From a case-control survey to a diagnostic viral gastroenteritis panel for testing of general practitioners' patients.

OBJECTIVE: 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). METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Prevalence of respiratory viruses using polymerase chain reaction in children with wheezing, a systematic review and meta-analysis.

INTRODUCTION: 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. METHODS: 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. RESULTS: 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%). CONCLUSIONS: 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. REVIEW REGISTRATION: PROSPERO, CRD42018115128.

Inside the lungs of COVID-19 disease.

In the setting of the coronavirus disease 2019 (COVID-19) pandemic, only few data regarding lung pathology induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is available, especially without medical intervention interfering with the natural evolution of the disease. We present here the first case of forensic autopsy of a COVID-19 fatality occurring in a young woman, in the community. Diagnosis was made at necropsy and lung histology showed diffuse alveolar damage, edema, and interstitial pneumonia with a geographically heterogeneous pattern, mostly affecting the central part of the lungs. This death related to COVID-19 pathology highlights the heterogeneity and severity of central lung lesions after natural evolution of the disease.

Oxymatrine Inhibits Bocavirus MVC Replication, Reduces Viral Gene Expression and Decreases Apoptosis Induced by Viral Infection.

Oxymatrine (OMT), as the main active component of Sophoraflavescens, exhibits a variety of pharmacological properties, including anti-oxidative, anti-inflammatory, anti-tumor, and anti-viral activities, and currently is extensively employed to treat viral hepatitis; however, its effects on parvovirus infection have yet to be reported. In the present study, we investigated the effects of OMT on cell viability, virus DNA replication, viral gene expression, cell cycle, and apoptosis in Walter Reed canine cells/3873D infected with minute virus of canines (MVC). OMT, at concentrations below 4 mmol/L(no cellular toxicity), was found to inhibit MVC DNA replication and reduce viral gene expression at both mRNA and protein levels, which was associated with the inhibition of cell cycle S-phase arrest in early-stage of MVC infection. Furthermore, OMT significantly increased cell viability, decreased MVC-infected cell apoptosis, and reduced the expression of activated caspase 3. Our results suggest that OMT has potential application in combating parvovirus infection.

Etiology and Clinical Characteristics of Community-Acquired Pneumonia with Airway Malacia in Children.

Objective: The objective of this article is to study the etiology of community-acquired pneumonia in children with airway malacia. Methods: We retrospectively reviewed the medical records of 428 pneumonia patients. All patients underwent bronchoscopy, and bronchoalveolar lavage samples were processed for microbiological assessment. Results: In a total of 428 cases reviewed, 60 were found to have airway malacia. Pathogens were identified in 44 of the 60 specimens (73.3%), with 32 being single-pathogen infections. The most common pathogen was respiratory syncytial virus (RSV; 20%). Mixed-pathogen infections were observed in 12 patients. Airway malacia patients were younger than those without malacia (10.5 vs. 50 months, respectively; p < 0.001). Compared with those without airway malacia, wheezing, cyanosis and admission to the pediatric intensive care unit were more common in children with airway malacia and their hospital stay was longer. Conclusion: RSV was the most common pathogen in those with airway malacia. Airway malacia was found to aggravate infectious pneumonia.

Human Bocavirus Capsid Messenger RNA Detection in Children With Pneumonia.

