Mispronounced consonants and short-term memory among children with developmental speech sound disorder / 中华物理医学与康复杂志

Objective:To observe the pronunciation of consonants among children with developmental speech sound disorder and explore the correlation between mispronounced consonants and short-term memory so as to determine the pathogenesis of the disorder.Methods:Thirty-six children with developmental speech sound disorder and aged 4 to 13 years were evaluated. Their pronunciation of consonants at the phoneme and lexical levels was tested to record the error types and error rate. Twelve of the children were then randomly chosen to form a voice disorder group. Another 10 healthy counterparts constituted a control group. The short-term memory of both groups was assessed and any correlation between pronunciation and short-term memory was analyzed.Results:The children with a developmental speech sound disorder differed significantly from the controls in terms of the numbers of errors in articulating blade-alveolar, blade-palatal and velar consonants. On the phoneme level, the highest substitution error rate occurred when pronouncing lingua-palatal consonants (42.86%), followed by supradental consonants (32%). The highest distortion and non-acquisition error rates were with blade-palatal consonants (14%) and lingua-palatal consonants (9.5%). On the vocabulary level, the highest substitution, distortion, ellipsis and non-acquisition error rates appeared when pronouncing lingua-palatal and velar consonants, velar and blade-palatal consonants, supradental consonants as well as blade-palatal consonants. Significant differences were found between the phoneme and lexical levels in the substitution of supradental and blade-palatal consonants as well as in the ellipsis of blade-alveolar consonants. They were moderately associated with pronunciation level. There was, however, no significant difference in working memory span between the two groups, and no significant correlation was observed between working memory span and pronunciation level.Conclusion:The mispronunciation of consonants by children with developmental speech disorders is higher at the lexical than at the phoneme level. They mainly substitute lingua-palatal and velar consonants and elide supradental consonants, which may be related to short-term memory span.

Establishment of recombinase polymerase amplification assay for five hemorrhagic fever-related viruses / 解放军医学杂志

Objective To establish a one-step recombinase polymerase amplification (RPA) method for pathogen screening and rapid detection in the field targeting for five hemorrhagic fever related viruses (Zaire ebola virus, Sudan ebola virus, Marburg virus, Lassa virus and Yellow fever virus). Methods The specific nucleic acid (NA) fragments of each virus were selected as target genes by genome sequence analysis, and the primers and probes for RPA assays were designed according to the sequence. A series of diluted template genes were used for RPA detection to determine the sensitivity. The hemorrhagic fever-related viral nucleic acids were used for RPA detection to determine the specificity. The amplification experiments were carried out at different temperature ranging from 37℃ to 42℃ to validate the reaction temperature range. Results The RPA reaction systems of the five hemorrhagic fever viruses could effectively amplify the target genes, the sensitivities were between 1.5×102 and 1.5×103 copies. No cross reactions existed with the other hemorrhagic fever-related viral genes. Meanwhile, RPA assay could effectively amplify the target genes at 37-42℃. Conclusion The isothermal RPA assays of five hemorrhagic fever viruses are established, which may amply target genes fast and react at a wide temperature range, and be potentially useful for in field pathogens detection.

Construction of pseudovirus reference standard containing five kinds of hemorrhagic fever virus / 军事医学

Objective To construct the pseudovirus containing nucleic acid(NA)fragments of Marburg virus,Zaire Ebola virus,Sudan Ebola virus,Lassa fever virus and Yellow fever virus by using a lentiviral vector system in order to provide a reference standard for the detection of the five viruses.Methods The gene fragments of the above five viruses were synthesized in vitro,connected into a single gene by fusion PCR technique,and cloned into lentiviral vectors with its auxiliary vector.After co-transfecting into 293T cells,the supernatants were collected on 48 h and 72 h post transfection. The naked NA was cleaned from the supernatants using DNase and RNase digestion before pseudotype virus was purified and concentrated.After the NA of the pseudotype virus,were extracted normal PCR and real-time PCR were conducted. Results Sequence analysis showed that the five target genes in vitro synthesis were properly connected and inserted into lentivirus vectors.Using the NA of the pseudotype virus as the template,both normal PCR and real-time PCR could sensitively amplify the target gene with the primers and probes of the above five,viruses respectively.The result indicated that the pseudovirus particles containing the five kinds of hemorrhagic fever virus target genes were successfully packaged. Conclusion The pseudovirus particles containing gene fragments of five viruses are constructed,which can be used as a common reference standard for NA detection.

