Publications (57)

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    [Show abstract] [Hide abstract] ABSTRACT: Background: Human rhinoviruses (HRVs) are divided into three genetic species: HRV-A, HRV-B, and HRV-C. The association of different HRV species with asthma in children in China has not yet been evaluated. This preliminary study aimed to assess the associations between different HRV species, particularly HRV-C, and asthma in young children in China. Methods: A total of 702 nasopharyngeal aspirates were obtained from 155 children with asthma (asthma group), 461 children with acute respiratory infection (ARI) without asthma (nonasthma ARI group), and 86 children from the control group. Semi-nested polymerase chain reaction (PCR) was used to detect HRVs, and PCR products were sequenced for species identification. Epidemiological characteristics of HRV-positive cases were analyzed. Results: HRVs were the most common pathogen (15.4%; 108/702) in the patients in this study. The prevalence of HRV was significantly different (F = 20.633, P = 0.000) between the asthma (25.8%) and nonasthma ARI groups (11.1%). Phylogenetic analysis indicated that in the 108 cases positive for HRVs, 41 were identified as HRV-A, 8 as HRV-B, and 56 as HRV-C. Comparing the asthma with the nonasthma ARI group, Spearman's rank correlation analysis revealed an association between HRV-A (P < 0.05) and C (P < 0.01) and asthma, confirmed by regression analysis, with odds ratios of 2.2 (HRV-A) and 4.2 (HRV-C). Conclusions: Our data revealed a high prevalence of HRVs in children in China, regardless of clinical status. HRV-C was the dominant species and may be one of the key factors in the association of HRVs with asthma.
    Full-text available · Article · Jul 2016 · Chinese medical journal
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    Fang Wang · Lin-Qing Zhao · Ru-Nan Zhu · [...] · Yuan Qian
    [Show abstract] [Hide abstract] ABSTRACT: Background: Although human parainfluenza virus (HPIV) has been determined as an important viral cause of acute respiratory infections (ARIs) in infants and young children, data on long-term investigation are still lacking to disclose the infection pattern of HPIV in China. Methods: Nasopharyngeal aspirates were collected from 25,773 hospitalized pediatric patients with ARIs from January 2004 through December 2012 for respiratory virus screen by direct immuno-fluorescence assay. Results: Out of these specimens, 1675 (6.50%, 1675/25,773) showed HPIV positive, including 261 (1.01%, 261/25,773) for HPIV1, 28 (0.11%, 28/25,773) for HPIV2, and 1388 (5.39%, 1388/25,773) for HPIV3, 2 of the samples were positive for both HPIV1 and HPIV3, and 36 were co-detected with other viruses. The positive rates of HPIVs were higher in those younger than 3 years old. HPIV3 was detected from all age groups, predominantly from patients under 3 years of age, and the highest frequency was found in those 6 months to 1-year old (352/4077, 8.63%). HPIV3 was the dominant type in each of the years detected between May and July. HPIV1 showed a peak in every odd year, mainly in August or September. HPIV was detected most frequently from patients with upper respiratory infection (12.49%, 157/1257), followed by bronchitis (11.13%, 176/2479), asthma (9.31%, 43/462), bronchiolitis (5.91%, 150/2536), pneumonia (6.06%, 1034/17,068), and those with underlying diseases (1.0%, 15/1506). HPIV3 is the dominant type in these six disease groups referred above, especially in the asthma group. Conclusions: HPIV is one of the important viral causes of ARIs in infants and young children in Beijing based on the data from the hospitalized children covering a 9-year term. HPIV3 is the predominant type in all these years and in most of the disease groups. HPIVs with different types show different seasonality.