Background: The role of human bocavirus (HBoV) in respiratory illness is uncertain. HBoV genomic DNA is frequently detected in both ill and healthy children. We hypothesized that spliced viral capsid messenger RNA (mRNA) produced during active replication might be a better marker for acute infection. Methods: As part of the Etiology of Pneumonia in the Community (EPIC) study, children aged <18 years who were hospitalized with community-acquired pneumonia (CAP) and children asymptomatic at the time of elective outpatient surgery (controls) were enrolled. Nasopharyngeal/oropharyngeal specimens were tested for HBoV mRNA and genomic DNA by quantitative polymerase chain reaction. Results: HBoV DNA was detected in 10.4% of 1295 patients with CAP and 7.5% of 721 controls (odds ratio [OR], 1.4 [95% confidence interval {CI}, 1.0-2.0]); HBoV mRNA was detected in 2.1% and 0.4%, respectively (OR, 5.1 [95% CI, 1.6-26]). When adjusted for age, enrollment month, and detection of other respiratory viruses, HBoV mRNA detection (adjusted OR, 7.6 [95% CI, 1.5-38.4]) but not DNA (adjusted OR, 1.2 [95% CI, .6-2.4]) was associated with CAP. Among children with no other pathogens detected, HBoV mRNA (OR, 9.6 [95% CI, 1.9-82]) was strongly associated with CAP. Conclusions: Detection of HBoV mRNA but not DNA was associated with CAP, supporting a pathogenic role for HBoV in CAP. HBoV mRNA could be a useful target for diagnostic testing.

The impact of anthropogenic pressure on the virological quality of water from the Tiber River, Italy.

The objective of the present study was to assess the occurrence of major waterborne enteric viruses (enterovirus, norovirus, adenovirus, rotavirus, hepatitis A and E virus) along the Tiber River in Italy, in areas affected by different kinds of anthropogenic pressure (agricultural, urban, industrial and pristine). Moreover, in light of the recent abundant detection of human bocavirus in urban wastewater samples in Italy, the occurrence of this virus was also assessed. Virus detection was based on nested PCR followed by sequencing, and on real-time PCR. A correlation with anthropogenic pressure was observed. The urban and industrial areas were the most contaminated (100 and 75% of samples were positive for at least one virus respectively). The agricultural area was less contaminated, with 50% of samples positive. None of the samples collected in a pristine area were positive for viruses. The most frequently detected virus was human bocavirus, identified in 37·5% of samples, followed by norovirus and enterovirus (28% each) and adenovirus (21·6%). Rotavirus, and hepatitis A and E viruses were less common (<9%). Although Human Bocavirus is not considered a waterborne pathogen, the widespread contamination of river waters suggests that virus transmission via the water route should not be neglected. SIGNIFICANCE AND IMPACT OF THE STUDY: To the best of our knowledge, this study constitutes the first attempt to assess the occurrence of enteric viruses in river waters, in areas differentially influenced by anthropogenic pressure. Enteric viruses (enterovirus, norovirus, adenovirus, rotavirus, hepatitis A and E viruses, and bocavirus) were widespread in the industrial and urban areas, and were less frequently detected in the agricultural area. Interestingly, human bocavirus was the most frequently detected virus, outnumbering even adenoviruses, known to be widespread in water environments. The widespread presence of bocavirus in surface waters suggests that a potential role of water in its transmission should not be excluded.

Virome of US bovine calf serum.

Using viral metagenomics we analyzed four bovine serum pools assembled from 715 calves in the United States. Two parvoviruses, bovine parvovirus 2 (BPV2) and a previously uncharacterized parvovirus designated as bosavirus (BosaV), were detected in 3 and 4 pools respectively and their complete coding sequences generated. Based on NS1 protein identity, bosavirus qualifies as a member of a new species in the copiparvovirus genus. Also detected were low number of reads matching ungulate tetraparvovirus 2, bovine hepacivirus, and several papillomaviruses. This study further characterizes the diversity of viruses in calf serum with the potential to infect fetuses and through fetal bovine serum contaminate cell cultures.

A Novel High-Throughput Method for Molecular Detection of Human Pathogenic Viruses Using a Nanofluidic Real-Time PCR System.

