Objective To develop a real-time quantitative PCR(RQ-PCR) assay based on TaqMan technology for rapid detection and quantification of tick-born encephalitis virus (TBEV) RNA. Methods According to all the TBEV genome sequences in GenBank, RQ-PCR primers and probes were designed in the conservative regions of TBEV C gene and NS5 gene. In addition, primers for conventional PCR were designed using E gene as target. The detective system was established and validated by using TBEV MDJ01 strain. In order to examine the specificity of the system, other viruses of flavivirus were assayed with the RQ-PCR simultaneously. The TBEV standard curve was drawn respectively by measuring TCID_ 50 titre and copy number. The sensitivity of RQ-PCR and the conventional PCR assays were compared, and TBEV infected mice model was reproduced for evaluation. Results The sensitivity of RQ-PCR assay was 100copies/reaction or 1 TCID_ 50 , which was 10 fold higher than conventional PCR. The results were all negative when used to detect other flavivirus including the yellow fever virus, dengue virus type 1, 2, 3 and 4, Japanese encephalitis virus, West Nile virus. The coefficient of variability was less than 5% from inter- and intra-assay showing that both the repeatability and stability of the system were good. Conclusion A sensitive, specific and convenient RQ-PCR method has been established, which is valuable for early detection of TBEV.

Objective To evaluate the diagnostic value of TSSC1 in glioma patients and its influence on cell biologi-cal behavior of glioma U87 cells. Methods RT-PCR and Western blot were used to examine the expression of TSSC1 in glioma samples, including 80 normal paraneoplastic tissues and 80 primary tumors. MTT and transwell were used to analyze the effect of TSSC1 knockout on proliferation, migration, and invasion in U87 cells. Results TSSC1 is down-regulated in glioma compared to its paraneoplastic counterparts and negatively related to higher grade. Furthermore, knockdown of TSSC1 expression results in increased proliferation, migration and invasion in U87 cells in vitro. Conclusion Our results may worked as a marker for early diagnosis and prognosis of glioma.

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.
Animals ; Arachnid Vectors ; virology ; China ; Encephalitis Viruses, Tick-Borne ; classification ; genetics ; isolation & purification ; Encephalitis, Tick-Borne ; transmission ; virology ; Genome, Viral ; Humans ; Ixodes ; virology ; Molecular Sequence Data ; Phylogeny

Objective To detect human metapneumovirus (hMPV)in respiratory intection rapidly and perform molecular analysis of hMPV.Methods Seven respiratory tract virus(11 subtypes)were assessed using multiplex PCR technology and flexible Multi-Analyte Profiling(suspension array).Human metapneumovirus was confirmed by using a real.Time reverse ranscriptase CR(RT-PCR)assay followed by sequencing.The cladogram analysis was performed further.Results The virus were detected in 40.2%(19/47)samples collected from clinicsl respiratory tract infections,including 8(42.1%)HRSV,7(36.8%)influenza virus,1(5.3%)parainfluenza virus,1(5.3%)rhinovirus,1(5.3%) coxsackievirus and 1(5.3%)human etapneumovirus infections.This is the first time that hMPV was deteced from clinical samples in Shenzhen.The sequencing of specific fragment of neucleoprotein of hMPV showed this hMPV shares over 98% homology with Beijing strain.Japan strain and Thailand strain.The cladogram analysis showed that they were in the same cluste.Conclusions Human etapneumovirus is a maior cause of children respiratory tract disease. Multiplex PCR technology and nexible Multi-Analyte Profiling were hish sensitive and high-throughput for detection of human metapneumovirus.They axe very robust and applicable in etiology analysis.

Objective To express the capsid proteins of WU polyomavirus(WUPyV) for research and find antigen for diagnostic value. Methods Coding sequences of capsid proteins of WU polyomavirus by PCR were cloned in prokaryotic expression vector PGEX-20T. Recombinant plasmids were transformed into E. coli BL21(DE3) and induced by IPTG for proteins expression. Recombinant proteins were identified by Western blot. Results SDS-PAGE proved that recombinant proteins showed three bands with molecular relative mass of 69×103, 63×103 and 56×103. The recombinant proteins were recognized by anti-GST McAb. The antigenicity was tested by Western blot using 16 WU polyomavirus positive and 70 negative sera. Conclusion Recombinant VP1, VP2 and VP3 expressed in E. coli can combine with WUPyV-Ab and have good antigenicity. They can be used for further research.

