Objective To investigate the sequences of the 3' end of genomes from enterovirus 71 isolated from pediatric patients with different symptoms in Beijing during 2008-2009.Methods Clinical specimens were collected from pediatric patients suffering from hand,foot and mouth disease(HFMD)and/or with neurological complications who visited the affiliated Children's Hospital during the epidemic seasons of 2008 and 2009 in Beijing.The samples were inoculated into the Vero cell line,and the virus isolates were further identified by nested reverse transcription-nested PCR(RT-nPCR)assay using universal enterovirus primers,type specific primers for EV71 and CA16.The 3' end of genomes(including 3D and 3' UTR regions)from 10 EV71 strains derived from various clinical presentations were amplified and sequenced.Results Analysis of the nucleotide sequences of the amplified fragments showed that the 3' end of genomes of 10 EV71 isolates include the 3D region of 1386 nucleotides(nt)encoding the 3D polymerase(3Dpol,462 amino acids),the terminator codon TGA and 3' UTR of 81 nt.Nucleotide sequence identities among 3D regions from these 10 EV71 isolates were in the range of 95.8%-99.6%,while the nucleotide sequence identities for 3' UTR were 96.3%-100%.The majority of nucleotide changes were located at the third codon positions which caused silent mutations,thus the amino acid sequence changes of 3Dpol among those 10 EV71isolates were scanty.The residues 140 and 263 which were R and 1 were substituted by K and V,respectively in 3 of 4 neurovirulent strains,whereas only 1 of 6 strains from mild cases had these 2 amino acid changes.The sequences of the 3D and 3' UTR regions of 10 EV71 isolates were compared to the representative strains of known genotypes from the GenBank.The nucleotide and amino acid sequences of 10 EV71 isolates in the 3D region exhibited highest homology to the subgenotype C4 of EV71(92.7%-94.2%and 96.8%-97.6%.respectively).However,3' UTR of 10 EV71 isolates shared the highest nucleotide identity with CA16/G10(88.9%-91.4%).The phylogenetic analysis based on the 3D regions demonstrated that 10 EV71 isolates had the closest genetic relationship with the representative isolate of CA sub-genotype of EV71 and shared more homology with CA16/G10 than other known genotypes of EV71.Conclusion Genetic analysis of the 3' end of genomes from 10 EV71 strains indicated that the 3' end of genome may play a role in the evolution of EV71.

Objective To develop a convenient reverse transcription PCR(RT-PCR)method for identifying genotypes of human metapneumovirus(hMPV)from clinical samples.Methods According to the gene sequences of hMPV G with different genotypes,the A and B genotype specific primers were designed.A diplex RT-PCR was applied to identify different genotypes according to the molecular weight of PCR products in agarose gel.37 clinical samples were detected through this method.Results It was convenient to distinguish different genotypes of hMPV(383 bp for A and 284 bp for B)by the diplex RTPCR,and there was no non-specific amplification for common respiratory viruses.so it meant that the specificity of primers was good.The results of genotyping 37 clinical samples showed that 20 samples were identified as genotype A by both sequence analysis of M gene and diplex RT-PCR,whereas 17 samples were identified as genotype B by sequence analysis of M gene.but in these 17 samples 14 samples were identified as genotype B by the diplex RT-PCR and remaining 3 samples could not be genotyped because there was no PCR product after amplification.The consistency rate for these two methods Was 91.9%[(20+14)/37].Conclusion The method of diplex RT-PCR Was developed successfully and can be used for identify genotypes of hMPV.