    Full-text available · Article · Oct 2015 · Chinese medical journal
  • [Show abstract] [Hide abstract] ABSTRACT: Norovirus is a major cause of diarrheal disease with epidemic, outbreak or sporadic patterns in humans of all ages worldwide. This study aimed to determine the genotypic characteristics of noroviruses from infants and children in Beijing. Stool samples (n = 1128) were collected from patients with symptoms of acute gastroenteritis in the past 3 years from 2010 to 2012. The norovirus positivity rate was 16.1% (182/1128) by using RT-PCR, including 122 with primer set covering polymerase region, 177 with primer set covering capsid region, and 117 with both polymerase and capsid regions. By sequence analysis for capsid genes, all the noroviruses identified were belonging to genogroup II (GII). Among these positive samples, GII.4 (61.0%) was the most common genotype detected, followed by GII.3 (35.0%). The new variant GII.4 Sydney_2012 strains emerged in this study in September and became the predominant genotype later. Those 117 from 182 RT-PCR positive samplers were able to be genotyped based on the sequences of both polymerase and capsid genes. The result was interesting that 59 out of these 117 positive specimens (50.4%) had mismatched genotypes between polymerase and capsid genes, including 7 suspected recombinants patterns. Among them, GII.P12/GII.3 was the most common combination which accounts for 54.2% (32/59), followed by GII.Pe/GII.4 Sydney_2012 which was 23.7% (14/59). Two novel recombinants, GII.P22/GII.5 and GII.21/GII.3 were first detected in this study. In summary, this study provides a detailed description based on laboratory data of the genetic diversity of norovirus in young children with acute gastroenteritis in Beijing. Moreover the data revealed that in the evolution of norovirus, new variant and novel recombination emerged frequently.
    Article · Sep 2014 · Infection, genetics and evolution: journal of molecular epidemiology and evolutionary genetics in infectious diseases
  • Ru-Nan Zhu · Lei Luo · Yuan Qian · [...] · Ya-Xin Ding
    [Show abstract] [Hide abstract] ABSTRACT: 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.
    Article · Sep 2014 · Bing du xue bao = Chinese journal of virology / [bian ji, Bing du xue bao bian ji wei yuan hui]
  • [Show abstract] [Hide abstract] ABSTRACT: To understand the clinical characteristics of different groups human rhinovirus (HRV)-A, B and C infection in children with acute respiratory tract infections(ARI) in Beijing. Respiratory tract specimens(n = 1412) collected from children with ARI during Jan. 2011 to Dec. 2012 were tested for HRV by using semi-nested PCR.Gene fragments of VP4/VP2 capsid protein amplified from HRV positive specimens were sequenced for HRV genotype confirmation. Then epidemiological characteristics of these HRV-positive cases were analyzed. Among these 1412 specimens tested, 103(7.3%) were HRV positive, including 54(52.4%) positive for HRV-A, 14(13.6%) for HRV-B, 35(34.0%) for HRV-C determined by sequence analysis. The positive rates of HRV-A, B and C(2.5%, 16/638; 0.3%, 2/638 and 1.3%, 8/638) in children with acute upper respiratory tract infections(URI) were lower than those(5.8%, 36/623; 1.8%, 11/623 and 3.9%, 24/623) in children with acute lower respiratory tract infections(LRI) (P = 0.003, 0.011, 0.003).In children with LRI, the positive rates of HRV-A, C were similar to each other(P = 0.112), and both were higher than that of HRV-B(P = 0.000, P = 0.026). The severity of ARI among children positive for different groups HRV showed no significant difference evaluated by Kruskal-Wallis H test(Hc = 0.044, P > 0.05), as well as that between children co-infected with HRV and other viruses and those infected with HRV only evaluated by Wilcoxon rank sum test(Zc = 0.872, P > 0.05). HRV is one of important pathogens for children with ARI, especially LRI in Beijing. The positive rates of HRV-A and HRV-C are similar to each other, and both are higher than that of HRV-B. No significant difference was shown among children with different HRV genotypes by evaluation of the severity of ARI, and co-infections of HRV with other viruses do not significantly increase the severity of ARI.
    Article · Dec 2013 · Zhonghua er ke za zhi. Chinese journal of pediatrics
  • [Show abstract] [Hide abstract] ABSTRACT: To 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. According 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. Sensitivity 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. A 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.
