Objective To elucidate the prevalence and clinical characteristics of human metapneumovirus(hMPV)in hospital- ized children with respiratory infection.Methods A total of 452 hospitalized children with lower respiratory tract infection were observed from Aug 2004 to Jan 2005.Respiratory tract aspirates were collected from all patients within 48 hours after admis sion.The specimens were routinely tested for respiratory syncytial virus,influenza virus A and B,parainfluenza virus 1 to 3 and adenovirus by direct fluorescent assay(DFA).The 245 specimens negative by DFA were tested for hMPV by RT-PCR. PCR products of hMPV M gene from some patients were randomly selected for sequencing analysis.Results hMPV was identi- fied in 59(24.1%)of the 245 specimens tested,hMPV infection alone accounted for 13.1% of the infections in the 452 chil- dren under study,The prevalence of hMPV was higher than other respiratory viruses in winter.The mean age of hMPV-infec- ted children(n=59)was 27.7 months.There was no significant difference between age groups in terms of the prevalence of hMPV(P＞0.05).There were no statistically significant difference in demographics and clinical symptoms between hMPV in- fection and other common respiratory virus infection.Genotyping for the hMPV M gene from 23 Shanghai patients showed two distinct hMPV genotypes.Sequence analysis of these hMPV M genes showed 82.8%-100% homology to the registered se- quence in GenBank.There was no significant difference in clinical characteristics between the 2 genotypes.Conclusions hMPV plays an important pathogenic role in lower respiratory tract infection of children,hMPV prevailed in the winter of 2004.Clini- cally,hMPV infection can not be discriminated from the infection of other respiratory viruses.Clinical manifestation is similar between the two hMPV genotypes.

OBJECTIVE: To study the forensic application of Goldeneye DNA ID 26Y Kit in the She nationality. METHODS: Through capillary electrophoresis, the genotype of 26 Y-STR loci were analyzed in 53 unrelated male individuals from Fujian She nationality. The population genetics parameters such as allele frequency and haplotype diversity were calculated. The comparisons among the She nationality and the other nationalities were analyzed. RESULTS: A total of 126 alleles were observed on the 26 Y-STR loci of 53 unrelated male individuals. The allele frequencies and GD value ranged from 0.010 1 to 0.886 8 and 0.211 2 to 0.846 2, respectively. The GD value was greater than 0.5 in the 19 loci. A total of 47 haplotypes were observed. Based on R(ST), multidimensional scaling plot indicated that the genetic relationship among Fujian She nationality and Minnan Han nationality was closest, followed by Southern China Han nationality and Northern China nationality. CONCLUSION Goldeneye™ DNA ID 26Y Kit including 26 Y-STR loci has good polymorphism in the She nationality. As an additional system, it has forensic application value in some special cases.
Asian People/genetics* ; China ; Chromosomes, Human, Y/genetics* ; Ethnicity/genetics* ; Forensic Genetics ; Genetic Markers ; Genetics, Population ; Humans ; Polymerase Chain Reaction ; Polymorphism, Genetic ; Population Groups

OBJECTIVETo establish the triple-transgenic mouse model and study their biological characteristics by molecular biology, behavior and pathology.

METHODSHybrid the Tau and amyloid precursor protein (APP)/presenilins (PS1) transgenic mouse, the genotype of offspring mice were identified by PCR. Transcribed target genes were detected by RT-PCR. The protein expression of exogenous genes was detected by Western-blot. The pathological change of neurofibrillary tangles and senile plaque were observed by Bielschowsky silver staining and ABC immunohistochemical method. The changes time of learning and memory were observed by Morris water maze.

RESULTSAPP, PS1 and Tau genes were transcript in Tau/APP/PS1 mice. In 6 to 8 months old Tau/APP/PS1 mice, the neurofibrillary tangles and senile plaque could be found in cortex and hippocampus. In 6 months old Tau/APP/PS1 mice, the learning and memory abilities were worse.

CONCLUSIONWith the behavior change and pathological changes in Tau and beta-amyloid protein (AP), the Tau/APP/PS1 triple-transgenic mice can be used as a further study animal model of AD's pathogenesis and the target of drug treatment.

