Objective To find out the importance of human bocavirus (HBoV) as an infectious agent for population in Beijing, China. Seroprevalence study was conducted by using expressed recombinant major capsid VP2 protein as an antigen.Methods Serum specimens collected from infants and children who visited the Children's Hospital Affiliated to the Capital Institute of Pediatrics for health check-up and adults visiting the Xuanwu Hospital, Beijing for diseases other than respiratory infections from April 1996 to March 1997 were used for the investigation. The major capsid protein VP2 from HBoV was expressed in E. coli strain BL21 (DE3) with the transformed PET30b vector inserted with full-length VP2 gene of HBoV and the specific antigenicity of this expressed protein was validated by previous study. Western blotting was used to detect specific IgG antibody against HBoV in collected serum specimens diluted to 1:200. Mock expressed protein was E. coli cells strain BL21 (DE3) with the transformed PET30b vector without insert. Anti-His monoclonal antibody and rabbit anti-HBoV VP2 polypeptides hyper-immune serum were used as positive control for antibody detection.Results Out of 677 serum specimens tested, 400 (59.1% ) were positive for HBoV by Western blotting. About 45.3% (34/75) of the newborns under 1 month of age had anti-HBoV antibodies, and antibody positive rates were decreased in age groups of 1 and 2 months (41.4% and 31.3%, respectively) then increased in the following ages from 6 months to 7 years old ( from 45.6% to 69.7% ). The antibody positive rates were maintained at a relatively constant level ( about 70% ) in the age groups from 7 years to 40 years of age and became lower ( 61.8% - 62. 8% ) in those over 50 years.Conclusions The high seroprevalence of antibody against recombinant HBoV VP2 protein and early age antibody acquisition indicate that HBoV has been circulating in population of Beijing, China as early as in 1996 and most of children had been exposed to HBoV by the age of 7 years. Infants under the age of 6 months were susceptible to this virus.

Background Our previous study determined that expressions of matrix metalloproteinases (MMPs) and tissue matrix metalloproteinase inhibitors (TIMPs)change in the conjuntival fibroblasts of conjunctivochalasis in vitro.To seek a suitable drug is very important in the prevention and treatment of conjunctivochalasis.Objective This study was to explore the effect of qijingmingmu soup on the expressions of MMPs and TIMPs in human conjunctival fibroblasts of conjunctivochalasis.Methods Twenty-four SD rats were randomized into two groups.Qijingmingmu soup was administration gastrically for consecutive 3 days,and normal saline solution was given in the same way in the control group.The blood was collected from aortaventralis and drug serum was prepared.Human conjunctival samples were obtained during the surgery of conjunctivochalasis relaxation and cultured in the DMEM containing 10％ fetal bovine serum,20％,15％,10％,5％ of drug serum and 8 ml/L epidermal growth factor(EGF) was added into the medium respectively.Enzyme-linked immunosorbent assay(ELISA)was used to detect the expressions of MMP1,MMP3,TIMP1 and TIMP3in conjunctival fibroblasts.Results Cultured cells grew well with the fusiform shape and showed the positive response for vimentin.The expression value of MMP1 (A value)in the cells was declined after administration of qijingmingmu soup.A significant difference was found in the expression of MMP1 among the control group,20％,15％,10％,5％ drug serum groups and EGF group(F=466.664,P＜0.05),and that in the 10％ ([9.92±0.14] mg/L) and 20％ ([11.87 ±0.11] mg/L) drug serum groups was significantly lowed in comparison with the control group([16.31±0.10] mg/L)(t=99.974,87.394,P＜0.05).The expression value of the MMP3in the cells in the various drug serum groups,EGF group and the control group was significantly different(F=158.168,P＜0.05),with a lower value in the 20％ drug serum group compared with the control group ([3.50±0.03] mg/L vs.[4.44 ± 0.11] mg/L) (t =21.991,P ＜ 0.05).Also,the significantly different expressing value of TIMP1 was seen among all the groups (F=183.508,P＜0.05),and expressing value of TIMP1 in the 15％ drug serum group was(1.88±0.06)mg/L,which was lower than(3.20±0.32) mg/L of the control group(t=10.353,P＜0.05).Furthermore,the expressing value of the TIMP3 in the cells was significantly different among the various groups(F=54.503,P＜0.05),and that of the 20％ drug serum group was (1.743±0.065)mg/L and it was significantly higher than (1.54 ± 0.05) mg/L of the control group (t =5.046,P =0.004).However,the expressing value of TIMP3of the 15％,10％ and 5％ drug serum groups was lower than that of the control group,respectively all at(P＜0.05).Conclusions Qijingmingmu soup drug serum at the concentration of 20％ can down-regulate the expressions of MMP1,MMP3,TIMP1 and up-regulate the expression of TIMP3 in human conjunctivochalasis bulbar conjunctival fibroblastsin vitro,which probably plays preventive and therapeutic effects on conjunctivochalasis.

