The aim of this study was to obtain the capsid protein VP2 of human bocavirus (HBoV) identified in Beijing recently and construct virus-like particles (VLPs) in insect cells for further study of this virus. The full-length VP2 gene of HBoV from BJ3722 was inserted into the baculovirus expression transfer vector (pFastBac 1) to obtain the recombinant Bacmid, and generation of recombinant baculoviruses was followed by transfection of the recombinant Bacmid into insect cells. Then the recombinant VP2 protein was recognized by SDS-PAGE using Coomassie-blue staining and Western blot using hyper-immune serum against VP2 of HBoV from rabbit. The recombinant baculoviruses were harvested and amplified to gain large amounts of viruses with high titers to infect insect cells at a multiplicity of infection (MOI) of 0. 5. After 7-10 days or 4-5 days of the infection, the supernatants of culture or the cell lysates treated with lysing solution were harvested, and ultracentrifuged twice through 40% sucrose cushion to obtain purified VLPs, which were followed by Western blot and IFA for VLPs' composition and specificity analysis, by electron microscopy for VLPs' morphologic structure. The recombinant VP2 protein with molecular weight of approximately 61 kD expressed in recombinant baculoviruses was recognized by SDS-PAGE using Coomassie-blue staining and Western blot. The presence of VP2 on VLPs was demonstrated by Western blot and IFA from samples collected during the purification of VLPs from the supernatants of culture or the cell lysates, and the expression of VP2 in insect cells led to the formation of VLPs which formed the typical icosahedral appearance of parvoviruses with a diameter of approximately 20 nm. In conclusion, the recombinant baculoviruses were constructed, the HBoV VP2 protein was expressed in insect cells with high specific antigenicity and VLPs was formed successfully.
Animals ; Blotting, Western ; Capsid Proteins ; genetics ; metabolism ; Cell Line ; Electrophoresis, Polyacrylamide Gel ; Fluorescent Antibody Technique, Indirect ; Human bocavirus ; genetics ; metabolism ; Microscopy, Electron, Transmission ; Polymerase Chain Reaction ; Spodoptera ; Virion ; genetics ; metabolism ; ultrastructure

OBJECTIVEThe present study was designed to explore the practical application of the rapid etiological diagnosis by detecting specific IgM antibody against common respiratory viruses in children with acute lower respiratory infections (ALRI).

METHODClinical specimens including nasopharyngeal aspirates and serum of acute phase from hospitalized children were collected from 207 infants and children with acute lower respiratory infections from March 2009 to September 2010. Seven common respiratory virus antigens were identified from the collected nasopharyngeal aspirates by direct immunofluorescence assay (DFA). ELISA was used to detect specific IgM antibody against RSV, ADV, IFVA, IFVB and PIV, while indirect immunofluorescence assay (IFA) was used to detect specific IgM antibody against RSV, ADV, IFVA, IFVB, PIV1, PIV2 and PIV3 in collected acute phase serum.

RESULTThe overall positive rates to detect viral antigen by using DFA, ELISA and IFA was 67.6%, 57.5% and 39.6%, respectively. The consistent rate of ELISA and IFA versus accepted DFA were 21.7% and 31.4%, respectively. The average days from onset of the symptoms to blood sample collection for those with the consistent results by ELISA and DFA were 12.0 d for ADV, 9.6 d for PIV2, 9.5 d for IFV, and 5.3 d for RSV, respectively, and by IFA and DFA were 15.0 d for PIV3, 9.2 d for ADV, and 7.4 d for RSV, respectively. Among all age groups, the consistent rate of serum viral IgM and antigen detections was highest in children younger than 3 years old.

CONCLUSIONAlthough there were differences between serum IgM antibody and viral antigen detections, specific IgM antibody detection was of value in early and rapid etiological diagnosis of pediatric ALRI, especially for young children. It could provide serologic evidence of respiratory virus infection. The diagnostic rate of pathogen could be improved if it was used in combination with viral antigen diagnostic methods.