Detection of the Siberian Tick-borne Encephalitis Virus in the Xinjiang Uygur Autonomous Region, northwestern China / 病毒学报

Until the recent emergence/re-emergence of human-pathogenic viruses in ticks, tick-borne viruses have been neglected as causative agents of human disease (particularly in China). To gain insight into the diversity of tick-borne viruses in Xinjiang Uygur Autonomous Region (northwestern China), we conducted illumina deep sequencing-based screening for virus-derived small RNAs in field-collected Ixodes persulcatus ticks. We found 32, 631 unique virus-matched reads. In particular, 77 reads mapped to the tick-borne group within the genus of Flavivirus, and covered 3.8%-2.4% viral genomes. In addition, 32 unique reads were specific to the Siberian subtype of tick-borne encephalitis viruses (TBEV-Sib) which have never been reported in Chinese TBE loci. We confirmed the potential existence of TBEV-Sib by amplification (using reverse transcription-polymerase chain reaction) of genomic fragments from the envelope gene or 3' genomic terminus from the pools of examined ticks. Both sequences demonstrated high homology to TBEV-Sib strains attached geographically to southern Siberia with nucleotide identity of 97.2%-95.5% and aminoacid identity of 99.4%-98.3%, respectively. In conclusion, we report, for the first time, detection of TBEV-Sib in the natural TBE loci of China. These novel data may provide genetic information for further isolation and epidemiologic investigation of TBEV-Sib.

Identification of Culex Flavivirus by deep sequencing approach in Xinjiang, China / 中华微生物学和免疫学杂志

Objective To investigate the diversity of mosquito-borne viruses in Xinjiang , China, and to identify mosquitos-borne viruses of medical importance rapidly .Methods The virus-derived RNAs in mosquitos captured in wild were screened and confirmed by using Illumina deep sequencing approach and reverse transcription PCR , respectively .The alignment analysis was performed by using gene sequences from GenBank.Results One hundred and forty-four Culex Flavivirus ( CxFV, Flavivirus genus, Flaviviridae) specific sequences were identified .The overlapping reads were assembled into 7 uncontinuous viral genomic contigs.The gaps between the contigs were further filled by RT-PCR products, which resulted in reconstruc-tion of viral genomic 5′and 3′terminus (687 nt and 411 nt).Phylogenetic analysis showed that the newly identified CxFV belonged to America/Asian genotype , which specifically clustered into a clade with other CxFV strains from China mainland ,sharing 98.2%-99.5%homologies in nucleotide sequences and 99.5%in amino acids sequences among them .Conclusion Illumina deep sequencing approach was successfully applied to arthropod-borne virus surveillance .The recently emerged Culex Flavivirus was detected for the first time in Xinjiang, China.

Expression and purification of recombinant JEV EDⅢ protein and its application in Array-ELISA assay / 中华微生物学和免疫学杂志

Objective To express and purify Japanese encephalitis virus ( JEV) EDⅢprotein and evaluate the possibility of using it as a candidate antigen in JEV diagnostic kit .Methods PCR primers spe-cific for the gene encoding JEV EDⅢprotein were designed and used to amplify the gene fragment by RT-PCR.The cloned gene fragment was then inserted into pET-30a (+) to construct the recombinant expression plasmid.The transformed E.coli BL21 carrying expression plasmid were induced by IPTG to express JEV EDⅢ protein.The expressed JEV EDⅢprotein and a control antigen of tick-borne encephalitis virus protein were deposited in small spots to set up ELISA microarray .The serum samples from patients with Japanese encephalitis and healthy people were detected by Array-ELISA.The results obtained by Array-ELISA were compared with those by using indirect immunofluorescence assay .Results The gene fragment encoding JEV EDⅢprotein was successfully cloned and expressed in E.coli BL21.The recombinant protein could be used in Array-ELISA assay for the detection of serum samples from patients with Japanese encephalitis and healthy subjects .The results were consistent with those by using indirect immunofluorescence assay.Conclusion The recombinant JEV EDⅢprotein can be used as a candidate antigen for the diagnosis of JEV infection .