Objective To analyze the clinical features,diagnosis and treatment of 2 pedigrees (5 patients) with familial hemangioblastoma. Methods The detailed clinical and imaging data of 2 pedigrees (5 patients) with familial hemangioblastoma,admitted to our hospital from September 2005 to May 2010, were retrospectively analyzed; their diagnosis and treatment were concluded. Results Among the 3 patients of the first pedigree,2 patients displayed cystic and solid tumor and 1 cystic tumor under cranial MRI; both 2 patients of the second pedigree displayed cystic solids.All the 5 patients had mutations and could be diagnosed as having yon Hippel-Lindau (VHL).No patients were combined with other parts of the lesion; after microscopic total/sub-total resection, no patients existed postoperative occurrence and no patient died. Conclusion MRI is the most important detective method in the diagnosis of patients with familiar hemangioblastomas; microsurgery is still the most important therapy method to familiar hemangioblastomas.

OBJECTIVETo isolate, identify and culture human spermatogonial stem cells (SSC) and then obtain purified and enriched human SSCs for research and application.

METHODSWe detected the expression of CD90 in the human testis using the immunofluorescence technique and isolated human testicular spermatogenic cells by two-step enzymatic digestion, followed by differential plating and magnetic-activated cell sorting (MACS) with CD90 as an SSC marker. Then we identified the isolated CD90-positive spermatogenic cells by RT-PCR and immunocytochemistry, and meanwhile cocultured them with Sertoli cells in SG medium in vitro.

RESULTSThe isolated CD90-positive cells showed a relatively homogeneous characteristic in size and morphology and expressed the genes specific for human SSCs, with high expressions (90.5%) of GFRA1, GPR125, and UCHL1. After coculture with Sertoli cells in the SG medium for 2 weeks, the isolated CD90-positive cells maintained a good activity.

CONCLUSIONCD90 can be regarded as a speci- fic marker for human SSCs and used to obtain highly enriched human SSCs by differential plating and MACS. Furthermore, the isolated human SSCs can be cultured in SG medium in vitro.

Adult Stem Cells ; cytology ; Biomarkers ; metabolism ; Cell Separation ; methods ; Cell Shape ; Cell Size ; Coculture Techniques ; Glial Cell Line-Derived Neurotrophic Factor Receptors ; metabolism ; Humans ; Immunohistochemistry ; Male ; Receptors, G-Protein-Coupled ; metabolism ; Sertoli Cells ; Spermatogonia ; cytology ; Testis ; metabolism ; Thy-1 Antigens ; isolation & purification ; metabolism ; Ubiquitin Thiolesterase ; metabolism

OBJECTIVEHuman respiratory syncytial virus (hRSV) is the leading cause of acute upper and lower respiratory tract infections in infants and young children worldwide. Pediatric RSV disease claims more than 1 million lives annually. With the rapid development of specific anti-RSV agents and the spread of respiratory infections, RSV detection techniques with higher sensitivity, specificity and quicker performance are badly needed. This study was designed to develop a real-time polymerase chain reaction (PCR) for detection of RSV in nasopharyngeal aspirates.

METHODS(1) The TaqMan probe and primers of real-time PCR for RSV subgroup A and subgroup B detection were designed from the conserved region in N protein encoding gene, respectively. The sensitivity of real-time PCR was evaluated by using the virus with known amount of PFU. The specificity of real-time PCR for RSV detection was assessed by cross testing 10 isolates of strains A, 10 isolates of strains B, and by testing a variety of other respiratory viruses positive samples. (2) Sixty-one stored RSV positive respiratory samples and 103 nasopharyngeal aspirates were detected by real-time PCR, virus isolation, immunofluorescence assay (IFA), and nested-PCR.