Antibodies, Viral ; analysis ; blood ; Antibody Specificity ; Antigens, Viral ; analysis ; Child ; Child, Preschool ; Enzyme-Linked Immunosorbent Assay ; Female ; Fluorescent Antibody Technique ; Humans ; Immunoglobulin M ; analysis ; blood ; Infant ; Male ; Nasopharynx ; virology ; RNA Viruses ; genetics ; isolation & purification ; Respiratory Syncytial Virus Infections ; diagnosis ; virology ; Respiratory Syncytial Viruses ; genetics ; isolation & purification ; Respiratory Tract Infections ; diagnosis ; immunology ; virology ; Sensitivity and Specificity

To characterize the genomic sequence and arrangement of WU polyomavirus (WU virus) identified in clinical specimens collected from children with acute respiratory infections in Beijing, China, the sequences of capsid proteins VP1, VP2, and the large tumor antigen (LTAg), as well as the 5'-terminal sequence of WU virus, were amplified from the clinical specimen with ID number of BJF5276 which was determined as WU virus positive by PCR amplification. The PCR amplicons were sequenced, and genomic sequence analysis was performed by using the software DNAStar. In addition, VP2 coding-region sequences were amplified from other 21 clinical specimens identified as WU virus positive to investigate the gene diversity of WU virus. The genomic sequence of WU virus BJF5276 with accession number of HQ218321 in GenBank was 5,229 base pairs in length with 3 major coding domain sequences (CDS) sited on one strand coding for capsid proteins VP2, VP3 and VP1, and two CDS sited on the complementary strand coding for small tumor antigen (STAg) and LTAg; These 22 VP2 CDS sequences including 5 sequences submitted to GenBank were compared with 64 corresponding sequences downloaded from GenBank by MegAlign of DNAStar software, indicated that these sequences coming from children in Beijing shared high homology (over 98.8%) with those from GenBank. Phylogenetic analysis of these VP2 CDS by using Neighbor-joining (NJ) analyses with 2,000 bootstraps (Mega 4.0) showed that 20 sequences out of 22 belonged to clade Ia, and other 2 of them belonged to clade III, including 1 clustered in IIIa and 1 in a novel cluster proposed as IIIc. In conclusion, the genomic sequence of WU polyomavirus detected from clinical specimens from children in Beijing is closely related to other WU polyomaviruses in the feature of genomic coding region arrangement. Overall variation of VP2 CDS was very low, and there were different clades circulating in Beijing with a dominant clade Ia, which is different from dominated Ib circulating in other parts of the world reported previously, and a novel clade IIIc was proposed.
Acute Disease ; Child, Preschool ; China ; Female ; Genome, Viral ; Humans ; Infant ; Male ; Molecular Sequence Data ; Phylogeny ; Polyomavirus ; classification ; genetics ; isolation & purification ; Respiratory Tract Infections ; virology ; Viral Proteins ; genetics

OBJECTIVETo study the role of a pathologic niche inducing mouse embryonic stem cells (ESC) to express hepatic cell functions in vitro.

METHODSEmbryoid bodies were developed from 5 to 7 day hanging-drop culture of mouse ESC, and their dissociated cells were planted in three differential systems: nothing added; with 20 ng/ml hepatocyte growth factor (HGF); and 5% rat cholestatic serum plus 20 ng/ml HGF added. Their differentiation was observed with inverted microscopes daily, and their hepatic functions were analyzed against their synthesis of glycogen, triglycerides, albumin, and urea nitrogen, and by their staining of indocyanine green (ICG) and fluorescein diacetate (FDA).

RESULTSESC spontaneous differentiation was hardly being controlled to form three germ layers. HGF prompted the ESC to develop further into visceral endoderm and mesoderm (myocardium), but both of them only expressed a low level of hepatocyte-specific metabolic functions. With cholestatic serum added into the HGF-induced system, differentiated cells grew into similar angular cells, and had a higher level synthesis of glycogen, triglycerides, albumin and urea nitrogen with positive ICG and FDA staining.

