Rapid Internal Control Reference Recombinase-Aided Amplification Assays for EBV and CMV Detection / 生物医学与环境科学（英文）

Epstein-Barr virus (EBV) and cytomegalovirus (CMV), two of the most prevalent human herpesviruses, cause a wide spectrum of diseases and symptoms and are associated with serious health problem. In this study, we developed an internal control reference recombinase-aided amplification (ICR-RAA) assay for the rapid detection of EBV and CMV within 30 min. The assay had a sensitivity of 5 and 1 copies/test for EBV and CMV, respectively, with no cross reaction with other pathogens. In comparison with those of the commercial quantitative polymerase chain reaction (qPCR), the sensitivity of the EBV and CMV ICR-RAAs using extracted DNA was 93.33% and 84.84%, respectively; the specificity was 98.75% and 100.00%, respectively; and the Kappa values were 0.930 and 0.892 (

Association of Different Human Rhinovirus Species with Asthma in Children: A Preliminary Study / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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).</p><p><b>CONCLUSIONS</b>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.</p>

Parainfluenza Virus Types 1, 2, and 3 in Pediatric Patients with Acute Respiratory Infections in Beijing During 2004 to 2012 / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSIONS</b>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.</p>

Characterization of the whole genome from a human parechovirus type 3 detected from the serum of a child with sepsis in Beijing, China / 病毒学报

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.

Evaluation of serum specific IgM detection in diagnosis of respiratory viral infections in children / 中华儿科杂志

<p><b>OBJECTIVE</b>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).</p><p><b>METHOD</b>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.</p><p><b>RESULT</b>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.</p><p><b>CONCLUSION</b>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.</p>

Genomic characterization of WU polyomavirus identified from pediatric patients with acute respiratory infections in Beijing, China / 病毒学报

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.

The expression of the capsid protein VP2 from human bocavirus identified in Beijing and the formation of virus-like particles (VLPs) in insect cells / 病毒学报

The aim of this study was to obtain the capsid protein VP2 of human bocavirus (HBoV) identified in Beijing recently and construct virus-like particles (VLPs) in insect cells for further study of this virus. The full-length VP2 gene of HBoV from BJ3722 was inserted into the baculovirus expression transfer vector (pFastBac 1) to obtain the recombinant Bacmid, and generation of recombinant baculoviruses was followed by transfection of the recombinant Bacmid into insect cells. Then the recombinant VP2 protein was recognized by SDS-PAGE using Coomassie-blue staining and Western blot using hyper-immune serum against VP2 of HBoV from rabbit. The recombinant baculoviruses were harvested and amplified to gain large amounts of viruses with high titers to infect insect cells at a multiplicity of infection (MOI) of 0. 5. After 7-10 days or 4-5 days of the infection, the supernatants of culture or the cell lysates treated with lysing solution were harvested, and ultracentrifuged twice through 40% sucrose cushion to obtain purified VLPs, which were followed by Western blot and IFA for VLPs' composition and specificity analysis, by electron microscopy for VLPs' morphologic structure. The recombinant VP2 protein with molecular weight of approximately 61 kD expressed in recombinant baculoviruses was recognized by SDS-PAGE using Coomassie-blue staining and Western blot. The presence of VP2 on VLPs was demonstrated by Western blot and IFA from samples collected during the purification of VLPs from the supernatants of culture or the cell lysates, and the expression of VP2 in insect cells led to the formation of VLPs which formed the typical icosahedral appearance of parvoviruses with a diameter of approximately 20 nm. In conclusion, the recombinant baculoviruses were constructed, the HBoV VP2 protein was expressed in insect cells with high specific antigenicity and VLPs was formed successfully.