    Article · Apr 2013 · Zhonghua er ke za zhi. Chinese journal of pediatrics
  • [Show abstract] [Hide abstract] ABSTRACT: To establish a rapid and reliable loop-mediated isothermal amplification (LAMP) method for detecting adenoviruses (ADV)in respiratory samples collected from children with acute respiratory infections. According to the sequences of hexon genes of common adenovirus serotypes (Ad3, Ad7, and Ad14) downloaded from GenBank, primers were designed and LAMP method for detecting adenovirus DNA was developed. Sensitivity of the LAMP method was evaluated by using constructed recombinant plasmid DNA with gene fragment from hexon of ADV3, and specificity was tested through cross-reaction with other viruses. Then 11 ADV strains isolated from clinical specimens using tissue cultures were tested by LAMP method. A total of 108 nasopharyngeal aspirates from hospitalized patients with acute respiratory infections which had been tested by direct immunofluorescence assay (DFA), including 36 for ADV positive and 72 for ADV negative, were tested by both LAMP method and multiplex nested PCR. Analysis for sensitivity indicated that this LAMP method can detect 1.9×10(2)copies/ml of DNA, and no amplification was shown in DNA or cDNA of other viruses, which revealed that the specificity of the LAMP method is high. For 108 specimens which had been tested by DFA, 34 out of the 36 ADV positive specimens showed positive signal within 90 minutes using LAMP. Five out of 72 negative specimens by DFA were positive using LAMP; 39 out of the 41 ADV positive specimens by multiplex nested PCR showed positive signal using LAMP, including 19 for Ad3 and 20 for Ad7; 67 negative specimens confirmed by multiplex nested PCR showed negative signal using LAMP. The total consistency rate of DFA and LAMP method for detecting ADV was 93.5%, and the total coincidence rate of multiplex nested PCR and LAMP method for detecting ADV was 98.1%. A new, sensitive, accurate and rapid method for detecting human adenovirus from nasopharyngeal aspirates by LAMP was developed, which should be a potential method for rapid detection of ADV from respiratory tract of children in clinical diagnosis of ADV infection.
    Article · Mar 2013 · Zhonghua er ke za zhi. Chinese journal of pediatrics
  • [Show abstract] [Hide abstract] ABSTRACT: To understand the infections and molecular biological characteristics of different human rhinovirus (HRV) genotypes -A, B, C, especially C in children with acute respiratory tract infections (ARI) in Beijing. Seven hundreds and three respiratory tract specimens were collected from children with ARI during Jan. 2011 to Dec. 2011. Semi-nested PCR was developed for detecting HRVs. Gene fragment of VP4/VP2 capsid protein amplified from HRV positive specimens was sequenced and analyzed by software DNAStar, the phylogenetic tree was then constructed by MEGA 5. 05. Among these 703 specimens tested, 54 (7.7%, 54/703) were HRV positive, including 25 (46.3%, 25/54) positive for HRV-A, 8 (14. 8%, 8/54) for HRV-B, 21 (38. 9%, 21/54) for HRV-C determined by sequence analysis. Most of these children (94. 4%00, 51/54) infected with HRVs were younger than 5 years old, and the highest positive rate was shown in group younger than 1 year (11. 4%). These patients positive for HRVs were diagnosed as bronchiolitis (23.1%), asthma (20.0%), pneumonia (1.0%), bronchitis (4.4%) and upper respiratory tract infections (4. 1%). Sequence analysis of VP4/VP2 gene fragment revealed that 70. 0% to 100. 0% nucleotide identity was shown among the sequences within the same HRV genotype, and 55. 5% to 65. 8% nucleotide identity among the sequences from different HRV genotypes. In conclusion, HRVs, especially HRV-C, are important pathogens for children with ARI in Beijing. The prevalence of HRV-C is similar to that of HRV-A, higher than that of HRV-B. High sequence variation among different HRV genotypes was indicated in this study.
    Article · Mar 2013 · Bing du xue bao = Chinese journal of virology / [bian ji, Bing du xue bao bian ji wei yuan hui]
  • [Show abstract] [Hide abstract] ABSTRACT: WU polyomavirus (WU virus) was identified as a novel polyomavirus in 2007 from specimens of pediatric patients with acute respiratory infection (ARI). A lack of permissive cell lines has limited investigations into WU virus pathogenesis and prevalence. The encoding region of the capsid protein VP2 gene was amplified from a WU virus DNA-positive clinical specimen and expressed as a recombinant Histagged protein in Escherichia coli BL21 (DE3). The expressed VP2 was identified by expected molecular weight and immunoreactivity with anti-His monoclonal antibody in Western blotting assay. Serum samples collected from 455 individuals of all ages in Beijing without symptoms of ARI were tested for IgG antibodies against the affinity-purified recombinant VP2 protein by Western blotting to investigate the prevalence of natural WU virus infection. In addition, serum samples from four ARI pediatric patients, whose nasopharyngeal aspirates were positive for WU virus DNA and negative for all other respiratory-related viruses, were tested for IgM antibody against the recombinant VP2. Of the 455 serum samples, 238 reacted with the recombinant VP2, yielding an overall positive rate of 52.3% for IgG against VP2 of WU virus. The positive rate was the highest in serum samples from infants and children between 1 to 4 years of age. One of four ARI pediatric patients was positive for IgM against WU virus VP2, implicating WU virus as the causative disease agent. The high prevalence of IgG against WU polyomavirus in Beijing-based study population indicates that WU virus infection is common in Beijing. WU virus may be responsible for some pediatric ARI cases, and primary infection of this virus may occur mostly in childhood.
