OBJECTIVE: Newly identified human rhinovirus C (HRV-C) and human bocavirus (HBoV) cannot propagate in vitro in traditional cell culture models; thus obtaining knowledge about these viruses and developing related vaccines are difficult. Therefore, it is necessary to develop a novel platform for the propagation of these types of viruses. METHODS: A platform for culturing human airway epithelia in a three-dimensional (3D) pattern using Matrigel as scaffold was developed. The features of 3D culture were identified by immunochemical staining and transmission electron microscopy. Nucleic acid levels of HRV-C and HBoV in 3D cells at designated time points were quantitated by real-time polymerase chain reaction (PCR). Levels of cytokines, whose secretion was induced by the viruses, were measured by ELISA. RESULTS: Properties of bronchial-like tissues, such as the expression of biomarkers CK5, ZO-1, and PCK, and the development of cilium-like protuberances indicative of the human respiration tract, were observed in 3D-cultured human airway epithelial (HAE) cultures, but not in monolayer-cultured cells. Nucleic acid levels of HRV-C and HBoV and levels of virus-induced cytokines were also measured using the 3D culture system. CONCLUSION Our data provide a preliminary indication that the 3D culture model of primary epithelia using a Matrigel scaffold in vitro can be used to propagate HRV-C and HBoV.
Collagen ; Drug Combinations ; Enterovirus ; growth & development ; isolation & purification ; Enterovirus Infections ; virology ; Enzyme-Linked Immunosorbent Assay ; Epithelial Cells ; virology ; Human bocavirus ; growth & development ; isolation & purification ; Humans ; Laminin ; Parvoviridae Infections ; virology ; Primary Cell Culture ; methods ; Proteoglycans ; Real-Time Polymerase Chain Reaction ; Respiratory Mucosa ; virology ; Virus Cultivation

OBJECTIVETo investigate the prevalence of human bocavirus (HBoV) in children with acute lower respiratory tract infection and to explore the relationship between the viral load of HBoV and the clinical characteristics of acute lower respiratory tract infection in children.

METHODSA total of 1 554 nasopharyngeal aspirates from children who were hospitalized due to acute lower respiratory tract infection between March 2011 and March 2014 were collected. Quantitative real-time PCR was used to detect 12 RNA and 2 DNA viruses, adenovirus (ADV) and HBoV, and to measure the viral load of HBoV in HBoV-positive children. A comprehensive analysis was performed with reference to clinical symptoms and indicators.

RESULTSIn the 1 554 specimens, 1 212 (77.99%) were positive for viruses, and 275 (17.70%) were HBoV-positive. In HBoV-positive cases, 94.9% were aged <3 years, and there were more males than females. In the 275 HBoV-positive cases, 45 (16.36%) had single infection, and 230 (83.64%) had mixed infection. There was no significant difference in viral load between children with single infection and mixed infection (P>0.05). The patients with fever had a significantly higher viral load than those without fever (P<0.05). The children with wheezing had a significantly higher viral load than those without wheezing (P<0.05). There was no significant difference in viral load between children with mild, moderate, and severe acute lower respiratory tract infection (P>0.05).

CONCLUSIONSHBoV is one of the important pathogens of acute lower respiratory tract infection in children. Children with a higher viral load of HBoV are more likely to experience symptoms such as fever and wheezing. However, the severity of disease and mixed infection are not significantly related to viral load.

Acute Disease ; Adolescent ; Child ; Child, Preschool ; Female ; Human bocavirus ; isolation & purification ; Humans ; Infant ; Infant, Newborn ; Male ; Respiratory Tract Infections ; virology ; Viral Load

We studied the epidemiological characteristics of human bocavirus 4 (HBoV4) in children with a- cute gastroenteritis in Lanzhou (China). A total of 331 stool specimens were collected from children aged < 5 years with acute diarrhea at the First Hospital of Lanzhou University between July 2012 and June 2013. Specimens of HBoV were identified by nested polymerase chain reaction assays. Compared with related sequences in GenBank, the HBoV-positive strain isolated in the present study was,quite surprisingly, a rare genotype named HBoV4. This strain was a typical HBoV4,with high levels of nucleotide and amino acid homology to the Thailand strain, JQ267789 (98.9% and 98.7%, respectively), and the USA strain, GQ506568 (97.6% and 97.4%, respectively). This is the first report of HBoV4 as the causative agent for acute gastroenteritis in pediatric patients in China. This strain is one of two genotypes of HBoV that are currently circulating.
Child, Preschool ; China ; Feces ; virology ; Female ; Gastroenteritis ; virology ; Human bocavirus ; classification ; genetics ; isolation & purification ; Humans ; Infant ; Male ; Molecular Sequence Data ; Parvoviridae Infections ; virology ; Phylogeny