Human enteric viruses are recognized as the main causes of food- and waterborne diseases worldwide. Sensitive and quantitative detection of human enteric viruses is typically achieved through quantitative RT-PCR (RT-qPCR). A nanofluidic real-time PCR system was used to develop novel high-throughput methods for qualitative molecular detection (RT-qPCR array) and quantification of human pathogenic viruses by digital RT-PCR (RT-dPCR). The performance of high-throughput PCR methods was investigated for detecting 19 human pathogenic viruses and two main process controls used in food virology. The conventional real-time PCR system was compared to the RT-dPCR and RT-qPCR array. Based on the number of genome copies calculated by spectrophotometry, sensitivity was found to be slightly better with RT-qPCR than with RT-dPCR for 14 viruses by a factor range of from 0.3 to 1.6 log10. Conversely, sensitivity was better with RT-dPCR than with RT-qPCR for seven viruses by a factor range of from 0.10 to 1.40 log10. Interestingly, the number of genome copies determined by RT-dPCR was always from 1 to 2 log10 lower than the expected copy number calculated by RT-qPCR standard curve. The sensitivity of the RT-qPCR and RT-qPCR array assays was found to be similar for two viruses, and better with RT-qPCR than with RT-qPCR array for eighteen viruses by a factor range of from 0.7 to 3.0 log10. Conversely, sensitivity was only 0.30 log10 better with the RT-qPCR array than with conventional RT-qPCR assays for norovirus GIV detection. Finally, the RT-qPCR array and RT-dPCR assays were successfully used together to screen clinical samples and quantify pathogenic viruses. Additionally, this method made it possible to identify co-infection in clinical samples. In conclusion, given the rapidity and potential for large numbers of viral targets, this nanofluidic RT-qPCR assay should have a major impact on human pathogenic virus surveillance and outbreak investigations and is likely to be of benefit to public health.

Prevalence and genotypes of the adenovirus infection as well detection of co-infection with bocavirus in Mexican immunosuppressed and non-immunosuppressed children with pneumonia.

BACKGROUND: Adenovirus (AdV) causes respiratory infection; recent observations suggest that some subtypes have more ability to develop fatal disease. AdV infection has been associated with co-infection with human bocavirus (HBoV). We analysed the frequency of AdV infection, its subtypes and the presence of co-infection with HBoV, as well the clinical characteristics of such co-infection in Mexican paediatric immunosuppressed (IP) and non-immunosuppressed patients (non-IP) diagnosed with pneumonia. METHODS: A total of 5185 nasopharyngeal swabs from two groups of children with pneumonia, one IP and the other non-IP, were analysed for the detection of AdV by immunofluorescence and confirmed by PCR and culture. HBoV was identified by PCR. Positive samples for AdV and AdV/HBoV were typed using PCR sequencing, the clinical characteristics of the AdV/HBoV co-infection were analysed. RESULTS: Thirty-seven of the 5185 (0.71%) samples were positive for AdV, of those 27/37 (73%) were detected in non-IP and 10/37 (27%) in the IP group. Twelve were typed as follows: 9/12 (75%) as Species B1 subtype 3, of those 8/9 (88.9%) in non-IP and 1/9 in the IP group. One of twelve AdV2 subtype B11a was identified in one non-IP and the remaining two out of 12 successfully typed, were identified as Species C subtypes 2 and 6 in the group of non-IP. The presence of both AdV and HBoV1 in co-infection was observed in 2/37 (5.4%) non-IP with a syndrome like influenza. CONCLUSIONS: In this 5 year analysis of samples from non-IP and IP hospitalized paediatric patients with a diagnosis of pneumonia, a low incidence of AdV was found. B1 was the most frequent subtype and frequently found in non-IP, and two cases of co-infection AdV/HBoV1 were detected in two non-IP with a influenza-like syndromes. This is the first report of HBoV and AdV co-infection in Mexico. The frequency of AdV and HBoV co-infection was lower than that reported in other populations.

Clinical epidemiology of bocavirus, rhinovirus, two polyomaviruses and four coronaviruses in HIV-infected and HIV-uninfected South African children.