RESULTS(1) The sensitivity of the real-time PCR developed in this study for RSV subgroup A detection was 5.25 pfu, and for subgroup B was 3.75 pfu, the same as that of nested-PCR. (2) No positive results were found in cross testing of other viruses positive specimens. (3) Twenty-seven out of 30 (90%) of RSV A stored samples and 27 out of 31 (87.1%) of RSV B stored samples were positive by the real-time PCR. (4) Thirty-five (34.0%) out of the 103 specimens were found RSV positive by real-time PCR (7 of them were subgroup A and 28 subgroup B); 31 (30.1%) specimens were positive by nested-PCR (6 of them were subgroup A and 25 subgroup B); 22 (21.4%) were found positive for RSV with IFA (5 of them were subgroup A and 17 subgroup B); RSV was isolated from 9 (8.7%) specimens (6 of them were subgroup A and 3 subgroup B). All the specimens found to be negative by real-time PCR were negative by rest of the methods used in this study.

CONCLUSIONThe real time PCR method developed in this project with the TaqMan probe and primers is sensitive and specific for detecting RSV subgroup A and B in nasopharyngeal aspirates.

Child ; DNA, Complementary ; isolation & purification ; Fluorescent Antibody Technique ; Humans ; Nasopharynx ; secretion ; virology ; Polymerase Chain Reaction ; methods ; RNA, Viral ; isolation & purification ; Respiratory Syncytial Virus Infections ; genetics ; Respiratory Syncytial Virus, Human ; genetics ; isolation & purification

OBJECTIVEA new human coronavirus, HCoV-NL63, was identified recently from two Dutch children with acute respiratory infection (ARI) by two scientists in the Netherlands in 2004. To investigate if this newly discovered virus is associated with acute respiratory infections in pediatric patients in Beijing, tests were developed to detect HCoV-NL63 gene fragments from throat swab and nasopharyngeal aspirates collected from children in outpatient and inpatient departments with ARI in Beijing from Dec. 2003 to Mar. 2004.

METHODSA total of 245 clinical samples, which were negative either for diagnostic tests of human respiratory syncytial virus, influenza virus A and B, adenovirus, parainfluenza virus 1, 2 and 3 by indirect immunofluorescence assay or human metapneumovirus by RT-PCR, were screened for HCoV-NL63 by nested PCR amplifying gene fragments located on the 1b and 1a genes. Amplicon of PCR from 1a gene of HCoV-NL63 was sequenced and the sequences were compared with those in GenBank nucleotide sequence database.

RESULTSThree (1.2%) out of the 245 samples were positive for HCoV-NL63 by nested-PCR using primers on 1b gene. These three samples also showed positive results on nested PCR in which primers were designed with sequences complementary to 1a gene segments. These positive samples were collected from hospitalized children under 2 years of age with pneumonia, bronchiolitis and bronchitis, respectively. The partial 1a gene sequences from two positive samples (BJ3140 and BJ3787) of HCoV-NL63 showed 100% homology between each other and high homology (98%-99%) with the sequences of 1a gene of HCoV-NL63 reported from different countries in GenBank. Phylogenetic analysis showed that BJ3140 and BJ3787 fell into the same genetic cluster (group 1).

CONCLUSIONSThese data suggest that some of acute respiratory infections in young children in Beijing area are related to the newly identified HCoV-NL63.

Acute Disease ; China ; Coronavirus ; genetics ; isolation & purification ; Databases, Nucleic Acid ; Humans ; Infant ; Phylogeny ; Polymerase Chain Reaction ; Respiratory Tract Infections ; virology ; Sequence Homology, Nucleic Acid