Background Acute respiratory infection (ARI) is one of the most common infectious diseases in infants and young children globally.This study aimed to determine the virus profile in children with ARI presenting with different severities.Methods Clinical specimens collected from children with ARI in Beijing from September 2010 to March 2011 were investigated for 18 respiratory viruses using an xTAG Respiratory Viral Panel Fast (RVP Fast) assay.The Pearson chisquare analysis was used to identify statistical significance.Results Of 270 cases from three groups of ARI patients,including Out-patients,In-patients and patients in the intensive care unit (ICU),viruses were detected in 176 (65.2％) specimens with the RVP Fast assay.The viral detection rate from the Out-patients group (50.0％) was significantly lower than that from the In-patients (71.1％) and ICU-patients (74.4％) groups.The virus distribution was different between the Out-patients group and the other hospitalized groups,while the virus detection rate and distribution characteristics were similar between the In-patients and ICU-patients groups.The coinfection rates of the Out-patients group,the In-patients group,and the ICU-patients group were 15.6％,50.0％ and 35.8％,respectively.In addition to respiratory syncytial virus (RSV) and adenovirus (ADV),human rhinovirus (HRV) was frequently detected from children with serious illnesses,followed by human metapneumovirus (hMPV),human bocavirus (HBoV) and coronaviruses.Parainfluenza virus 3 (PIV3) was detected in children with lower respiratory illness,but rarely from those with serious illnesses in the ICU-patient group.Conclusion In addition to so-called common respiratory viruses,virus detection in children with ARI should include those thoucht to be uncommon respiratory viruses,especially when there are severe ARI-related clinical illnesses.

OBJECTIVETo analyze the genotype, epidemic pattern and the characteristics of the disease of enteroviruses during the epidemic season of hand, foot and mouth disease (HMFD) in children from 2013 to 2014 in Beijing to provide the scientific evidence for prevention and treatment of HFMD.

METHODDuring April to September in 2013 and March to October in 2014, a total of 977 throat swabs were collected from children who visited the Children's Hospital Affiliated to Capital Institute of Pediatrics, including 147 from patients with HFMD in 2013, 343 with HFMD, 201 with atypical HFMD, 83 with herpangina, 25 with fever with convulsions, 64 fever with rash and 114 with rash in 2014. Enteroviruses universal type (EV), Enteroviruses type 71 (EV71) and Coxsackievirus group A 16 (CA16) were detected by real-time RT-PCR respectively. The nucleic acid of specimens which were identified with non-EV71, non-CA16 was tested by nested PCR and analyzed by VP1 sequencing. The detection rate and epidemic pattern of different genotypes of enterovirus were analyzed among different age groups and between 2013 and 2014.

RESULTOf 977 throat swabs, 80. 1% samples were detected positive for enteroviruses. The positive rates of CA16, EV71, CA6, CA10, CA4 and other EVs were 25. 6% (250/977), 18. 9% (185/977), 20. 0% (195/977), 5. 0% (49/977), 1.5% (15/977) and 9.1% (89/977), respectively. Twenty six of the 89 other EVs included CA2, CA5, CA8, CA9, CA12, CA14, CB2, CB5, E6, E9 and E25, each genotype of which was no more than 3. The nucleotide homologies shared among CA6, CA10 and CA4 strains between 2013 and 2014 were 94. 3% - 100%, 93. 8% - 99. 1% and 92.7% - 99. 8%, respectively. The positive rates of ≤1 year group were 71. 1% (106/149), which was lower than that of other age groups (all P <0. 05), but similar to that of >5 year group (χ2 =1. 181,P = 0. 277). In 2013, the positive rate of EV was 85. 7% (126/147) and the predominant genotype was CA6 54. 8% (69/126), followed by CA16 20. 6% (26/126) and EV71 11. 9% (15/126). In 2014, the positive rate of EV was 85. 4% (293/343) in the 343 children with HFMD, the predominant genotypes were CA16 with the positive rate of 42. 7% (125/293), EV71 with 38. 2% (112/293) and CA6 with only 11. 3% (33/293). In 2014, the positive rates of EV in 201 atypical HFMD, 83 herpangina, 25 fever with convulsions, 64 fever with rash and 114 rash were 83. 6% (168/201), 80. 7% (67/83), 76. 0% (19/25), 64. 1% (41/64) and 60. 5% (69/114), respectively. All genotypes of enteroviruses peaked mainly during May to August every year, but there were no obvious epidemiological pattern about each genotype.