CONCLUSIONSSpontaneous or HGF-induced ESC differentiation has only limited hepatic functions expressed. A pathologic niche in vitro induces ESC to develop into hepatic lineages, with a higher level of hepatic metabolic functions.

Animals ; Cell Differentiation ; physiology ; Cells, Cultured ; Cholestasis ; blood ; Culture Media ; pharmacology ; Embryo, Mammalian ; Hepatocytes ; cytology ; Mice ; Serum ; Stem Cells ; cytology

OBJECTIVETo determine the incidence and clinical significance of chromosome 13q14 deletion in multiple myeloma (MM).

METHODSBone marrow samples were collected from 132 newly diagnosed MM patients referred to our hospital. Interphase fluorescence in situ hybridization (i-FISH) combined with magnetic activated cell sorting (MACS) were performed on chromosome 13q14 (RB-1).

RESULTS(1) i-FISH was used to investigate CD138-enriched bone marrow MM cells and revealed a 13q14 deletion rate of 51.5% (68/132), while conventional cytogenetic (CC) analysis revealed 13q deletions/monosomy 13 (Δ13) only of 5.0%(6/120). (2) Univariate analysis showed that 13q14 deletion rate by i-FISH > 25%, bone marrow plasma cells > 50%, ISS stage and β(2)-MG ≥ 5.5 mg/L were associated with shorter overall survival (OS). Multivariate analysis revealed that 13q14 deletion rate by i-FISH > 25% was an independent unfavorable factor (P = 0.042). (3) Patients treated with bortezomib had a much better response than those treated with traditional chemotherapy (P = 0.001). There was no significant difference in OS between patients received bortezomib with and without 13q14 deletion (P > 0.05), indicating that bortezomib could reverse the poor prognosis of 13q14 deletion.

CONCLUSION(1) i-FISH followed CD138 cell sorting appears to be a highly sensitive method for detecting 13q14 deletion. (2) 13q14 deletion rate by i-FISH > 25% is an independent unfavorable factor. (3) Bortezomib could reverse the poor prognosis of 13q14 deletion.

Adult ; Aged ; Aged, 80 and over ; Antineoplastic Agents ; therapeutic use ; Boronic Acids ; therapeutic use ; Bortezomib ; Chromosome Deletion ; Chromosome Disorders ; Chromosomes, Human, Pair 13 ; Female ; Flow Cytometry ; Humans ; In Situ Hybridization, Fluorescence ; Male ; Middle Aged ; Multiple Myeloma ; diagnosis ; drug therapy ; genetics ; pathology ; Prognosis ; Pyrazines ; therapeutic use

OBJECTIVEThe aim of the study was to evaluate the anti-HBs persistence and the long term preventive efficacy after vaccination 23 years with plasma-derived hepatitis B vaccine.

METHODSThe study consisted of 261 children who were 5 - 9 years aged, from two primary schools in two townships of Xi'an. 126 children were randomly selected as vaccine group, and 135 children in control group. These children were followed up again in 2009. Excluding self-inoculation, the vaccine and control groups were 81 and 75, who was used to ask to recall details of their experience for vaccination and liver-related illnesses during past twelve years. Individuals who had anti-HBs titers less 10 mIU/ml, HBsAg, anti-HBc and HBV-DNA all were negative, were given a booster dose vaccine and retest for anti-HBs titer after one month.

RESULTSAfter eliminated the interference of an early booster dose and vaccination outside the study, the positive rate of anti-HBs was 48.1% (39/81) in the vaccine group at year 23, higher than 34.7% (26/75) in control group. At year 23 after primary vaccination, 84.0% (21/25) individuals in the vaccine group whose anti-HBs and anti-HBc both are negative showed a stronger anamnestic response after received a booster dose, while 7.5% (3/40) in the control group. At year 23 after primary vaccination, none clinical case of hepatitis B was found among 194 individuals. However, anti-HBc positive rate in the vaccine group was 16.0% (13/81), while the rate in the control group was 30.7% (23/75) (χ(2) = 4.687, P < 0.05).

