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

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

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

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

OBJECTIVETo determine whether the GABA-containing neurons in rat central nucleus of amygdala (CeA) can be activated by acute sodium deprivation.

METHODSAcute sodium depletion was induced by subcutaneous injection of furosemide in rats followed by 24 h of dietary sodium deprivation. The rats underwent 0.3 mol/L NaCl/distilled water two bottle choice test, and the activated neurons were labeled and identified with GABA/Fos-double labeling immunohistochemistry.

RESULTSThe rats with acute sodium depletion exhibited significantly more numerous c-fos-positive neurons and GABA/Fos double-labeled neurons in the CeA than the control group (P<0.01, P<0.05). Consumption of 0.3 mol/L NaCl significantly increased the number of c-fos and GABA/Fos double labeled neurons compared to the distilled water group (P<0.001, P<0.01).

CONCLUSIONGABAergic neurons in the CeA may play an inhibitory role in the regulation of sodium intake in rats with acute sodium depletion.

Amygdala ; cytology ; metabolism ; Animals ; GABAergic Neurons ; metabolism ; Male ; Rats ; Rats, Sprague-Dawley ; Sodium Chloride, Dietary ; metabolism ; Sodium, Dietary

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

BACKGROUNDHuman bocavirus (HBoV) is a newly identified human parvovirus that was originally detected in the respiratory secretions of children with respiratory infections. This study aimed to learn about the importance of HBoV infections by revealing the prevalence of serum antibodies against HBoV in Beijing population.

METHODSTwo batches of serum specimens collected in different periods were tested by Western blotting for specific IgG against HBoV using recombinant VP2 as antigen.

RESULTSOut of 677 serum specimens collected during April 1996 to March 1997, 400 (59.1%) were positive and antibody positive rate for another batch of 141 serum specimens collected in August, 2005 from adults aged from 20 years to over 60 years was 78.7% (111/141). Comparison of the sero-prevalence profiles for serum specimens collected during 1996 - 1997 to those collected in 2005 indicated that the antibody positive rate for specimens collected in 2005 was higher than that of the corresponding age groups collected during 1996 - 1997.

CONCLUSIONSThe data suggest that HBoV has been circulating in Beijing population for at least over 10 years, and most of children had been exposed to HBoV by age of 7 years. Higher HBoV antibody positive rate shown in the serum specimens collected in 2005 suggested that infections by HBoV have been increased in Beijing population in recent years.

Adult ; Antibodies, Viral ; blood ; Blotting, Western ; Bocavirus ; pathogenicity ; China ; epidemiology ; Humans ; Immunoglobulin G ; immunology ; Middle Aged ; Parvoviridae Infections ; blood ; epidemiology ; immunology ; Seroepidemiologic Studies ; Viral Proteins ; immunology ; Young Adult

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

OBJECTIVETo obtain isolated human metapneumovirus (HMPV) strains from clinical specimens collected from infants and children in Beijing and to promote the investigation on this important respiratory pathogen.

METHODClinical specimens including throat swabs from outpatients and nasopharyngeal aspirates from hospitalized children were collected from infants and children visited the affiliated children's hospital for acute respiratory infections during May 2008 to April 2009. HMPV positive specimens identified by RT-PCR and/or direct immunofluorescent assay with monoclonal antibody against HMPV were inoculated to LLC-MK(2) cells and incubated at 37°C and 33°C, respectively. The replication of the virus in the cells was detected by direct immunofluorescent assay followed by RT-PCR. The genotypes of the isolated virus strains were identified by RT-PCR.

RESULTOut of 1092 clinical specimens, 81 were HMPV positive by RT-PCR, the positive rate was 7.4% (81/1092). Among these positive specimens, 33 were inoculated to LLC-MK(2) cells and the replication of HMPV was revealed by antigen detection and RT-PCR from 5 out of these 33 inoculates. These isolated viruses could be passed in LLC-MK(2) cells and were not cross-reacted with other common respiratory viruses, such as ADV, RSV and Parainfluenza viruses 1/2/3 by monoclonal antibodies against these viruses in direct immunofluorescent assay. The HMPV was more likely to be isolated from fresh specimens within 24 hours after the collection of specimens which were not frozen. Four of the 5 isolated strains were identified as genotype A and 1 as genotype B. Unlike other respiratory viruses, these isolated HMPV did not show specific CPE in cell culture and the replication of the virus was identified by antigen detection and RT-PCR.

CONCLUSIONHMPV of both genotypes were isolated from infants and children with acute respiratory infections in Beijing which will accelerate the investigation of this important virus.

Acute Disease ; Child ; China ; Genes, Viral ; genetics ; Genotype ; Humans ; Infant ; Metapneumovirus ; isolation & purification ; Respiratory Tract Infections ; virology