BACKGROUND: Advances in molecular diagnostics have implicated newly-discovered respiratory viruses in the pathogenesis of pneumonia. We aimed to determine the prevalence and clinical characteristics of human bocavirus (hBoV), human rhinovirus (hRV), polyomavirus-WU (WUPyV) and -KI (KIPyV) and human coronaviruses (CoV)-OC43, -NL63, -HKU1 and -229E among children hospitalized with lower respiratory tract infections (LRTI). METHODS: Multiplex real-time reverse-transcriptase polymerase chain reaction was undertaken on archived nasopharyngeal aspirates from HIV-infected and -uninfected children (<2 years age) hospitalized for LRTI, who had been previously investigated for respiratory syncytial virus, human metapneumovirus, parainfluenza I-III, adenovirus and influenza A/B. RESULTS: At least one of these viruses were identified in 274 (53.0%) of 517 and in 509 (54.0%) of 943 LRTI-episodes in HIV-infected and -uninfected children, respectively. Human rhinovirus was the most prevalent in HIV-infected (31.7%) and -uninfected children (32.0%), followed by CoV-OC43 (12.2%) and hBoV (9.5%) in HIV-infected; and by hBoV (13.3%) and WUPyV (11.9%) in HIV-uninfected children. Polyomavirus-KI (8.9% vs. 4.8%; p = 0.002) and CoV-OC43 (12.2% vs. 3.6%; p<0.001) were more prevalent in HIV-infected than -uninfected children. Combined with previously-tested viruses, respiratory viruses were identified in 60.9% of HIV-infected and 78.3% of HIV-uninfected children. The newly tested viruses were detected at high frequency in association with other respiratory viruses, including previously-investigated viruses (22.8% in HIV-infected and 28.5% in HIV-uninfected children). CONCLUSIONS: We established that combined with previously-investigated viruses, at least one respiratory virus was identified in the majority of HIV-infected and HIV-uninfected children hospitalized for LRTI. The high frequency of viral co-infections illustrates the complexities in attributing causality to specific viruses in the aetiology of LRTI and may indicate a synergetic role of viral co-infections in the pathogenesis of childhood LRTI.

Detection of a porcine boca-like virus in combination with porcine circovirus type 2 genotypes and Torque teno sus virus in pigs from postweaning multisystemic wasting syndrome (PMWS)-affected and non-PMWS-affected farms in archival samples from Great Britain.

In this study we detail the detection and genetic analysis of a novel porcine boca-like virus (PBo-likeV) in archival sera and tissue samples from pigs from farms in Great Britain. We also investigate the distribution of porcine circovirus type 2 (PCV2) genotypes and Torque teno sus virus (TTSuV) genogroups 1 and 2 in combination with this novel PBo-likeV. PBo-likeV was detected in over 70% of all tissues investigated. Over 24% of all tissues recovered from PMWS-affected animals had all viruses present and 25% of tissues recovered from non-PMWS-affected pigs were positive for all 4 viruses.

Detection of human bocavirus in children with upper respiratory tract infection by polymerase chain reaction.

BACKGROUND: The aim of this study was to investigate whether in children with middle ear effusions (MEE), adenoid and tonsil tissues are associated with human bocavirus (HBoV). MATERIALS AND METHODS: A total of 124 patients (56 females (45.2%) and 68 males (54.8%)) with chronic adenotonsillitis and serous otitis media under the age of 15 were recruited. Two hundered four samples (113 adenoid (55.4%), 68 tonsil (33.3%), and 23 middle ear effusion (11.3%)) were analyzed for the presence of HBoV using polymerase chain reaction (PCR). RESULTS: HBoV was detected in only 6 (4.8%) adenoid tissue samples each belonging to a different patient. CONCLUSIONS: Our findings are consistent with the results of other studies, reporting approximately 5 - 10% of the samples being positive for HBoV. To understand the detailed role of HBoV in the etiology of RTI in children, further studies would be needed.