Human metapneumovirus (hMPV) is associated with acute respiratory tract infections (ARTI) in all age groups. However, there is limited information of genetic analysis of hMPV circulating in Beijing. To learn the characteristics of structural protein genes of human metapneumovirus circulating in children in Beijing, sequence analysis of matrix (M), small hydrophobic (SH) and attachment (G) proteins of hMPV from 2006 to 2010 was performed. Phylogenetic analysis of nucleotide sequences of 42 full length M genes, 49 SH gene and 55 G gene revealed that the hMPVs from pediatric patients were divided into sub-genotypes A2, B1 and B. There were highly conserved identities among M gene, with 7 conserved mutations of amino acids between A and B genotypes which were fairly conserved in the same genotype A or B. The amino acid identities of SH were 60.7% to 64.4% between different genotypes, 93.3% - 100% among same sub-genotype and 84.7% - 88.7% between different sub-genotypes. Use of alternative transcription-termination codon, nucleotide deletion and insertion resulted in variable length of nucleotide and deduced amino acid of G protein. Amino acid identities within same genotype ranged from 81.5% - 100%, whereas sequence identities between two genotypes ranged from 34.0% - 38.6% at the amino acid level. A new cluster of G genes in sub-genotype B2 appeared due to the same mutations and insertion of two amino acids in G protein encoding genes amplified from specimens collected from 2008 to 2010. Prediction of antigen sites of SH and G protein indicated that the variation of antigen sites between different sub-genotypes existed.
Child ; China ; epidemiology ; Genetic Variation ; Genotype ; Humans ; Metapneumovirus ; genetics ; Paramyxoviridae Infections ; blood ; epidemiology ; virology ; Phylogeny ; Retroviridae Proteins, Oncogenic ; blood ; genetics ; Viral Envelope Proteins ; blood ; genetics ; Viral Matrix Proteins ; blood ; genetics

OBJECTIVETo understand the impact of human parainfluenza virus (HPIV) on acute respiratory infections in infants and young children in Beijing.

METHODSMultiplex reverse transcription-PCR was used to amplify the hemagglutinin (HA) gene fragment of HPIV from clinical specimens. Primer pairs derived from a conserved region of the HA genes of HPIV were used to develop the multiplex RT-PCR for detecting and typing HPIV. The sensitivity and specificity of the method were determined by using various RNA and DNA viruses as controls. Specimens collected from 3519 children with acute respiratory infections from Aug. 2003 to Apr. 2006 were analyzed for HPIV by the multiplex RT-PCR as well as for other respiratory viruses by virus isolation and/or indirect immunofluorescent assay (IFA). Ten amplicons with expected molecular weight matching different types of HPIV were randomly selected for sequence analysis.

RESULTSOnly the cDNA from the isolated strains of HPIV 1 and 3 was positive by the multiplex RT-PCR. Phylogenetic analysis for those 10 amplicons' sequences which belong to HPIV 1 - 4 types respectively as determined by multiplex-PCR indicated that these specimens were truly HPIV positive. These 10 HPIV positive specimens included two specimens of type 4 which was further subtyped as HPIV4A and 4B by sequence analysis. With the multiplex RT-PCR, HPIV were detected in 349 out of 3519 specimens with the positive rate of 9.9% (349/3519), which is higher than 4.8% by the methods of virus isolation and/or IFA. And the HPIV positive rates were high in patients with not only acute upper but also lower respiratory tract infection. No regular seasonality distribution of HPIV infection was found. HPIV 1 and 3 were more common than HPIV 2 and 4.

CONCLUSIONWith higher sensitivity and specificity than virus isolation and IFA, multiplex RT-PCR is beneficial for the etiologic and epidemiologic studies on HPIV, as well as for HPIV typing. The data from this study indicate that HPIV is one of the important etiological viruses of acute respiratory tract infections in infants and young children in Beijing.

Child, Preschool ; China ; epidemiology ; Genes, Viral ; HN Protein ; genetics ; Humans ; Infant ; Paramyxoviridae Infections ; epidemiology ; virology ; Phylogeny ; Prevalence ; Respiratory Tract Infections ; epidemiology ; virology ; Respirovirus ; genetics ; isolation & purification ; Reverse Transcriptase Polymerase Chain Reaction ; Sensitivity and Specificity

OBJECTIVETo characterize the prevalence and occurrence of subgroups of human respiratory syncytial virus (RSV) in infants and young children with acute respiratory infections (ARI) in Beijing area.

METHODSRSVs were identified from nasopharyngeal aspirates and throat swabs collected from infants and children with ARI who visited the Children's Hospital Affiliated to Capital Institute of Pediatrics during the period of November 2000 to March 2006, by virus isolation in Hep-2 cells and antigen detection by indirect immunofluorescence assay (IFA). RT-PCR was used to differentiate subgroups A and B of RSV from part of the positive specimens.