Alzheimer Disease ; pathology ; Amyloid beta-Protein Precursor ; genetics ; Animals ; Brain ; pathology ; Disease Models, Animal ; Learning ; Male ; Memory ; Mice ; Mice, Transgenic ; Neurofibrillary Tangles ; pathology ; Plaque, Amyloid ; pathology ; Presenilin-1 ; genetics ; tau Proteins ; genetics

The aim of this study was to characterize the N and E protein encoding genes of a new human coronavirus (HCoV-NL63) which was identified from one of the clinical specimens (BJ8081) collected from a 12 years-old patient with acute respiratory infection in Beijing. The complete N and E gene sequences of HCoV-NL63 were amplified from clinical sample by RT-PCR, then were cloned into the pCF-T and pUCm-T vectors respectively and sequenced. The complete sequences of N and E genes were submitted to GenBank by Sequin and compared with N and E genes of prototype HCoV-NL63 and the other coronaviruses published in GenBank. The secondary structure and the characteristics of sample BJ8081 N and E proteins were predicted by bioinformatics. It was indicated that the N and E genes amplified from sample BJ8081 were 1134 bp and 234 bp in length and the predicted proteins including 377 amino acids and 77 amino acids, respectively. The data suggested that the region of amino acids 78-85 within N protein probably was the conserved region for all coronaviruses identified so far including HCoV-NL63. The region of amino acids 15-37 for E protein was probably the transmembrane domain. In conclusion, the recombinant plasmids pCF-T-8081 N and pUCm-T-8081 E were successfully constructed and sequenced, and the data predicted by bioinformatics are helpful for the further analysis of HCoV-NL63.
Amino Acid Sequence ; Child ; China ; Computational Biology ; methods ; Coronavirus ; classification ; genetics ; metabolism ; Coronavirus Infections ; virology ; Humans ; Molecular Sequence Data ; Nucleocapsid Proteins ; chemistry ; genetics ; metabolism ; Phylogeny ; Protein Structure, Secondary ; Sequence Analysis, DNA ; Sequence Homology, Amino Acid ; Viral Envelope Proteins ; chemistry ; genetics ; metabolism

OBJECTIVEAn outbreak of acute respiratory infections in children occurred in Beijing from November to December, 2002. To investigate the etiological agents of affected children who were in day care centers and primary schools.

METHODSThroat swab specimens were collected from one primary school children with acute respiratory infections visiting one outpatient department. After centrifuging, supernatant from the specimens were inoculated into MDCK and Hep-2 cells for virus isolation and pallets for viral antigen detection and using indirect immunofluorescent assay on common respiratory viruses. Nested polymerase chain reaction was used at the same time for detection of respiratory viruses and Mycoplasma pneumoniae (Mp).

RESULTSA total number of 80 specimens were collected during the outbreak. Among them influenza B virus were detected from 18 specimens, with a positive rate of 22.5% (18/80) while Mp were detected from 13 specimens, with a positive rate of 16.3% (13/80). Influenza A3 were also detected from 2 patients (2.5%, 2/80). However, influenza A1, RSV, adenovirus and parainfluenza viruses were not found from these specimens. Influenza B virus and Mp were detected simultaneity in two specimens and influenza A3 virus and Mp were detected in one specimen.

CONCLUSIONThe outbreak of acute respiratory infection in children during the period of investigation was caused by both influenza B virus and Mp.

Adolescent ; Child ; Child, Preschool ; China ; epidemiology ; Disease Outbreaks ; Female ; Humans ; Infant ; Influenza B virus ; isolation & purification ; Influenza, Human ; epidemiology ; Male ; Mycoplasma Infections ; epidemiology ; Mycoplasma pneumoniae ; isolation & purification ; Respiratory Tract Infections ; epidemiology ; microbiology

OBJECTIVESTo develop techniques which can be used to detect genetic material of measles virus from clinical samples to make diagnosis and differentiate atypical measles from other exanthematous infections early in clinical course.

METHODSA nested reverse transcriptase-polymerase chain reaction (RT-PCR) was developed to amplify gene fragment with the size of 301 bps from N gene of measles virus in throat swabs and urine samples collected from infants and children who were suspected measles cases. Before the test was used for clinical samples, preliminary tests were performed to determine the sensitivity and specificity of the test. The sensitivity of the test was determined by plaque assay using measles virus strain Edmonton and the specificity of the test was determined by cross-reaction with rubella virus, respiratory syncytial virus, influenza A and B viruses, enterovirus, adenovirus, human cytomegalovirus (hCMV), EB virus, and herpes simplex virus I. Serum specific IgM antibody against measles virus was also tested by ELISA.