Detection of novel porcine bocaviruses in fecal samples of asymptomatic pigs in Cameroon.

Improvements and widespread use of nucleic acid amplification and sequencing methods have led to the recognition of new virus diversity in various domestic animals, including pigs. In this study we utilized either virus species specific or broadly reactive PCR assays to describe the occurrence and genetic diversity of selected DNA viruses belonging to families Adenoviridae, Circoviridae, Anelloviridae and Parvoviridae in Cameroonian pigs. Fecal specimens were collected during spring of 2011. No adenoviruses, circoviruses and anelloviruses were detected, however, high prevalence and remarkable genetic diversity within the identified parvoviruses and, particularly, within bocaviruses was observed. PPV4 was the most prevalent virus (20%), followed by PBoV3 (18%), PBoV4 (18%), PBoV5 plus 6V/7V (16%), and PBoV1 plus PBoV2 (6%). The frequency of mixed infections with various combinations of these virus species reached 20%. Genetic analysis of the identified viruses showed that the capsid gene of PBoV1 and PBoV2 strains shared up to 91% and 94%nt sequence similarities to reference PBoV1 and PBoV2 strains, respectively. The identified PBoV3 and PBoV4 strains shared ≤ 95% and ≤ 98%nt identities with reference PBoV3 and PBoV4 strains, respectively, along the NS gene, whereas the PBoV5 strains shared 86%nt identities with Hungarian and 87%nt identities with Chinese PBoV5 strains along the capsid gene. In addition, a single PBoV5-like strain shared ≤ 71%nt sequence identity with other PBoV5 strains. This is the first study to report evidence of the circulation of bocaviruses in Africa and contributes to our understanding of the impact of globalization on the dispersal of new and emerging viruses.

A novel bocavirus in canine liver.

BACKGROUND: Bocaviruses are classified as a genus within the Parvoviridae family of single-stranded DNA viruses and are pathogenic in some mammalian species. Two species have been previously reported in dogs, minute virus of canines (MVC), associated with neonatal diseases and fertility disorders; and Canine bocavirus (CBoV), associated with respiratory disease. FINDINGS: In this study using deep sequencing of enriched viral particles from the liver of a dog with severe hemorrhagic gastroenteritis, necrotizing vasculitis, granulomatous lymphadenitis and anuric renal failure, we identified and characterized a novel bocavirus we named Canine bocavirus 3 (CnBoV3). The three major ORFs of CnBoV3 (NS1, NP1 and VP1) shared less than 60% aa identity with those of other bocaviruses qualifying it as a novel species based on ICTV criteria. Inverse PCR showed the presence of concatemerized or circular forms of the genome in liver. CONCLUSIONS: We genetically characterized a bocavirus in a dog liver that is highly distinct from prior canine bocaviruses found in respiratory and fecal samples. Its role in this animal's complex disease remains to be determined.

SMC1-mediated intra-S-phase arrest facilitates bocavirus DNA replication.

Activation of a host DNA damage response (DDR) is essential for DNA replication of minute virus of canines (MVC), a member of the genus Bocavirus of the Parvoviridae family; however, the mechanism by which DDR contributes to viral DNA replication is unknown. In the current study, we demonstrate that MVC infection triggers the intra-S-phase arrest to slow down host cellular DNA replication and to recruit cellular DNA replication factors for viral DNA replication. The intra-S-phase arrest is regulated by ATM (ataxia telangiectasia-mutated kinase) signaling in a p53-independent manner. Moreover, we demonstrate that SMC1 (structural maintenance of chromosomes 1) is the key regulator of the intra-S-phase arrest induced during infection. Either knockdown of SMC1 or complementation with a dominant negative SMC1 mutant blocks both the intra-S-phase arrest and viral DNA replication. Finally, we show that the intra-S-phase arrest induced during MVC infection was caused neither by damaged host cellular DNA nor by viral proteins but by replicating viral genomes physically associated with the DNA damage sensor, the Mre11-Rad50-Nbs1 (MRN) complex. In conclusion, the feedback loop between MVC DNA replication and the intra-S-phase arrest is mediated by ATM-SMC1 signaling and plays a critical role in MVC DNA replication. Thus, our findings unravel the mechanism underlying DDR signaling-facilitated MVC DNA replication and demonstrate a novel strategy of DNA virus-host interaction.

