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

OBJECTIVETo characterize the HA1 regions of hemagglutinin gene of influenza viruses (H3N2) isolated from children in Beijing from 1998 - 2004.

METHODSThe HA1 regions of hemagglutinin gene were amplified by RT-PCR from the viruses isolated and identified as A3 (H3N2) from clinical samples collected from infants and children during the peak seasons of influenza between 1998 and 2004. PCR products were sequenced or cloned into T-A vector and were analyzed after being sequenced.

RESULTSThe HA1 regions of hemagglutinin genes amplified from those isolates were 987 bp in length, encoding a protein of 329 amino acids in length. The identities of nucleotides and amino acids among these H3N2 isolates in Beijing and vaccines strains from 1998 - 2004 were 95.5% - 100.0% and 93.0% - 100.0%, respectively. The homology of the HA1 regions were related to the date of virus isolation, meaning the homology was higher among those strains isolated in nearer dates than others. Seven potential N-linked glycosylation sites in the HA1 regions located at amino acid positions 8, 22, 38, 63, 126, 165 and 285 were conserved in all the viruses analyzed. Two sites at 122 and 133 were inserted in those virus isolated after 1997, and another site at 144 appeared in those isolated after 1999. More amino acid substitutions located in the five putative antigenic sites or receptor binding sites were found more in the isolates than the isolates from previous year. Phylogenetic analysis showed new branches appeared continuously during 1998 - 2004. The strains isolated during winter in 2004 belonged to different branches, suggesting the appearance of new variants.

CONCLUSIONAmino acid substitutions continuously occurred in the HA1 regions of hemagglutinin genes in influenza virus (H3N2) isolated from children in Beijing from 1998 - 2004, which might have resulted in antigenic drift and led to the appearance of new variants.

Amino Acid Substitution ; China ; DNA, Viral ; analysis ; Gene Amplification ; Hemagglutinins ; genetics ; Humans ; Influenza A Virus, H3N2 Subtype ; genetics ; Influenza, Human ; virology ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Analysis, DNA

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

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 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

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

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