Prokaryotic expression and antigenic activity analysis on the matrix protein genes of two strains of human metapneumovirus recently identified in Beijing / 病毒学报

Human metapneumovirus (hMPV) is a recently identified respiratory virus more like human respiratory syncytial virus in clinical symptoms. Matrix protein (M) is one of the most important structural proteins. For further studying of hMPV, the full length of M genes from the recombinant plasmid pUCm-M1816 and pUCmM1817 were cloned by PCR and sub-cloned into the pET30a(+) vector, which is a prokaryotic expression vector, after dual-enzyme digestion with Bam HI and Xho I. The positive recombinated plasmids were transformed into E. coli BL21 (DE3) and expressed under the inducing of IPTG. Target proteins were characterized by SDS-PAGE and Western blotting. In this article, we' ve successfully constructed the recombinated plasmids pET30a-M1816 and pET30a-M1817 which have correct open reading frames confirmed by dual-enzyme digestion analysis and sequencing. The fusion proteins with 6 x His-N were highly produced after inducing by 1mmol/ L IPTG at 37 degrees C. A unique protein band with approximate 27.6 kD was characterized by SDS-PAGE. Most of the target protein existed in inclusion body. Western blot analysis showed that the target protein has specific binding reaction to rabbit antiserum against polypeptides of the matrix protein of hMPV. So the M genes were highly expressed in the prokaryotic system and the expressed M proteins have specific antigenic activities. It can be used for further studying of hMPV infections in Beijing.

Molecular epidemiologic survey of rotaviruses from infants and children with diarrhea in Shanghai / 中华儿科杂志

<p><b>OBJECTIVE</b>To investigate molecular epidemiologic features of rotaviruses circulating in Shanghai, China.</p><p><b>METHODS</b>Stool samples were collected from 1230 hospitalized children with community-acquired and nosocomially acquired diarrhea in Children's Hospital Affiliated to Fudan University between November 1, 1999 and December 31, 2001. Polyacrylamide gel electrophoresis (PAGE) was used to detect rotavirus genomic RNA and identify electropherotypes of group A rotavirus RNAs. Reverse transcription polymerase chain reaction (RT-PCR) was performed to amplify full length VP7 gene and dot blot hybridization was performed to identify rotavirus G serotypes using digoxigenin-labelled variable regions from VP7 genes as probes. These probes were amplified by PCR from recombinant plasmids containing full length G1, G2, G3 and G4 VP7 genes from rotavirus field strains detected in Beijing and digoxigenin labelled dUTP was integrated into the PCR products. The Kruskal-Wallis analysis of variance was employed to analyze whether there were significant differences in variables.</p><p><b>RESULTS</b>Out of 1230 samples investigated, 493 (40.1%) were group A rotavirus gene positive by PAGE, among which 397 (80.5%) showed long electropherotypes, 55 (11.2%) showed short electropherotypes, 18 (3.7%) showed mixed electropherotypes which suggested that the children were co-infected by rotaviruses with different electropherotypes, 23 (4.7%) were non-typable because of degradation of some of the genomic RNA fragments. No group B or group C rotavirus was found. RT-PCRs were performed for 328 fecal specimens containing sufficient rotavirus RNAs and VP7 gene products were obtained from 254 (77.4%) samples. Dot blot hybridization showed serotype G1 accounted for 55.5% (141) of these samples, serotype G3 accounted for 27.6% (70), serotype G2 accounted for 9.4% (24), co-infection by 2 rotaviruses with different G types accounted for 6.3% (16), only 1 G4 was detected and 2 were non-typable. The genomic RNA patterns of all G2 strains were short and those of G1, G3 and G4 strains were long. There were no statistically significant differences for age distribution and clinical manifestations among those infants and children infected by rotaviruses with different G serotypes.</p><p><b>CONCLUSION</b>Group A rotavirus is the major pathogen for diarrhea in infants and children in Shanghai during the period of Nov. 1999 to Dec. 2001. Rotaviruses with long electropherotype were dominant during these years. Serotypes G1 to G3 constituted 98.8% of all 254 strains tested, and G1 was the most common serotype followed by G3 and G2, whereas serotype G4 was seldom found. Some of the children were co-infected by rotaviruses with different G serotypes. Clinical manifestations were not related to the infecting rotavirus with different G serotypes.</p>