CONCLUSIONCA6 became the main causative agent of HFMD in 2013, however, CA16 and EV71 predominated again in 2014 in Beijing. The clinical manifestations caused by CA6, CA10, CA4 and other genotype of enteroviruses differed from EV71 and CA16. Besides EV71 and CA16, more attention should be paid to CA6, CA10, CA4 and other type of enteroviruses.

Beijing ; epidemiology ; Child, Preschool ; Enterovirus A, Human ; classification ; Enterovirus Infections ; epidemiology ; virology ; Exanthema ; Fever ; Genotype ; Hand, Foot and Mouth Disease ; epidemiology ; virology ; Humans ; Infant ; Real-Time Polymerase Chain Reaction

Objective To investigate the etiological agents of hand, foot and mouth disease (HFMD) in children in spring and summer from 2007 to 2008 in Beijing and the characteristics of the disease by virus isolation and to provide the scientific evidence for prevention and treatment for HFMD. Methods During April to August, 2007 and May to September, 2008, 356 clinical specimens including 255 throat swabs and 101 vesicle fluids were collected from 256 patients with HFMD who visited the Children's Hospital Affiliated to Capital Institute of Pediatrics and children with severe HFMD with neural system complications from Ditan Hospital and Youan Hospital All of the specimens were inoculated into Vero cells for virus isolation. After the cell pathogenic effects (CPE) appeared, the isolates were identified by RT-PCR with the universal primers within 5'untranslated region of enterovirus and typed by specific primers for VP1 gene of EV71 and CA16, respectively. The throat swabs from all of 10 severe HFMD were tested for enterovirus by RT-PCR addition to virus isolation. Results Out of 256 patients, 188 were positive for enterovirus by virus isolation, with the overall positive rate of 73.4%. Among the 356 clinical specimens collected from these 256 patients, 239 enterovirus strains were isolated with the overall positive rate of 67.1%. The positive rate for virus isolation from vesicle fluid samples was 75.2% which was higher than the positive rate of isolation from throat swabs (63.9%), but the time for CPE appearing in cell culture showed no significant difference. The positive rate of virus isolation from throat swabs from children with severe HFMD was 50% (5/10) which was lower than overall positive rate (73.4%) from regular HFMD. The RT-PCR typing for virus isolates revealed that among 45 enterevirus strains isolated from the specimens collected in 2007 by the universal primer pairs, 43 were CAI6 (95.6%, 43/45) and 2 were EV71 (4.4%, 2/45), whereas for the specimens collected in 2008, out of 143 enterovirus isolates by PCR with universal primers, 117 were EV71 (82.4%, 117/142) and 24 were CA16 (16.8%, 24/142). All of 10 severe cases were positive for EV71 by RT-PCR directly from clinical specimens. Conclusion CA16 and EVT1 were the etiological pathogens of HFMD in Beijing during 2007 to 2008 HFMD seasons. The dominant type of enterovirus was different between 2007 and 2008. Enterovirus type CA16 was predominant in 2007, whereas EV71 was predominant in 2008. All of severe cases of HFMD in children in this study were caused by EV71.

Objective:Based on gene expression omnibus(GEO),differential expressed genes,gene set enrichment analysis(GSEA)and immune cell infiltration analysis were performed on microarray data of chronic spontaneous urticaria(CSU)expression profile,to gain more insight into the pathogenesis of CSU.Methods:The GSE72541 raw data were obtained from the GEO.Differential expressed genes were screened using R software.String database were used to construct the the protein-protein interaction(PPI)net-work.Gene ontology(GO)and Kyoto encyclopedia of gene and genomes(KEGG)enrichment analysis were performed using GSEA software.The ssGSEA method was used to analyze the infiltration of immune cells in the expression profile.Results:Genes closely related to platelet activation and its function were up-regulated in CSU serum,while genes related to Th1 cell chemotaxis were down-regulated in CSU serum.Biological processes and signal pathways related to coagulation cascade reaction,regulation of vascular per-meability,immune and inflammatory reactions,and mood-modulating were up-regulated in CSU group.Immunized cell infiltration analysis showed that activated B cells,immature B cells,follicular helper T cells,and Th2 cells were down-regulated in the CSU serum.Conclusion:Platelet activation,coagulation cascade reaction and the imbalance of Th1/Th2 immunity play important roles in the pathogenesis of CSU.