Objective To study the relationship of immunophenotype and prognosis of acute leukemia(AL). Methods 75 patients with AL were analyzed immunophenotype expression by FCM and evaluated the effect of differ-ent immunophenotype to prognosis. Results (1) The incidence of CD13, CD33, CD64, CD117 expression in AML was 82%. The incidence of CD2, CD3, CD7, CD19, CD20 expression in ALL was 88%. The incidence of lymphocytic lineage antigen expression in AML(Ly + AML) was 13% and myeloid lineage antigen expression in ALL(My + ALL) was 11%. (2)According to the antigen expression, AL could be classified into three subgroups:lineage-specific expres-sion;mixture-lineage expression and null type. The lineage-specific expression was the highest in AML and ALL, and had a better clinical prognosis. The null type was the lowest neither in AML nor ALL and had a poorer clinical progno-sis. In mixture-lineage expression the CR rate of AML with CD7+ was the lowest than those with lineage-specific ex-pression and had poorer prognosis. Conclusions AL immtmophenotype might be devided into three subgroups:line-age-specific expression; mixture-lineage expression and null type. In the patients with CD7+ AML and null type ex-pression,lower CR rate and poorer prognosis were seen than those with lineage-specific expression. It needed to ex-plore new treatment methods.

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.

Objective To investigate the application and effect of aerobic exercise of pulmonary rehabilitation in patients with chronic obstructive pulmonary disease (COPD).Methods Ninety-four hospitalized patients with COPD from June 2011 to June 2012 were enrolled.The clinical curative effect and safety of aerobic exercise of pulmonary rehabilitation in patients with COPD were observed and compared.Results After 3 months treatment,first second forced expiratory volume (FEV1) and forced vital capacity (FVC) were increased compared with those before treatment [(3.25 ± 0.49) L vs.(2.59 ± 0.55) L,(1.95 ± 0.41) L vs.(1.44 ± 0.48) L],and there were significant differences (P ＜ 0.05).There was no significant difference in FEV1/FVC before and after treatment (P ＞ 0.05).After 3 months treatment,arterial partial pressure of oxygen and arterial oxygen saturation were increased compared with those before treatment [(87.61 ± 8.56) mm Hg(1 mm Hg =0.133 kPa) vs.(63.88 ± 8.79) mm Hg,0.9648 ±0.0449 vs.0.7632 ± 0.0477],partial pressure of arterial carbon dioxide was decreased compared with that before treatment [(30.57 ± 9.47) mm Hg vs.(49.23 ± 9.54) mm Hg],and there were significant differences (P ＜ 0.05).There was no signifi.cant difference in blood pressure and heart rate before and after treatment (P＞ 0.05).No obvious adverse reactions were observed.Conclusions The aerobic exercise of pulmonary rehabilitation can improve significantly lung function.It is safe and rehable,and has less adverse reaction in patients with COPD.It is worthy of promotion and use.

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.

Objective: To test the effectiveness of psychological flexibility training on career adaptability among middle school students who undertook psychological courses based on acceptance commitment therapy and the adolescent mental flexibility model(DNA-V), and to provide a reference plan to improve the mental health of middle school students. Methods: This study recruited 110 junior high school students (60 boys and 50 girls) from a middle-school in Beijing. The students were randomly divided by class into a DNA-V face-to-face course group(offline group n=33), a DNA-V online course group(online group n=40), and a regular school psychology course group(control group n=37). Louise Hayes DNA-V intervention program was condensed into a six-hour middle-school DNA-V psychology curriculum. Using the Avoidance and Fusion Questionnaire for Youth and the Career Adaptability Scale, changes in psychological flexibility and career adaptability were measured before(T1), one week after(T2), and two months after (T3) the intervention. Results: Linear mixed models were used for the analysis, while controlling for demographic variables. Psychological flexibility and career adaptability in the offline group were higher at T2 and T3 than at T1(psychological flexibility t=4.22, 3.11; career adaptablity t=3.05, 4.16, P<0.01), while the difference between T2 and T3 was not statistically significant. The psychological flexibility and career adaptability of the online group were not statistically significant at T1, T2, and T3. The psychological flexibility and career adaptability of the control group increased from T1 to T2(t=4.64, 2.47, P<0.05), but T3 decreased back to a level close to T1. Conclusion In terms of both psychological flexibility and career adaptability, the DNA-V face-to-face psychology course resulted in a retention period of at least two months.