This study aimed to study the epidemiological and clinical characteristics of human bocavirus 1-4 (HBoV1-4) in children with acute diarrhea in Lanzhou and to investigate the association between HBoV and acute gastroenteritis. A total of 331 stool samples were collected from children aged under 5 years with acute diarrhea at the Department of Pediatrics, the First Hospital, Lanzhou University, between July 2012 and June 2013. Nested PCR was used to screen for HBoV and a general PCR was employed to screen other common diarrhea viruses. We found human bocavirus 1, 2, 3 and 4 in 26, 15, 7 and 1 cases, respectively. There was no specific seasonal distribution of HBoV, with infections occurring throughout the year. HBoV was mostly found in children aged between 7 and 12 months, with a mean age of 11.04 months (+/- 6.92 months), and 93.88% of affected children were aged under 2 years. Overall, 71.3% of mixed infections were mixed and the majority of other infections were caused by rotavirus. There was no statistical difference in the incidence of fever and vomiting associated with HBoV infection. A rare virus strain, HBoV4 (LZFB086), was identified, which showed highest levels of nucleotide sequence identity (99.0%) with a single Thai HBoV strain (JQ267789). No case of HBoV2B was found. In conclusion, HBoV1 was a major etiological pathogen of HBoV in pediatric cases in Lanzhou. HBoV4 was detected in feces for the first time in China. The rate of mixed infections was high and rotavirus was dominant. The data presented suggests that HBoV is not a major causative agent of gastroenteritis.
China ; epidemiology ; Diarrhea ; epidemiology ; virology ; Feces ; virology ; Human bocavirus ; classification ; genetics ; isolation & purification ; Humans ; Infant ; Molecular Sequence Data ; Parvoviridae Infections ; epidemiology ; virology ; Phylogeny ; Seasons

OBJECTIVEThe aim of this study was to explore the prevalent characteristics of HBoV1 and its co-infection.

METHODSPCR was used to detect HBoV1-DNA (HBoV1) and other viruses. A multivariate logistic regression model was used to explore possibility of co-detected for related viruses.

RESULTSThe positivity rates in Nanjing and Lanzhou were 9.38% (74/789) and 11.62% (161/1386), respectively (P>0.05). The HBoV1 positive group was younger than negative group (P<0.05). Seasonal differences were noted, with a higher frequency of infection in December and July. HBoV1-positive children [72.34% (169/235)] were co-infected with other respiratory viruses. Multifactorial analysis showed no correlations between HBoV1 and the clinical classification, region, gender, age, or treatment as an outpatient or in a hospital. Correlations were identified between HBoV1 infections with ADV (OR=1.53, 95% CI 1.03-2.28), RSV (OR=0.71, 95% CI 0.52-0.98), and IFVA (OR=1.77, 95% CI 1.00-3.13).

CONCLUSIONPresence of HBoV1 in nasopharyngeal aspirates did not correlate with region or gender, although the prevalence of HBoV1 was higher in younger children. There were no correlations between HBoV1 and other variables, except for the season and ADV, RSV, or IFVA infections.

Acute Disease ; Child, Preschool ; China ; epidemiology ; Comorbidity ; DNA, Viral ; genetics ; Female ; Human bocavirus ; genetics ; isolation & purification ; Humans ; Logistic Models ; Male ; Multivariate Analysis ; Parvoviridae Infections ; epidemiology ; virology ; Prevalence ; Respiratory Tract Infections ; epidemiology ; virology

OBJECTIVETo study the application of serodiagnosis of human bocavirus (HBoV) lower respiratory tract infection in children.

METHODFrom January to April, 2013, samples including serum, sputum and bronchoalveolar lavage fluids (BALFs) were obtained from 714 children hospitalized with ALRI. Serums were tested for HBoV-specific IgG and IgM antibodies by ELISA and all kinds of samples were tested for HBoV DNA by quantitative real-time fluorescent PCR. The results of HBoV serologic tests, viral DNA in sputum and their combination were compared with those of HBoV DNA in serums and/or BALFs, which was considered as the "standard". Their consistence and differences were evaluated, and the diagnostic parameters including sensitivity, specificity, positive predictive value, negative predictive value, consistency rate, Kappa value and J value were calculated. Age distributions of the HBoV positive patients tested by the latter two methods were also compared.