RESULTSOut of 10 048 specimens including 7176 nasopharyngeal aspirates from inpatients and 2872 throat swabs from outpatients, 2286 (22.8%) were RSV positive. The positive rate for RSV identification were 30.0% (2153/7176) in specimens from the hospitalized patients, which was higher than that from outpatients (4.6%, 133/2872). The youngest of the RSV positive patients was 1 day after birth and the oldest was 15 years of age, with 73.0% younger than 1 year. Among those RSV positives, only 1.6% were older than 5 years. The ratio of male to female who were RSV positive was 2.4:1 (1598:674). The clinical diagnosis for 91.2% (1991) of those RSV positive patients was severe lower respiratory infections including bronchiolitis and pneumonia, whereas in only 8.8% (192) the diagnosis was upper respiratory infections. The data revealed that RSV started to be detected in October each year during the survey period and November to next April was the RSV season. The detection rate declined in May and almost no RSV could be found in summer. Positive rates for RSV detection were 42.3%, 41.0% and 40.5% in the seasons of 2001 - 2002, 2003 - 2004, 2005 - 2006, which were higher than those in seasons of 2000 - 2001 (14.0%), 2002 - 2003 (18.2%), 2004 - 2005 (20.4%). Subtyping of A and B during the surveillance period showed that 73.7% (691/938) were subgroup A and 26.3% (247/938) were subgroup B. Subgroup B was predominant in the 2000 - 2001 and 2004 - 2005 seasons, whereas subgroup A predominated in the 2001 - 2002, 2002 - 2003 and 2003 - 2004 seasons. Almost equal proportions of subgroup A and B appeared in 2005 - 2006 seasons.

CONCLUSIONThe data indicate that RSV is an important etiological agent for lower respiratory infections in infants and young children in winter and spring during the survey period. The pattern of RSV circulation varied alternately with higher rate every other year. The predominant subgroup changed between A and B, and co-circulated in equal proportion in some years.

Adolescent ; Cell Line, Tumor ; Child ; Child, Preschool ; China ; epidemiology ; Female ; Humans ; Infant ; Infant, Newborn ; Male ; Population Surveillance ; Prevalence ; Respiratory Syncytial Virus Infections ; diagnosis ; epidemiology ; Respiratory Syncytial Virus, Human ; genetics ; isolation & purification ; Respiratory Tract Infections ; diagnosis ; epidemiology ; virology ; Seasons

OBJECTIVETo establish a rapid, sensitive and specific reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for detecting human respiratory syncytial virus (RSV) in respiratory samples collected from children with acute respiratory infections.

METHODAccording to the conserved matrix gene sequences of respiratory syncytial virus subtypes A and B downloaded from GenBank, primers were designed and RT-LAMP assay was developed to detect RNA of RSV sensitivity of the RT-LAMP method was evaluated by using ten-fold serially diluted in vitro-transcribed matrix RNA fragments from RSV A and RSV B, respectively. Specificity of the RT-LAMP method was tested through cross-reaction with other RNA and DNA viruses. Then 5 RSV strains isolated from clinical specimens using tissue cultures were tested by RT-LAMP assay. A total of 101 nasopharyngeal aspirates from hospitalized patients with acute respiratory infections which had been tested by direct immunofluorescence assay (DFA), including 40 positive for RSV and 61 negative for RSV, were tested by RT-LAMP assay and by RT-nested PCR.

RESULTSensitivity analysis indicated that this RT-LAMP method was able to detect 1 copy/µl of RSV A and RSV B RNA, no amplification was shown in RT-LAMP with DNA or cDNA from other viruses in 60 min, revealed that the RT-LAMP assay is highly specific. Five RSV isolates confirmed as 4 RSV A and 1 RSV B previously were detected by RT-LAMP method as positive in 30 min. For those 101 specimens tested, 37 were RSV positive determined by RT-LAMP assay, as well as 35 RSV positive by RT-nested PCR. The total coincidence rate of RT-LAMP assay with DFA and RT-nested PCR in detecting RSV is 95.0%, 94.1% with Kappa value 0.895 and 0.871, respectively.

CONCLUSIONA new, sensitive, accurate and rapid method, RT-LAMP assay for detecting human respiratory syncytial viruses from nasopharyngeal aspirates was developed, which should be helpful in rapid detection of RSV from respiratory tract samples of children.

Acute Disease ; Child ; Child, Preschool ; DNA Primers ; Humans ; Infant ; Molecular Diagnostic Techniques ; Nasopharynx ; virology ; Nucleic Acid Amplification Techniques ; RNA, Viral ; isolation & purification ; Respiratory Syncytial Virus Infections ; diagnosis ; Respiratory Syncytial Virus, Human ; isolation & purification ; Reverse Transcriptase Polymerase Chain Reaction ; Sensitivity and Specificity