Objective To analyze the status of occupational hazards in key positions of the small and micro book and newspaper printing enterprises in Beijing City. Methods A total of 16 small and micro printing enterprises in Beijing City were selected as the research subjects using the judgment sampling method. The status of occupational hazard was assessed using on-site occupational health investigations. The volatile chemical components in organic solvents such as inks and cleaning agents used in the workplace, the level of chemical hazardous agents in the air, and noise intensity were detected and analyzed. Results A total of 1 105 workers from 16 small and micro printing enterprises were included. The occupational hazard exposure rate was 22.2% (245/1 105), with chemical hazardous agents and noise exposure rates of 13.5% and 22.2%, respectively. The rate of compliance with occupational health training among the head of the enterprise and the occupational health management personnel, the regular detection rate of occupational hazards in last year, and the rate for the occupational health examination in last year were both 100.0%. The rate for annual update of occupational hazard project reporting was 93.8% (15/16). The usage rates of gas mask and noise-proof earplug were 68.8% (11/16) and 50.0% (8/16), respectively. The effectiveness rates of anti-toxic and noise-reduction facilities were 87.5% (14/16) and 62.5% (10/16). The sign setting rates of chemical hazardous agents and noise warning were 93.8% (15/16). Acetone and isopropanol were found in ink and cleaning agents in 15 printing enterprises, while toluene, xylene, and ethylbenzene were found in three, two, and one enterprise, respectively. Benzene, 1,2-dichloroethane, n-hexane, and trichloroethylene were not found in all 16 enterprises. Both the exposure concentration of short term and exposure concentration of time weighted average of the above chemical hazards were lower than the lower limit of quantitation of the detection method in the workplace and work site air among the 16 printing enterprises, and none of concentration was exceeded the national standard. A total of 240 noise work sites were detected, and the national noise-exceeding rate was 11.2%(27/240). Conclusion Noise hazards are prominent in the small and micro printing enterprises in Beijing City, and attention should be paid to the prevention and protection of noise-exposure. The usage rate of personal protective equipment is not high, and the training and on-site supervision management of protective equipment wearing in workers should be strengthened.

Objective To investigate the prevalence of influenza virus infections in infants and young children during the pandemic period of 2009 influenza A(H1N1)in Beijing.Methods Throat swabs were collected from children visited the affiliated Children's Hospital to Capital Institute of Pediatrics for influenza-like illness from June 1,2009 to February 28,2010.The specific gene segments of 2009 pandemic influenza H1N1 and seasonal influenza viruses were amplified from samples by real-time RT-PCR recommended by WHO and National Influenza Reference Center of China.Results Out of 4363 clinical samples tested by real-time RT-PCR,the total positive rate of influenza A viruses was 29.3%,including 623(14.3%)identified as 2009 pandemic influenza A(H1N1)and 657(15.1%)influenza A viruses without subtype identity.Among those pandemic influenza H1N1 positive,23 were severe cases with 5 deaths.The ages for 618 pandemic influenza H1N1 infected children with completed information were from 14 days to 16 years.The ratio of male to female wag 1.3:1.Among them,25.2% were patients in age group of 1 to 3 years old and distribution of children in age groups of 3 to 6 years old and 6 to 12 years old were similar(about 30.0%).During the survey period,it appeared only one prevalence wave of pandemic influenza H1N1.The positive rate of pandemic H1N1 increased in September and the peak(36.5%of positive rate)was in November and then declined to 2.7%in February 2010.The data from routine influenza virus surveillance from 20-30 clinical samples collected each week indicated an alternative prevalence of seasonal H3N2,pandemic H1N1 and influenza B during this study period.Respiratory syncytial virus(RSV)became predominant in children after the circulating of pandemic H1N1.Conclusion There was an epidemic of pandemic influenza H1N1 in children in Beijing from June 2009 to February 2010,especially in those of preschool and school aged children.Seasonal influenza viruses and pandemic influenza H1N1 were contributed alternatively.