Application of an array-ELISA for evaluation of diagnostic antigens of HFRS virus / 中华微生物学和免疫学杂志

Objective To express and purify the recombinant nucleoprotein fragments of hemor-rhagic fever with renal syndrome ( HFRS) virus and to evaluate their diagnostic efficacy by using array-ELISA technology .Methods The target genes encoding nucleoprotein fragments of HFRS virus were amplified by PCR, and then inserted into prokaryotic expression vectors to construct the recombinant plasmids of pET -32a (+)/Pn and pET-32a(+)/Pc.The plasmids were transformed into E.coli BL21 ( DE3) to induce the ex-pression of nucleoprotein fragments by IPTG and the expressed products were purified by affinity chromatog -raphy using Ni-NTA agarose.The specificity and sensitivity of the recombinant antigens were evaluated by the assay of array-ELISA using commercial colloidal gold assay kit as a comparison .Results The recombi-nant nucleoprotein fragments of HFRS virus were correctly expressed in E.coli and highly purified by affinity chromatography .Array-ELISA showed that 13 of 16 suspected serum samples were positive by using the His-Pn protein as diagnostic antigen , consistency with the commercial colloidal gold assay kit reaching 94%. Conclusion The recombinant His-Pn protein expressed in E.coli cells could be used for specific serodiag-nosis of HFRS virus as its high antigenicity and sensitivity .The array-ELISA is an effective assay for the de-tection of virus at protein level .

Studying the pathogenicity of TBEV to human neuroblastoma tumor cells / 中华微生物学和免疫学杂志

Objective To identify the infection and the replication of Tick-borne encephalitis virus(TBEV) in human neuroblastoma cells.Methods After being inffected with TBEV,the cell culture supernatant of human neuroblastoma cell line SK-N-SH was collected and assayed at different time points.Byusing real-time RT-PCR and plaque assay to measure the titer of virus in the supernatant,the replication andproliferation of TBEV in human neuroblastoma cell was identified.Meanwhile,the morphological change of SK-N-SH after TBEV infection was also visualized by observation under microscope and immunmquorescenceassay.Results Real-time RT-PCR and plaque assay both demonstrated that TBEV could replicate effectively in SK-N-SH cells,the peak titer could reach 2.92× 107 PFU/ml on 3 days post-inoculation.And significant morphological change occured on infected SK-N-SH cells after 2 days post inoculation.By immunofluorescence assay,the virus particles could be detected and visualized.Conclusion TBEV can replicate andproliferate effcctively and cause significant cell morphological changes in human neuroblastoma cell SK-N-SH,which demonstrated that SK-N-SH could be a suitable cell model for TBEV culture.