    Article · Feb 2013 · World Journal of Pediatrics
  • [Show abstract] [Hide abstract] ABSTRACT: Objective: To explore the clinical features of types 3 and 7 adenovirus pneumonia in children and compare the difference between two types. Methods: A total of 47 patients with adenovirus pneumonia at our hospital from January 2009 to June 2011 were reviewed. According to the serological types, they were divided into two groups: type 3 (n = 19) and type 7 (n = 28). Two groups were analyzed statistically with regards to age, gender, clinical presentation, critical scores, laboratory examinations, radiographic findings, lung function changes, complications and prognosis respectively. Results: For types 3 and 7 adenovirus pneumonia, the median age of onset were 1.8 and 1.1 years old respectively. The male-to-female ratio were 14:5 and 20:8 respectively. As compared with type 3, type 7 had the longer fever time ((18 ± 10) vs (11 ± 7)days, P = 0.010), the higher critical proportion (13/28 (46.4%) vs 3/19 (15.8%), P = 0.031), the longer hospital stays ((28 ± 17) vs (15 ± 6) days, P = 0.003)), the higher proportion of involved lung ≥ 3 lobes (22/28 (78.6%) vs 4/19 (21.1%), P = 0.000), the lower oxygenation index ((192 ± 85) vs (256 ± 79) mm Hg,1 mm Hg = 0.133 kPa, P = 0.011), the higher proportion of mechanical ventilation (17/28 (60.7%) vs 5/19 (26.3%), P = 0.020) and the higher proportion of multiple organ dysfunction syndrome (16/28 (57.1%) vs 5/19 (26.3%), P = 0.037). In type 7, organ dysfunction was more obvious, particularly in nervous system, blood system and enzyme changes. Three cases of type 7 had pulmonary sequela with small airway disease. Conclusion: With more severe clinical manifestations, laboratory parameters and imaging data than type 3, type 7 adenovirus pneumonia in children is more likely to cause pulmonary sequela.
    Article · Dec 2012 · Zhonghua yi xue za zhi
  • [Show abstract] [Hide abstract] ABSTRACT: To investigate the viral etiology and clinical features of hospitalized children with acute respiratory tract infections in Tibet. Nasopharyngeal aspirate samples were collected from children with acute respiratory tract infection hospitalized at the department of Pediatrics, Tibet Autonomous Region People's Hospital from April to July, 2011. The specimens of nasopharyngeal aspirate were screened for antigens of 7 common respiratory viruses by direct immunofluorescence (DIF) [respiratory syncytial virus (RSV), adenovirus (ADV), parainfluenza viruses type I-III, influenza virus A and B] and human metapneumovirus. Clinical data of the children were analyzed by statistical software SPSS16. A total of 167 children with acute respiratory tract infections hospitalized from April to July 2011 were enrolled in this investigation. Sixty-five out of 167 specimens were positive for viral antigens. The virus positive rate for specimens was 38.9% (65/167). Two of 65 positive specimens were positive for 2 virus antigens (RSV + influenza B) and (hMPV + parainfluenza virus type III), respectively. RSV was detected in 45 cases (67.2%, 45/67) which was the most predominant, followed by parainfluenza virus type III detected in 7 cases (10.4%, 7/67), ADV in 6 cases (9.0%, 6/67), parainfluenza virus type I in 4 cases (6.0%, 4/67), influenza virus type B in 3 cases (4.5%, 3/67), and hMPV in 2 cases (3.0%, 2/67). In addition to clinical manifestations of pneumonia, such as cough and shortness of breath, only 3 virus positive cases (6.67%) presented with wheezing, but the signs of severe cyanosis, fine rales in lung were common. Most of the children in this study recovered soon, only a few younger children with underlying diseases or complications had severe illness. Virus is an important pathogen for acute respiratory infections for hospitalized children in Tibet. RSV was the most predominant etiological agent, especially for those younger than 3 years old.