Objective: To evaluate the clinical value of a rapid respiratory syncytial virus (RSV) antigen detection in point-of-care testing (POCT). Method: A total of 209 specimens, including 78 throat swabs (TS) and 131 nasopharyngeal aspirates (NPAs), were collected from inpatients who visited the Children′s Hospital Affiliated to the Capital Institute of Pediatrics and were diagnosed as acute respiratory infection from 5 January to 7 February, 2015. These specimens were tested for RSV by a rapid antigen detection kit which was compared with reverse transcription polymerase chain reaction (RT-PCR) and direct immunofluorescence assay (DFA) for RSV detection. Result: Compared with DFA for NPAs, the sensitivity and specificity of rapid antigen detection were 83.9% and 97.3%, respectively, with Kappa value of 0.86; Compared with RT-PCR, the sensitivity (NPAs, 74.2%; TS, 77.8%) and specificity (NPAs, 100.0%; TS, 92.0%) of rapid antigen detection were high, too, with Kappa value of 0.74 in NPAs and 0.62 in TS. However, the RSV positive rate of rapid antigen detection in TS (21.7%) from pediatric patients with acute lower respiratory tract infection was lower than that in NPAs (78.3%), as well as that of RT-PCR (7.3% in TS verse 78% in NPAs). The RSV rapid antigen detection kit can be finished in about 10 minutes. Conclusion With characteristics of high specificity, high sensitivity, being rapid, efficient and easy to operate in comparison with DFA and RT-PCR, RSV rapid antigen detection in this study is suitable for POCT. For pediatric patients with acute respiratory tract infection, NPA was better than TS for RSV detection.

Objective: To investigate the clinical characteristics of respiratory syncytial virus(RSV)bronchiolitis and molecular biological characteristics of RSV in children in Beijing. Method: In a systematic retrospective study, 2 296 nasopharyngeal aspirates (NPA) were collected from children diagnosed with bronchiolitis from July 2006 to June 2016 for respiratory virus screening using direct immunofluorescence assay (DFA). For specimens positive for RSV, subgroup A or B was confirmed by real time RT-PCR and genotype of RSV was determined by amplifying the full G glycoprotein gene and sequencing. Clinical data were evaluated by the modified Tal score to compare the severity between RSV subtypes, as well as genotypes. Statistical analyses were performed using t test, Mann-Whitney U test and χ² test. Result: In 2 296 bronchiolitis cases, 961(41.9%) were RSV positive, including 719(74.8%) RSV A and 236 (24.6%) RSV B. The dominant RSV subtype changed from year to year: A-A-B-B-A-A-B-AB-A-AB and more bronchiolitis cases were identified in RSV A dominant years. Six genotypes of RSV A (NA1, NA2, NA3, NA4, GA5 and ON1) and 5 genotypes of RSV B (BA3, BA7, BA9, BA10 and CB1) were prevalent in Beijing. The dominant genotypes of RSV A were NA1 (55.9%) with high rates (50.0%-100%) before 2014 and ON1 (39.1%), mainly detected after 2014, while BA9 (90.6%) was the absolute dominant RSV B genotype. No significant difference in the severity of bronchiolitis was shown between cases of RSV A and B. Children positive for NA1 were more likely to stay longer in hospital (Median time: 8 days) compared to the group positive for ON1(Median time: 6 days ) (U=1.035, P=0.005) and had higher proportion of moderate to severe degree symptoms (Moderate: 41.0%, Severe: 10.0%) compared with ON1 group (Moderate: 22.9%, Severe: 4.3%) (U=9.785, P=0.008). In the group positive for ON1, more children had fever (ON1: 38.6%, NA1: 15.0%) (χ²=11.064, P=0.001) and more were younger than 3 months(ON1: 54.3%, NA1: 33.0%) (χ²=77.408, P<0.001). Conclusion The dominant RSV subgroup changed from year to year with a shifting pattern. The correlation between RSV genotypes and the severity of disease was documented in the study.