CONCLUSIONAt 23 years after implemented a full course of plasma-derived hepatitis B vaccine, the recipients of vaccine were maintained anti-HBs at a high level or strong immunological memory.

Child ; Child, Preschool ; Follow-Up Studies ; Hepatitis B ; immunology ; prevention & control ; Hepatitis B Antibodies ; blood ; Hepatitis B Vaccines ; immunology ; Humans ; Immunization, Secondary ; Immunologic Memory ; immunology ; Plasma ; immunology

Human parechovirus type 3 (HPeV3) is an important pathogen of severe sepsis. HPeV3 is a non- enveloped, single-stranded, positive-sense RNA virus with a linear and continuous genomic RNA. The complete genome of a HPeV3 (BJ-C3174) strain was analyzed from the serum specimen from a child with sepsis hospitalized in Beijing, China, in 2012. The whole genome of BJ-C3174 was 7329 nucleotides (nt) in length excluding a poly (A) tail. One large open reading frame (ORF) of 6531 nt encoding a putative polyprotein precursor of 2177 amino acids (aa) was flanked by a 5' untranslated region (UTR) of 709 nt and 3' UTR of 91 nt. Phylogenetic analysis showed that BJ-C3174 belonged to HPeV3 and was closest to the HPeV3 strain BONN-2 from Germany. Compared with HPeV1-8 reference strains, BJ-C3174 shared the highest similarities with BONN-2 in full length and in each of the gene segments of the genome. The nucleotide and predicted amino acid identities of the whole genome between BJ-C3174 and BONN-2 were 99.3% and 99.8%, respectively, which were higher than those compared with HPeV3 prototype. Recom- bination of the gene segment with other HPeVs types was not identified.
Amino Acid Sequence ; Child ; Genome, Viral ; Humans ; Molecular Sequence Data ; Parechovirus ; classification ; genetics ; Phylogeny ; Sepsis ; blood ; virology

OBJECTIVETo investigate the association between natural-resistance-associated macrophage protein 1 (NRAMP1) gene polymorphisms and susceptibility to pulmonary tuberculosis (TB) in Han nationality from Northern part of China.

METHODSA 1:1 matched case-control study was adopted. Polymerase chain reaction and restriction fragment length polymorphism technique were used to type the two NRAMP1 polymorphisms: INT4 and 3'UTR. Information on environmental-related risk factors and pathological changes of tuberculosis was collected using a pre-tested standard questionnaire. Univariate and multivariate conditional logistic analyses were conducted using SPSS for window software.

RESULTSA sample consisting 124 pairs of cases and controls was studied. Univariate analysis demonstrated that the 3'UTR TGTG+/del genotype occurred more frequently in the cases than in the controls, with crude OR (95% CI) being 2.923 (1.557 - 5.487). No significant association was observed between TB and INT4 polymorphism. In multivariate analysis, associations of TB and 3'UTR TGTG+/del genotype remained, after adjusting for scar of bacillus Calmette-Guérin vaccine, marriage status, body mass index and exposure history. Adjusted OR (95% CI) was 2.955 (1.369 - 6.381). Again, no significant association between INT4 polymorphism and TB was found. Among different INT4 genotypes, the pathological characters of pulmonary tuberculosis were also found different (chi(2) = 9.634, P < 0.05).

CONCLUSIONPolymorphism of 3'UTR locus in NRAMP1 gene might affect their susceptibility to TB in Han nationality living in the northern part of China, and polymorphism of INT4 might affect the pathological characters of tuberculosis.