    Article · Oct 2012 · Zhonghua er ke za zhi. Chinese journal of pediatrics
  • Qin-wei Song · Ru-nan Zhu · Jie Deng · [...] · Yuan Qian
    [Show abstract] [Hide abstract] ABSTRACT: The 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). Clinical 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. The 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. Although 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.
    Article · Jun 2012 · Zhonghua er ke za zhi. Chinese journal of pediatrics
  • Jie Deng · Ru-Nan Zhu · Yuan Qian · [...] · Mei-Duo Deji
    [Show abstract] [Hide abstract] ABSTRACT: To understand the role of respiratory syncytial virus (RSV) in children with acute respiratory infections (ARI) in Tibet Autonomous Region and the contribution of two major groups of RSV, nasopharyngeal aspirates (NPA) were collected from hospitalized children with ARI in Department of Pediatrics, Tibet People's Hospital in Lasa, Tibet from April to July in 2011 and tested for seven common respiratory viruses and human metapneumovirus (hMPV) by direct immunofluorescence assay (DFA). Total RNAs were extracted from RSV positive samples by DFA and reverse transcripted to cDNA. Nested-PCR was employed to determine the genogroups of RSV, which were confirmed by real time-PCR and sequence analysis for G protein encoding gene. The Characteristics and variations of G genes from RSV in this project were identified by sequence comparison with those G genes in GenBank. Out of 167 samples, 65 were positive for respiratory viruses with a total positive rate of 38.9%, including 45 (69.2%, 45/65)positive samples for RSV. Among 42 samples that were positive for RSV and genotyped, 40 were identified as group A and 2 as group B. Sequence analysis of full-length G genes for 7 RSV of group A indicated that all of these belonged to subgroup GA2. The nucleotide identities between RSVs from Tibet and prototype A2 strain were 90.7%-91.8%, with 86.5%-87.2% identities of amino acid. The mutations of amino acids were mainly located in both ends of a highly conserved region in the ectodomain of the G proteins. The data indicated that RSV was the most important viral etiologic agent of ARI in spring of 2011 in Tibet and group A of RSV was predominant during the study period. High divergence existed in the ectodomain of G proteins of RSVs from Tibet.
    Article · Mar 2012 · Bing du xue bao = Chinese journal of virology / [bian ji, Bing du xue bao bian ji wei yuan hui]
  • [Show abstract] [Hide abstract] ABSTRACT: P[8]b is a newly discovered sub-genotype for VP4 gene of group A human rotaviruses (HRV) worldwide. This study was to develop an effective method to identify P[8]a, P[8]b, P[4] and P[6] (sub) genotypes of VP4 genes of HRV and to investigate the prevalence of P[8]b sub-genotype and its G/P combinations of HRV in outpatient and inpatient children with diarrhea in Children's Hospital affiliated to Capital Institute of Pediatrics from 2009 to 2010. By analyzing the collected nucleotide sequences of VP4 gene for all known P genotypes of HRV including P[8]b subtype from GenBank and using softwares of DNAS-tar and MegAlign to align and analyze multiple sequences, probes for P[8]a, P[8]b, P[4] and P[6] (sub) genotypes in the corresponding regions which are highly divergent among genes from different genotypes and conserved within genes of VP4s in same genotypes were designed. Then four sets of primers for PCR amplified DIG labeled probes were designed and corresponding DIG-labeled specific P genotype probes were synthesized with PCR by using VP8* genes of Beijing field HRV strains representing P-genotypes P[8]a, P[8]b, P[4] and P[6], respectively, as templates. Dot-blot hybridization was developed based on cDNA of VP4 genes. The dot-blot hybridization assay for P genotyping was reliable which was confirmed by sequencing of RT-PCR products of VP4 genes amplified from corresponding clinical samples. P genotyping for VP4 genes from 88 HRV positive specimens from the Outpatient Department (55%, 88/160) and 79 HRV positive specimens from the hospitalized (70.5%, 79/112) children with diarrhea indicated that P[8] a subtype was still the most prevalent sub-genotype, which was 96.6% (85/88) and 62.0% (49/79) respectively. The positive rate for P[8]b subtypes in hospitalized children with HRV diarrhea was higher (27.9%, 22/79) than that of in outpatient (2.3%, 2/88) HRV infected children. HRV with P[4] genotype was only found in one of the hospitalized children (1.3%, 1/79), and HRV with P[6] genotype was not detected from specimens either from outpatient or inpatient. G9P[8]b was the predominant combination among the P[8]b subtype of HRV positive specimens in this study. The results in this study indicated that G9P[8]b HRV circulated in children with diarrhea in Beijing.
