HMPV, detected from nasal swap by real-time polymerase chain reaction (PCR) during the period from January, 2013 to December, 2017. In total, there were 101 HBoV (group 1) and 23 HMPV (group 2) infections in our data. The patients were further divided into four subgroups according to the coinfection status: HoBV only (subgroup 1, n = 41), HMPV only (subgroup 2, n = 19), HBoV and coinfection with other respiratory viruses (subgroup 3, n = 60), and HMPV and coinfection with other respiratory viruses (subgroup 4, n = 4). RESULTS: The majority (88.8%) of the patients were aged 5 years or younger. Coinfections with other respiratory viruses were significantly more common in group 1 (P = 0.001). Among patients who had nosocomial pneumonia, patients with HBoV infections had significantly longer mean length of hospital stay (LOS) than those with HMPV infections (P = 0.032). The hospitalization and antibiotic requirements were significantly higher in subgroup 1 than subgroup 3 (P = 0.005, 0.039, resp.) According to the logistic regression analyses, the LOS increased by 21.7 times with HBoV infections (P = 0.006). CONCLUSIONS: Human bocavirus and HMPV infections are serious pathogens mostly seen in children and usually requiring hospitalization regardless of co-infection status. The HBoV infections caused longer LOS than the HMPV infections in patients with nosocomial infections.

A STUDY OF THE NOVEL WU AND KI POLYOMAVIRUSES, BOCAVIRUS ADENOVIRUS IN CHILDREN WITH UPPER RESPIRATORY TRACT INFECTIONS.

OBJECTIVE: The aim: To find out the frequency of WU and KI polyomaviruses, and Human Boca and Adenoviruses infections among children with different types of acute upper respiratory tract infections and to compare the frequency of these viruses among immune-competent and immune compromised patients. PATIENTS AND METHODS: Materials and methods: A case-control study conducted in children aged 3-18 years with acute upper respiratory tract infections. The samples were taken from: Group 1: 100 immuno competent children with acute upper respiratory tract infections. Group 2: 100 immuno compromised children (Leukemic, cancer, Nephrotic syndrome, chronic renal failure and children with renal transplant) with acute upper respiratory tract infections. Group 3: 100 apparently healthy children without respiratory infections as control group. Nasal swap samples were collected from children and then viral DNA extracted from these samples. Then detection of WU, KI polyomaviruses HBoV and HAdv was done by using real time PCR. RESULTS: Results: All of 300 samples were negative for WU and KI polyomaviruses. However, human Bocavirus was detected in the three groups (immunocompromised, immunocompetent and control group) and the positivity rates were 61.61%, 37.37% and 18.18%, respectively. While human adenovirus was found only in 2% of immunocompromised patients and 1.1% of immunocompetent patients also there were cases positive for both HBoV and HAdv in 5.5% of immunocompromised patients, and 8.8% of immunocompetent patients. CONCLUSION: Conclusions: High frequency of HBoV especially in immunocompromised patients while low number of positive cases for HAdv by using nasal swab samples, WU and KI polyomaviruses could not be detected in samples.

Role of Human Bocavirus Respiratory Tract Infection in Hematopoietic Cell Transplant Recipients.

BACKGROUND: Limited data exist regarding the impact of human bocavirus (BoV) in hematopoietic cell transplant (HCT) recipients. METHODS: 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 Hutchinson Cancer Research Center (2005-2010). Samples were tested by multiplex semiquantitative polymerase chain reaction (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. RESULTS: 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 = .04) and presence of respiratory copathogens (P = .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. CONCLUSIONS: 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.

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%).

Impact of Natural or Synthetic Singletons in the Capsid of Human Bocavirus 1 on Particle Infectivity and Immunoreactivity.

Human bocavirus 1 (HBoV1) is a parvovirus that gathers increasing attention due to its pleiotropic role as a pathogen and emerging vector for human gene therapy. Curiously, albeit a large variety of HBoV1 capsid variants has been isolated from human samples, only one has been studied as a gene transfer vector to date. Here, we analyzed a cohort of HBoV1-positive samples and managed to PCR amplify and sequence 29 distinct HBoV1 capsid variants. These differed from the originally reported HBoV1 reference strain in 32 nucleotides or four amino acids, including a frequent change of threonine to serine at position 590. Interestingly, this T590S mutation was associated with lower viral loads in infected patients. Analysis of the time course of infection in two patients for up to 15 weeks revealed a gradual accumulation of T590S, concurrent with drops in viral loads. Surprisingly, in a recombinant vector context, T590S was beneficial and significantly increased titers compared to that of T590 variants but had no major impact on their transduction ability or immunoreactivity. Additional targeted mutations in the HBoV1 capsid identified several residues that are critical for transduction, capsid assembly, or DNA packaging. Our new findings on the phylogeny, infectivity, and immunoreactivity of HBoV1 capsid variants improve our understanding of bocaviral biology and suggest strategies to enhance HBoV1 gene transfer vectors.IMPORTANCE The family of Parvoviridae comprises a wide variety of members that exhibit a unique biology and that are concurrently highly interesting as a scaffold for the development of human gene therapy vectors. A most notable example is human bocavirus 1 (HBoV1), which we and others have recently harnessed to cross-package and deliver recombinant genomes derived from another parvovirus, the adeno-associated virus (AAV). Here, we expanded the repertoire of known HBoV1 variants by cloning 29 distinct HBoV1 capsid sequences from primary human samples and by analyzing their properties as AAV/HBoV1 gene transfer vectors. This led to our discovery of a mutational hot spot at HBoV1 capsid position 590 that accumulated in two patients during natural infection and that lowers viral loads but increases vector yields. Thereby, our study expands our current understanding of HBoV1 biology in infected human subjects and concomitantly provides avenues to improve AAV/HBoV1 gene transfer vectors.

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.

High prevalence of SARS-CoV-2 and influenza A virus (H1N1) coinfection in dead patients in Northeastern Iran.

In the last months of 2019, an outbreak of fatal respiratory disease started in Wuhan, China, and quickly spread to other parts of the world. It was named COVID-19, and to date, thousands of cases of infection and death are reported worldwide. This disease is associated with a wide range of symptoms, which makes accurate diagnosis of it difficult. During previous severe acute respiratory syndrome (SARS) pandemic in 2003, researchers found that the patients with fever, cough, or sore throat had a 5% influenza virus-positive rate. This finding made us think that the wide range of symptoms and also relatively high prevalence of death in our patients may be due to the coinfection with other viruses. Thus, we evaluated the coinfection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with other respiratory viruses in dead patients in North Khorasan. We evaluated the presence of influenza A/B virus, human metapneumovirus, bocavirus, adenovirus, respiratory syncytial virus (RSV), and parainfluenza viruses in 105 SARS-CoV-2 positive dead patients, using polymerase chain reaction (PCR) and reverse transcription PCR tests. We found coinfection with influenza virus in 22.3%, RSV, and bocavirus in 9.7%, parainfluenza viruses in 3.9%, human metapneumovirus in 2.9%, and finally adenovirus in 1.9% of SARS-CoV-2 positive dead cases. Our findings highlight a high prevalence of coinfection with influenza A virus and the monopoly of coinfection with Human metapneumovirus in children.