Development of antibody-array for detection of six arboviruses / 中华微生物学和免疫学杂志

ObjectiveTo develop an antibody-array system for multiple detection of antibodies against Japanese B encephalitis virus (JEV),Tick-borne encephalitis virus (TBV),Dengue virus ( DENV ),West Nile virus (WNV),Western equine encephalitis virus (WEEV) and East Equine encephalitis virus (EEEV).MethodsRecombined antigens were spotted on array as capture antigens.Specific antibodies were detected by using a sandwich ELISA format.Rabbit antiserum was employed to select and confirm the specificity of antigens and to optimize the conditions of the assay.The detection efficiency of the system was validated by 40 clinical suspected serum samples and compared with the relative ELISA assays.ResultsEleven recombined antigens were selected as diagnostic antigens with high specificity.Better detection could be achieved when scale of antigen concentrations were within 0.125-0.900 mg/ml and the serum dilutions were 1:100-1:1000.When detecting the 26 clinical suspected TBE serum samples,20 were IgG positive (76.9％),and 17 were IgM positive (65.3％) which was 96.1％ and 84.6％ consistent with the relevant ELLSA tests,the 8 clinical suspected JEV serum samples,4 were IgG positive (50.0％),and 5 were IgM positive (62.0％),which was 86.3％ and 90.1％ consistent with the relevant ELLSA tests.As for the 22 DEN serum samples,13 were IgG positive (60％) and 15 were IgM positive (68％) which was 85％ and 93％ consistent with ELISA.The specificity of the assay was 100％ and the sensitivity was higher than the relative ELISAs.ConclusionThe developed antibody-array is highly specific and reliable,which could be used for the detection of antibodies against the 6 arboviruses.

The application of monoclonal antibody 2A10 on protein chip for detecting of Flavivirus / 中华微生物学和免疫学杂志

Objective To evaluate the Flavivirus specific monoclonal antibody(McAb) 2A10 as detective antibody for simultaneously identify tick borne encephalitis virus( TBEV), Japanese encephalitis virus( JEV), dengue ( DEN )-2, DEN-4 and yellow fever virus ( YFV ) by antibody microarray technique.Methods The antibody microarray was developed by spotting TBEV, JEV, DEN-2, DEN-4 and YFV specific McAb on chip as capture antibodies. After incubating with cultured viral supernatants of the above viruses, CY3 labeled detective antibody 2A10 was added to the chips. After reaction, the antibody microarray was scanned and the results were analyzed. By comparing the signal intensities of different spots on chips,the detecting titre and sensitivity of 2A10 for Flavivirus were determined, and the value of 2A10 in detection of Flavivirus was evaluated. Results The hybridization results demonstrated that the titre of 2A10 for Flavi2A10 was specific for Flavivirus and could be used as universal detective antibody for Flavivirus on antibody microarray.

Construction of cDNA library of the treated Changliver cell and quality analysis / 新乡医学院学报

Objective To construct cDNA library of the treated Changliver cell by switching mechanism at 5′ end of RNA transcript (SMART) technique and analyze its quality.Methods cDNA of Changliver cell was aquired with reverse transcription polymerase chain reaction (RT-PCR) and long-distance PCR (LD-PCR),then the cDNA library was constructed with SMART cDNA library construction kit.Results Through testing,the high quality cDNA library containing full length cDNA of Changliver cell had been constructed.The titer of the amplified cDNA library was 4.5 × 1010 pfu*ml-1 and the average exogenous inserts of the recombinants was 1.5 kb.Conclusion These results suggest that the Changliver cell cDNA library has a high quality and lays a solid foundation for researching on Changliver cell and screening

Identification and characterization of 177 unreported genes associated with liver regeneration / 基因组蛋白质组与生物信息学报·英文版

The mammalian liver has a very strong regeneration capacity after partial hepatectomy (PH). To further learn the genes participating in the liver regeneration (LR), 551 cDNAs selected from subtracted cDNA libraries of the regenerating rat liver were screened by microarray, and their expression profiles were studied by cluster and generalization analyses. Among them, 177 genes were identified unreported and up- or down-regulated more than twofold at one or more time points after PH, of which 62 genes were down-regulated to less than 0.5; 99 genes were up-regulated to 2-10 folds, and 16 genes were either up- or down-regulated at different time points during LR. By using BLAST and GENSCAN, these genes were located on responsible chromosomes with 131 genes on the long arms of the chromosomes. The cluster and generalization analyses showed that the gene expression profiles are similar in 2 and 4, 12 and 16, 96 and 144 h respectively after PH, suggesting that the actions of the genes expressed in the same profiles are similar, and those expressed in different profiles have less similarity. However, the types, characteristics and functions of the 177 genes remain to be further studied.