RESULTThe positive rate of HBoV serology was 13.2% (94/714) . The results of HBoV serology, its DNA in sputum and their combination were all consistent with those of HBoV DNA in serums and/or BALFs (χ(2) = 91.834, 124.662, 138.643, P < 0.001 for all comparisons) . Differences were significant by McNemar test (χ(2) = 23.547, 33.440, 12.410, P all <0.001) . All the diagnostic parameters for single HBoV serologic test or single viral DNA test in sputa were approximate. However, they were improved to 70.4%, 94.8%, 38.0%, 98.6%, 93.7%, 0.463(P < 0.001), 0.65 for sensitivity, specificity, positive predictive value, negative predictive value, consistency rate, Kappa value and J value, respectively, when the methods were combined. HBoV was found positive mainly in children under 3 years of age, especially in the 1 year group. The positive rates were the highest in both group -1 year, and group -3 years was the next. However, the rate was the lowest in group >3 years and in the group -6 months.

CONCLUSIONDiagnostic power can be improved and age distribution can be demonstrated when serologic tests were combined with traditional sputum DNA detection in children with HBoV lower respiratory tract infection.

Acute Disease ; Adolescent ; Age Distribution ; Antibodies, Viral ; analysis ; blood ; Antigens, Viral ; analysis ; Child ; Child, Preschool ; Enzyme-Linked Immunosorbent Assay ; Female ; Human bocavirus ; genetics ; isolation & purification ; Humans ; Immunoglobulin G ; blood ; Immunoglobulin M ; blood ; Infant ; Male ; Parvoviridae Infections ; diagnosis ; virology ; Real-Time Polymerase Chain Reaction ; Respiratory Tract Infections ; diagnosis ; virology ; Sensitivity and Specificity

Human bocavirus (HBoV) 1-4 have been detected both in respiratory and stool samples since the first HBoV was discovered in 2005. HBoV-1 is mostly associated with respiratory infection, while HBoV 2-4 are usually associated with intestinal tract infection. A variety of signs and symptoms have been described in patients with HBoV infection, including cough, wheezing, pneumonia, and diarrhea, but the research on pathogenic mechanism of HBoV is limited because HBoV cannot be cultured in vitro due to the lack of appropriate host cells. Three-dimensional epithelial cell culture, reverse genetics, and viral metagenomics are identified as novel tools that may promote the research on pathogenic mechanism of HBoV and the discovery of new viruses. This review summaries currently available diagnostic approaches such as electron microscopy, cell culture, PCR, and immunoassay in order to provide a method reference for indepth research on HBoV.
Animals ; Human bocavirus ; genetics ; growth & development ; isolation & purification ; pathogenicity ; Humans ; Parvoviridae Infections ; diagnosis ; virology ; Viral Proteins ; genetics ; metabolism ; Virology ; methods ; Virulence ; Virus Cultivation

Human bocavirus 1 (HBoV1) is a novel virus that mainly causes respiratory tract infection, and it has the characteristic of genome of Parvovirus, containing three open reading frames that encode non-structural proteins NS1 and NP1 and structural proteins VP1 and VP2. Circular episome is present during the rolling circle replication of HBoV1, which provides the possibility of full genome amplification and infectious clone construction to save HBoV1. The recombination between HBoV1 and HBoV2-4 occurs frequently. With the three-dimensional culture, in vitro culture of HBoV1 provides a powerful tool for research on the pathogenesis of HBoV1. This review focuses on the molecular characteristics, association with diseases, in vitro culture, diagnosis and treatment of HBoV1.
Diarrhea ; virology ; Genomics ; Human bocavirus ; genetics ; isolation & purification ; physiology ; Humans ; Meningitis ; virology ; Respiratory Tract Diseases ; virology

Human bocavirus (HBoV) is classified in the family of parvovirdae, genus bocavirus. Besides parvovirus B19 and human parvovirus 4 (PARV4), HBoV isone of the parvoviruses currently known to infect and cause illness in human. So far, four different HBoVs (HBoV1-4) have been successively reported. The incidence of HBoVs infection varies widely, the clinical presentations of patients are different, and HBoVs are often co-detected with other pathogens. There are already quite a few report of HBoVs infection, and this article reviews and discusses the biological characters, epidemic characters, pathogenic mechanism, phylogenetic analyses of HBoVs and the epidemiological situation in China.
China ; epidemiology ; Gastrointestinal Diseases ; etiology ; Human bocavirus ; classification ; genetics ; immunology ; isolation & purification ; Humans ; Parvoviridae Infections ; epidemiology ; Phylogeny

China ; epidemiology ; Epidemiologic Studies ; Human bocavirus ; isolation & purification ; Humans ; Molecular Epidemiology ; Parvoviridae Infections ; epidemiology ; Respiratory Tract Infections ; epidemiology ; virology