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

BACKGROUND: The incidence and etiology of hepatocellular carcinoma (HCC) vary widely according to race and geographic regions. The insertional mutagenesis of adeno-associated virus 2 (AAV2) has recently been considered a new viral etiology of HCC. The aim of this study was to investigate the frequency and clinical characteristics of AAV2 in Korean patients with HCC. METHODS: A total of 289 unrelated Korean patients with HCC, including 159 Hepatitis-B-related cases, 16 Hepatitis-C-related cases, and 114 viral serology-negative cases, who underwent surgery at the Samsung Medical Center in Korea from 2009 to 2014 were enrolled in this study. The presence of AAV2 in fresh-frozen tumor tissues was investigated by DNA PCR and Sanger sequencing. The clinical and pathological characteristics of AAV2-associated HCC in these patients were compared with previous findings in French patients. RESULTS: The AAV2 detection rate in Korean patients (2/289) was very low compared with that in French patients (11/193). Similar to the French patients, the Korean patients with AAV2-related HCC showed no signs of liver cirrhosis. The Korean patients were younger than the French patients with the same AAV2-associated HCC; the ages at diagnosis of the two Korean patients were 47 and 39 yr, while the median age of the 11 French patients was 55 yr (range 43-90 yr). CONCLUSIONS: AAV2-associated HCC was very rare in Korean patients with HCC. Despite a limited number of cases, this study is the first to report the clinical characteristics of Korean patients with AAV2-associated HCC. These findings suggest epidemiologic differences in viral hepatocarcinogenesis between Korean and European patients.
Adult ; Asian Continental Ancestry Group ; Capsid Proteins/genetics ; Carcinoma, Hepatocellular/etiology/*pathology/virology ; DNA, Viral/chemistry/genetics/metabolism ; DNA-Binding Proteins/genetics ; Dependovirus/*genetics/isolation & purification/pathogenicity ; Female ; Humans ; Incidence ; Inverted Repeat Sequences/genetics ; Liver Neoplasms/etiology/*pathology/virology ; Male ; Middle Aged ; Parvoviridae Infections/complications/epidemiology ; Polymerase Chain Reaction ; Republic of Korea ; Sequence Analysis, DNA ; Viral Proteins/genetics

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

Until now, more than seventeen parvovirus have been reported which can infect mammals and poultries. The infected cells appeared different properties of apoptosis and death, present a typical cytopathic effect. NS1 is a major nonstructural protein of parvovirus, with a conservative structure and function, which plays an important role in the viral life cycle. In addition to the influence on viral replication, the NS1 also participates in apoptosis induced by viruses. Parvovirus induced apoptosis which is mainly mediated by mitochondrial pathway, this review summarized the latest research progresses of parvovirus induced apoptosis.
Animals ; Apoptosis ; Humans ; Parvoviridae Infections ; physiopathology ; veterinary ; virology ; Parvovirus ; genetics ; metabolism ; Viral Nonstructural Proteins ; genetics ; metabolism

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

To obtain the genome sequence of human bocavirus 2 (HBoV2), different regions of HBoV2 genome were amplified through PCR in fecal specimens which had been identified as single-positive for HBoV2 in 2010. A genome sequence of HBoV2 (HBoV2-NC, 5444 bp) was obtained after sequence assembly. The phylogenetic analysis showed that HBoV2-NC had the closest evolutionary relationship with HBoV2 Lanzhou strain. The predication of inverted terminal repeats of HBoV2-NC by DINAMelt showed that inverted terminal repeats were contained in HBoV2-NC 5' terminal, which had the typical stem-loop structure in other parvoviruses. Finally, some flanking sequences of HBoV2-NC were amplified by linker-PCR.
Base Sequence ; Gene Amplification ; Genome, Viral ; Human bocavirus ; chemistry ; classification ; genetics ; Humans ; Molecular Sequence Data ; Nucleic Acid Conformation ; Parvoviridae Infections ; virology ; Phylogeny ; RNA, Viral ; chemistry ; genetics ; Terminal Repeat Sequences

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

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

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

To study the proliferation characteristics of PPV in differently infected way and the variance of concentrations in different cells. A strain of porcine parvovirus(PPV) was adapted to PK-15 cells, and a Real-time fluorescent quantitative PCR (FQ-PCR) assay was developed based on the specific region of the NS1 gene of PPV to quantify the PPV. The FQ-PCR was used to measure the viral concentration of virus-infected cells by simultaneous or step by step inoculation and plot one-step growth curves. The proliferation characteristics of PPV strain in different cells lines (HeLa, MDBK, PK-15 ,ST, F81, BHK-21 and Marc-145) was also compared. The results showed the PK-15 cell -adapted strain of PPV produced CPE after 12 passages, and maintained stable CPE at the following 10 messages. The one-step growth curve showed that the virus concentration of simultaneous inoculation was higher than that of the step-by-step inoculation, and the proliferation cycle of step-by-step inoculation was shorter. The proliferation ability of PPV strain in different cells showed that CPE appeared first inPK-15, followed by ST, HeLa and MDBK, and the virus concentration was highest in ST, followed byPK-15, MDBK and HeLa. NO proliferation was observed in F81, BHK-21 and Marc-145 cells. These findings lay a material foundation for the basic researches on PPV and the development of vaccine.
Animals ; Cell Line ; Cricetinae ; Cytopathogenic Effect, Viral ; DNA, Viral ; genetics ; Female ; Haplorhini ; Humans ; Male ; Parvoviridae Infections ; virology ; Parvovirus, Porcine ; genetics ; physiology ; Real-Time Polymerase Chain Reaction ; Reproducibility of Results ; Sensitivity and Specificity ; Swine ; Viral Proteins ; genetics ; Virus Replication