Bocavirus infection induces a DNA damage response that facilitates viral DNA replication and mediates cell death.

Minute virus of canines (MVC) is an autonomous parvovirus that replicates efficiently without helper viruses in Walter Reed/3873D (WRD) canine cells. We previously showed that MVC infection induces mitochondrion-mediated apoptosis and G(2)/M-phase arrest in infected WRD cells. However, the mechanism responsible for these effects has not been established. Here, we report that MVC infection triggers a DNA damage response in infected cells, as evident from phosphorylation of H2AX and RPA32. We discovered that both ATM (ataxia telangiectasia-mutated kinase) and ATR (ATM- and Rad3-related kinase) were phosphorylated in MVC-infected WRD cells and confirmed that ATM activation was responsible for the phosphorylation of H2AX, whereas ATR activation was required for the phosphorylation of RPA32. Both pharmacological inhibition of ATM activation and knockdown of ATM in MVC-infected cells led to a significant reduction in cell death, a moderate correction of cell cycle arrest, and most importantly, a reduction in MVC DNA replication and progeny virus production. Parallel experiments with an ATR-targeted small interfering RNA (siRNA) had no effect. Moreover, we identified that this ATM-mediated cell death is p53 dependent. In addition, we localized the Mre11-Rad50-Nbs1 (MRN) complex, the major mediator as well as a substrate of the ATM-mediated DNA damage response pathway to MVC replication centers during infection, and show that Mre11 knockdown led to a reduction in MVC DNA replication. Our findings are the first to support the notion that an autonomous parvovirus is able to hijack the host DNA damage machinery for its own replication and for the induction of cell death.

[Sequence the complete sequence of bocavirus I with SISPA-PCR].

OBJECTIVE: To sequence the complete sequence of bocavirus I with sequence independent single primer amplification (SISPA-PCR). METHODS: To exclude the co-effection samples, all clinical samples of diarrhea cases were screened with special primers of rotavirus, astrovirus, adenovirus, calicivirus and bocavirus I. The virus were enriched through ultracentrifugation. Other nucleic acids, such as human and bacteria genomes, were degradated by DNase I and RNase. DNA of bocavirus was Amplificated with SISPA-PCR, then purificated, cloned and sequenced. The sequences were alighmented in nr with blastn and assembled with DNAstar. RESULTS: A 4834bp sequence of bocavirus I were assembled. CONCLUSION: SISPA-PCR is an economical and efficient technique for sequence a virus complete genome.

Human bocavirus can be cultured in differentiated human airway epithelial cells.

In 2005, a human bocavirus was discovered in children with respiratory tract illnesses. Attempts to culture this virus on conventional cell lines has failed thus far. We investigated whether the virus can replicate on pseudostratified human airway epithelium. This cell culture system mimics the human airway environment and facilitates culturing of various respiratory agents. The cells were inoculated with human bocavirus-positive nasopharyngeal washes from children, and virus replication was monitored by measuring apical release of the virus via real-time PCR. Furthermore, we identified different viral mRNAs in the infected cells. All mRNAs were transcribed from a single promoter but varied due to alternative splicing and alternative polyadenylation, similar to what has been described for bovine parvovirus and minute virus of canines, the other two members of the Bocavirus genus. Thus, transcription of human bocavirus displays strong homology to the transcription of the other bocaviruses. In conclusion, we report here for the first time that human bocavirus can be propagated in an in vitro culture system and present a detailed map of the set of mRNAs that are produced by the virus.