    Article · Nov 2011 · Bing du xue bao = Chinese journal of virology / [bian ji, Bing du xue bao bian ji wei yuan hui]
  • Ru-Nan Zhu · Yuan Qian · Lin-Qing Zhao · [...] · Fang Wang
    [Show abstract] [Hide abstract] ABSTRACT: 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.
    Article · Nov 2011 · Bing du xue bao = Chinese journal of virology / [bian ji, Bing du xue bao bian ji wei yuan hui]
  • Li Sha · Ru-nan Zhu · Ling Cao · [...] · Jun-bao Du
    [Show abstract] [Hide abstract] ABSTRACT: The novel influenza A (H1N1) virus firstly detected in April 2009 in Mexico rapidly spread to many countries including the United States and Canada where humans were infected with the H1N1 virus and deaths were reported. The pandemic virus strain had never been detected in specimen of human beings and swine. It was so highly contagious and widely spread that threatened life of humans globally. This study aimed to analyze clinical data of hospitalized children patients with 2009 novel H1N1 influenza A virus infection confirmed by etiologic tests, and compared with that of seasonal influenza A. Clinical manifestations, laboratory and therapy data from the hospitalized children were collected by designed case report form and analyzed. All patients were enrolled from Capital Institute of Pediatrics from January 2003 to 2010. There were 152 cases in seasonal influenza A group, which was composed of 100 boys and 52 girls. Other 93 boys and 86 girls formed 2009 novel influenza A group. Influenza A was dominate from 2003 to 2008 and the peak season was December and January, while the peak hospitalized time of 2009 novel H1N1 influenza was from November 2009 to January 2010. The median age of seasonal influenza group was 35 months, which was lower than that of novel influenza group (Z = -6.702, P<0.01). Besides, 80.9% of the patients in seasonal influenza group were infants, while the novel influenza A group was mainly composed of infants and pre-school children (chi2 = 40.725, P<0.01). The cases of both groups had influenza-like symptoms at onset and the most common presentations were fever and cough. The duration of fever was much longer in 2009 novel influenza group (Z = -7.173, P<0.01). Patients in two groups nearly had the same symptoms except cough was more frequently presented by novel influenza A group cases (chi2 = 4.109, P<0.05). In laboratory examination, the novel influenza group had more cases with abnormality in blood platelet, CRP, ALT, and CK-MB than that of seasonal influenza group (chi2 = 7.562, 17.245, 4.398, 6.217, P<0.01). Patients in novel influenza A group had more changes in electrocardiogram (chi2 = 24.461, P<0.01). More patients had common underlying medical condition in novel influenza groups than those in seasonal influenza group (chi2 = 12.553, P<0.01). Furthermore, the groups had different age distribution in underlying medical diseases (chi2 = 7.231, P<0.05). Children with 2009 novel H1N1 virus infection tended to catch pneumonia (chi2 = 8.661, P<0.01) and became the severe cases (chi2 = 10.595, P<0.01). They had much higher ICU admission rate (chi2 = 12.873, P<0.01) and longer hospital stay (Z = -2.764, P<0.01). As a new variant of influenza virus A, 2009 novel H1N1 influenza A had stronger pathogenicity. Children with underlying medical conditions had the high risk to be infected and developed severe manifestations.