Analysis of Variance ; Asian Continental Ancestry Group ; genetics ; Case-Control Studies ; Cation Transport Proteins ; genetics ; China ; Gene Frequency ; Genetic Predisposition to Disease ; Genotype ; Humans ; Logistic Models ; Polymerase Chain Reaction ; Polymorphism, Genetic ; Polymorphism, Restriction Fragment Length ; Risk Factors ; Surveys and Questionnaires ; Tuberculosis, Pulmonary ; genetics

OBJECTIVETo investigate the effect of miRNA silencing HIF-1α gene on the proliferation of HepG2 cells.

METHODSThe eukaryotic expression plasmids of HIF-1α miRNA and report gene containing hypoxia-reponse element were constructed and transfected into HepG2 cells. The expressions of HIF-1α gene and protein were determined by real time-PCR and Western blotting. The expressions of HIF-1α, vascular endothelial growth factor (VEGF) and angiopoietin-2 (Ang-2) were quantitatively detected by ELISA. The alterations of cell cycles and apoptosis rate were quantitatively measured by flow cytometry and Annexin V-FITC/PI double dyeing assay.

RESULTS72 h after transfection the down regulations of HIF-1α mRNA and protein were 87% and 56% respectively, and the decrease of target gene was 46% in the report gene, 54% in VEGF and 36% in Ang-2, respectively. The apoptotic ratio of HepG2 cells was 22.46+/-0.61% (P < 0.01). The cell cycle changed greatly at the ratio of G1 (61.49+/-1.12%) and S (22.40+/-0.58%, P < 0.01). After being combined with doxorubicin, the apoptotic ratio increased to 36.99+/-0.88% and the ratios of G1 and S phases were upregulated to 65.68+/-0.91% and 19.47+/-1.34% respectively.

CONCLUSIONSHIF-1α miRNA or / and doxorubicin can regulate the growth cycles of HepG2 cells, promote the cell apoptosis and inhibit the cell proliferation.

Apoptosis ; Cell Cycle ; Cell Proliferation ; Gene Silencing ; Hep G2 Cells ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; MicroRNAs ; genetics ; RNA, Messenger ; genetics ; Transfection

OBJECTIVETo assess the prognostic value of biological features and therapy-related factors in multiple myeloma (MM).

METHODS123 patients with newly diagnosed MM between January 1998 and May 2005 were enrolled in this retrospective study. Biological features at presentation and therapy-related factors were analysed. The overall survival (OS) and time to progression (TTP) were estimated by Kaplan-Meier analysis and the distribution of OS and TTP were compared using log-rank test. Cox regression was used to identify the independent prognostic factors.

RESULTS(1) The univariate analysis indicated that more immature plasma cells in bone marrow biopsy, C-reactive protein >8. Omg/L, CD117 expression, serum beta2-microglobulin (beta2-MG) (3.5 approximately 5.5 mg/L), abnormal cytogenetics aberration of chromosome 13 (Delta13), hypodiploid, poor response to chemotherapy, interferon(IFN) therapy less than 6 months were associated with shorter OS(P <0.05). Lytic bone lesions at presentation, more immature plasma cells in bone marrow biopsy, serum beta2-MG (3.5 approximately 5.5 mg/L), poor response to chemotherapy, and IFN therapy less than 6 months as well as abnormal cytogenetics, hypodiploid and Delta13 were associated with shorter TTP (P <0.05). (2) Multivariable COX analysis indicated IFN therapy more than 6 months was a protective factor for OS and TTP, and more immature plasma cells in bone marrow biopsy was an independent poor prognostic factor for TTP.

CONCLUSIONThe morphology of myeloma cells is useful for assessing the prognosis. And IFN therapy more than 6 months could lengthen OS and TTP.

Adult ; Aged ; Female ; Follow-Up Studies ; Humans ; Male ; Middle Aged ; Multiple Myeloma ; diagnosis ; pathology ; therapy ; Prognosis ; Retrospective Studies ; Risk Factors ; Survival Analysis