RESULTSMeasles virus with the titer of 0.53 pfu could be detected by using the nested RT-PCR developed in this study. No amplification was found with the nested RT-PCR when rubella virus, respiratory syncytial virus, influenza A and B virus, enterovirus, adenovirus, hCMV, EB virus, and herpes simplex virus I were used as templates. Out of 116 throat swabs collected from suspected measles cases, 70 (60.3%) were measles RNA positive. For urine samples, 48 out of 74 (64.9%) were positive. Both throat swab and urine samples were collected simultaneously from 73 patients. Among those, 71 (97.3%) showed consistent results. Serum specimens were collected from 110 suspected patients. Among those, 65 (59.1%) and 61 (55.5%) were measles virus specific IgM antibody positive detected with ELISA kits from two different sources, respectively. Out of 110 sera samples, 106 (96.4%) showed consistent results. The consistency of the gene amplification and specific IgM antibody detection was 80.8% as shown by 84 out of 104 patients from whom throat swab and sera were collected at the same time.

CONCLUSIONThe data indicate that the nested RT-PCR developed in this study is sensitive and specific for detection of gene fragment of measles virus from clinical samples. The test is superior to the commonly used specific IgM antibody detection because of identifying gene material in early clinical stage, and even single clinical sample can be tested.

Humans ; Measles ; diagnosis ; genetics ; Measles virus ; genetics ; isolation & purification ; RNA, Viral ; genetics ; isolation & purification ; Reverse Transcriptase Polymerase Chain Reaction ; Sensitivity and Specificity

OBJECTIVETo establish a rapid, specific and effective technique for identifying subtyping A(1), A(3) and B of influenza virus isolates and clinical specimens as well as to analyze the sequences of nucleotides and deduced amino acids of HA1 regions from isolates of influenza virus A(3) isolated from 1996 to 2002.

METHODSSix inner and outer sets of oligonucleotide primers were designed to detect, type and subtype human influenza A and B. The first two corresponding sets differentiate type A and B of matrix (M) gene while, the second two corresponding sets identify the H(1) and H(3) subtypes of type A virus HA gene. To type and subtype influenza viruses in clinical isolates, a mixture of inner primer sets specific for H(1), H(3) and B were used in a single PCR reaction tube. To detect influenza viruses in clinical specimens, a mixture of the outer primer sets were used in a single primary PCR tube, and the inner ones in a single second PCR reaction tube. Amplified products were visualized in 1.2% agrose gel containing ethidium bromide. HA1 regions of hemagglutinin of 5 field strains (H3N2) isolated from 1996 to 2002 in Beijing were amplified by RT-PCR and sequenced directly.

RESULTSThere was 100% correlation between multiplex RT-PCR and culture to type and subtype influenza viruses from clinical isolates. For typing and subtyping, 76.9%, 57.1% and 86.5% were positive for A(1), A(3) and B by multiplex nested-PCR compared within virus isolation on culture, respectively. The sequence data of HA1 of A(3) strains showed that there was a high homology of nucleotide and amino acid, and the closer the date of isolating was, the higher homology showed.

CONCLUSIONSMultiplex RT-PCR and nested-PCR for influenza viruses could provide a useful alternative to existing methods of influenza detected and identified from clinical isolate and specimens. There were certain, continuous mutations and increasing glycosylated sites which might cause the antigen drift in the A(3) strains during 1996-2002 in Beijing area.

Amino Acid Sequence ; Child ; China ; epidemiology ; Hemagglutinin Glycoproteins, Influenza Virus ; genetics ; Humans ; Influenza A virus ; classification ; genetics ; isolation & purification ; Influenza B virus ; classification ; genetics ; isolation & purification ; Influenza, Human ; epidemiology ; virology ; Molecular Sequence Data ; Point Mutation ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Sensitivity and Specificity

OBJECTIVETo 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.

METHODSSerum 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 visited the Xuanwu Hospital, Beijing for diseases other than respiratory infections from April 1996 to March 1997 were used for 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 blot 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.

RESULTSOut of 677 serum specimens tested, 400 (59.1%) were positive by Western blot. About 45.3% (34/75) of the newborns under 1 month of age had anti-HBoV antibodies, and antibody positive rates were lower in the age groups of 1 and 2 months (41.4% and 31.3%, respectively) and were higher in the following ages from 6 months to 7 years (from 45.6% to 69.7%). The antibody positive rates were at a relatively constant level (about 70%) in the age groups from 7 years to 40 years and became lower (61.8% - 62.8%) in groups of age over 50 years.

CONCLUSIONThe high seroprevalence against recombinant HBoV VP2 protein and early age antibody acquisition indicate that HBoV has been circulating in 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 infection with this virus.