    Article · Jul 2011 · Zhonghua er ke za zhi. Chinese journal of pediatrics
  • Ru-Nan Zhu · Yuan Qian · Lin-Qing Zhao · [...] · Rong-Yan Huang
    [Show abstract] [Hide abstract] ABSTRACT: Human metapneumovirus (hMPV) was discovered by scientists in the Netherlands as a novel respiratory virus in 2001 and had been found in children with acute respiratory tract infections (ARTI) in China. The objective of this study was to determine the importance of hMPV infection in children in Beijing and the genotypes of the circulating virus by the surveillance during a four-consecutive-year period. Clinical specimens collected from children with ARTI from January 2006 to December 2009 were tested for hMPV by RT-PCR using primers targeting the matrix (M) gene, followed by genotyping of hMPV directly from positive samples by diplex PCR with primers for glycoprotein (G) genes. Sequence analysis was used for genotyping of those un-typable samples. Common respiratory viruses in these clinical specimens were tested by virus isolation and antigen detection, in addition to hMPV detection. Of 4730 tested specimens, 191 (4.0%) were positive for hMPV and 62.8% of 191 were identified as genotype A. The positive rate of hMPV from hospitalized patients was higher than that from outpatients each year. Most of hMPV positive children were under five years old. The peak of hMPV activity mostly occurred in late spring and overlapped with or followed that of respiratory syncytial virus (RSV) and followed by parainfluenza virus 3. Of hMPV infected cases, 68.6% were lower respiratory tract infection, among which 79.4% were hospitalized, and upper respiratory tract infection was diagnosed for 31.4% of hMPV infected children. The 9.4% of hMPV positive samples were found to co-exist with other respiratory viruses. hMPV was an important pathogen for ARTI in pediatric patients, especially those under five years old. Both genotypes A and B circulated simultaneously in Beijing.
    Article · Jun 2011 · Chinese medical journal
  • Yu-Yun Li · Ru-Nan Zhu · Yuan Qian · [...] · Li-Ying Liu
    [Show abstract] [Hide abstract] ABSTRACT: In order to learn about the correlation between the sequences of VP4 of EV71 and clinical symptoms of patients and analyze the antigenicity of VP4 of EV71, as well as the cross-reactivity with VP4 of CA16, the sequences of VP4 gene from 10 EV71 strains isolated from infants and children with hand, foot and mouth diseases (HFMD) during 2007 to 2009 were determined through standard molecular cloning protocols, and the results were analyzed by EditSeq and MegAlign of DNAStar. Full-length genes of VP4s of EV71 and CA16 were amplified from virus isolates and expressed in E. coli. Then the expressed VP4s were used as antigens to detect IgG antibody in 189 sera samples from people taking health check up and patients of non-HFMD by Western-Blot. They were also used to detect IgM antibody in 14 of sera samples from infants and children with EV71 infection and 12 of sera samples from those with CA16 infection. The nucleotides identities among these 10 sequences of VP4s isolated in our lab were 94.20% - 100.00% and the deduced amino acids were identical. There was no consistent divergence between the sequences of serious cases and those from general HFMD cases. Phylogenetic analysis based on VP4s indicated that these 10 VP4s of EV71 belonged to C4. The nucleotide identities between EV71 VP4 (s67) and CA16 VP4 (s401) was 69.60% and the deduced amino acids identities was 78.60%. In the detection of IgG, the sera-positive rate for EV71 VP4 was 38.10% and the sera-positive rate of CA16 VP4 was 58.20%. The difference in the sera-positive rate between them was significant (chi2 = 15.30, P < 0.01), suggesting that the expressed VP4s of EV71 and CA16 were of good antigenicity and not cross-reactive. There was no positive reaction detected for IgM against VP4s for EV71 or CA16. The data from this study reveal important information for the further study of EV71.
    Article · May 2011 · Bing du xue bao = Chinese journal of virology / [bian ji, Bing du xue bao bian ji wei yuan hui]
  • Ru-nan Zhu · Yuan Qian · Lin-qing Zhao · [...] · Fang Wang
    [Show abstract] [Hide abstract] ABSTRACT: 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.
    Article · Mar 2011 · Bing du xue bao = Chinese journal of virology / [bian ji, Bing du xue bao bian ji wei yuan hui]
  • Lin-Qing Zhao · Yuan Qian · Ru-Nan Zhu · [...] · Ni-Na Zhang
    [Show abstract] [Hide abstract] ABSTRACT: 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.
    Article · Nov 2010 · Bing du xue bao = Chinese journal of virology / [bian ji, Bing du xue bao bian ji wei yuan hui]