Adolescent ; Adult ; Antibodies, Viral ; blood ; Blotting, Western ; Bocavirus ; immunology ; Capsid Proteins ; immunology ; Child ; Child, Preschool ; China ; epidemiology ; Female ; Humans ; Immunoglobulin G ; blood ; Infant ; Infant, Newborn ; Male ; Middle Aged ; Parvoviridae Infections ; epidemiology ; immunology ; Prevalence ; Seroepidemiologic Studies ; Young Adult

OBJECTIVETo characterize the prevalence of influenza B virus infection in infants and young children in Beijing.

METHODSMDCK cell culture, indirect fluorescence assay (IFA) and hemagglutination inhibition (HI) assay were used to isolate and identify type B influenza viruses from clinical samples collected from outpatients and inpatients who visited the Affiliated Children's Hospital because of acute respiratory infections from Nov. 2000 to Jun. 2006.

RESULTSOut of 10,770 clinical samples collected during this surveillance period, 384 (3.57%, 384/10,770) were positive for influenza B viruses. Circulation of influenza B viruses was revealed in the later epidemic season of influenza viruses each year. The detection rate for influenza B virus was higher than 10% each year during the survey, except in the period from 2003--2004 which was 2.91%. The highest detecting rate was 23.69% of the specimens collected in Mar. 2006. During the period of this study, most of the influenza B virus were identified from children who visited the outpatient department of the Affiliated Children's Hospital. Among those outpatients who were positive for influenza B, 77.6% (264/340) were older than 3 years of age, whereas the inpatients positive for influenza B, 66.0% (29/44) were under 3 years of age. Coinfection of influenza B virus with other respiratory viruses was not common, only one of the influenza B virus positive specimen was found also positive for influenza A3. There was no significant difference in positive rate between influenza virus B and A3. A significantly higher positive rate of influenza B virus than that of influenza A3 virus was seen from Sep. 2005 to May 2006 (23.9% vs 1.1%). B/Yamagata/16/168 lineage viruses were dominant during 2000--2002, and B/Victoria/2/87 lineage viruses became dominant during 2002--2003. After 2003, co-circulation of Victoria and Yamagata lineages of influenza B viruses was identified with predominance of Yamagata lineage viruses, while Victoria lineage viruses predominated during the 2005--2006 epidemic season.

CONCLUSIONInfluenza B viruses were identified from February to May in every influenza season during this surveillance period of 2000--2006. Most of the positive specimens were those collected from outpatient department. Victoria and Yamagata lineages of influenza B viruses co-circulated in Beijing, China in recent years.

Adolescent ; Age Distribution ; Child ; Child, Preschool ; China ; epidemiology ; Female ; Humans ; Infant ; Influenza B virus ; classification ; isolation & purification ; Influenza, Human ; epidemiology ; virology ; Male ; Prevalence

To understand the effectiveness of prokaryotic expression of fusion protein (F) of human metapneumovirus (hMPV) and its application as antigen, F proteins from different genotypes of hMPV were expressed in prokaryotic expression system and purified by Ni-NTA affinity chromatography column. According to the hydrophobicity, antigen index and surface probability of F protein, the subunit 1 (F1) region of F protein was generated and expressed in E. Coil. BL21(DE3). The 6-His-F1 proteins with molecular weight of approximately 37 kD generated from hMPV of two genotypes were expressed efficiently mainly in inclusion body. The antigenicity and specificity of the expressed proteins were tested and confirmed by Western Blot using polyclonal antibody against hMPV and one serum specimen from a patient with confirmed hMPV acute infection,and polyclonal antibodies against human respiratory syncytial virus and parainfluenza virus 2 and 3. The results of preliminary use of the expressed proteins for detecting antibodies against hMPV in 457 serum specimens collected from different age groups in Beijing indicated that 66%-67% of sera in all age groups were positive. The positive rate of antibodies declined in children in age groups from birth to 2-year-old and then rose along with the increase in age, in which the lowest was in age group from 1 to 2-year-old and the highest in newborn and people older than 60 years. The data indicated the existence of maternal transferred antibodies against hMPV in infants and the risk of hMPV infections in children younger than 2 years old.
Adult ; Antibodies, Viral ; immunology ; Antigens, Viral ; genetics ; immunology ; Escherichia coli ; genetics ; Gene Expression ; Humans ; Immunoglobulin G ; blood ; immunology ; Infant ; Infant, Newborn ; Metapneumovirus ; genetics ; Middle Aged ; Plasmids ; genetics ; Protein Engineering ; Protein Subunits ; genetics ; immunology ; Viral Fusion Proteins ; genetics ; immunology