Retrovirus (RV) is the most widely used vector for gene transfer, but its efficiency is very low for human cells. A Flow-through Transduction System made in General Hospital of PLA was used to deliver interleukin-12 RV vector into human leukemia cell line K562. Compared with static procedure, the efficiency could be significantly improved,and detected by clony forming units (CFU) for NeoR gene in semisolid culture medium with G418 (83.1%±3.9% vs 9.2%±2.3%, P＜0.01). No significant difference was found in the levels of IL-12 p70 protein expression detected by ELISA, by flow-through transducted cells without G418 selection and by statically-transducted cells with G418 selection (11.3±7.5ng/ml vs 12.0±6.4ng/ml, P>0.05), while statically transducted cells without G418 selection could express very low IL-12 (0.1±0.024ng/ml，P＜0.01)。The Flow-through Transduction System is efficient and easy for use, it can be widely used in research and clinic of gene therapy.

Retrovirus (RV) is the most widely used vector for gene transfer, but its efficiency is very low for human cells. A Flow-through Transduction System made in General Hospital of PLA was used to deliver interleukin-12 RV vector into human leukemia cell line K562. Compared with static procedure, the efficiency could be significantly improved,and detected by clony forming units (CFU) for NeoR gene in semisolid culture medium with G418 ( 83.1%?3.9% vs 9.2%?2.3%, P0.05), while statically transducted cells without G418 selection could express very low IL-12 (0.1?0.024ng/ml,P

C57BL/6 synergistical mice were divided into 4 groups (10 mice each). The first group was negative control without any interference. Each mouse in the other 3 groups was subcutaneously inoculated with 1 10 6 wide type (wt) EL4 tumor cells. Then each group was treated either with interleukin 12 (mIL 12) in vivo vaccine or NeoR control vaccine or PBS(positive control group) on day 1, 7, 14, 21, on the same place where wt EL4 tumor was inoculated. mIL 12 in vivo vaccine and NeoR control vaccine were package cells which can produce retrovirus (RV) with mIL 12 or NeoR gene, the vaccine was 60 Co irradiated and injected (1?10 7 cells/mouse). All mice in positive group and NeoR control group died of tumors in a month, while 5/10 mice in mIL 12 in vivo vaccine groups survived without tumors for more than 60 days. The 5 survived mice were rechallenged with 5?10 5 wt EL4 cells, 3/5 mice even survived without tumors for another 60 days. Among the mice with tumors in vivo vaccine group mice, compared with the controls, the development of tumors was delayed ( P

Objective To set up a new real-time quantitative PCR method for the detection of minimal residual disease in chronic myeloid leukemia patients, and to assess the bcr/abl fusion gene expression in chronic myeloid leukemia patients before and after treatment with imatinib mesylate by real-time quantitative PCR method. Methods The bcr/abl fusion gene expression in 30 patients with bcr/abl-positive chronic myeloid leukemia was analyzed by using real-time quantitative reverse transcription PCR (RQ-PCR) method. The patients treated with imatinib in a dose of 400mg/d for 1 year and 2 years were also examined (8 cases for each). In 19 new patients the same study was also conducted. Results The real time quantitative PCR method could detect 10 copies in the test. The average bcr/abl expression levels in new patients or patients who had been treated with imatinib for 1 year and 2 years were 68.18%?26.67%, 0.16%?0.15% and 0.04%?0.02%, respectively. The average logarithm reduction values after treatment were 2.82 in the first year and 3.36 in the second year. In 25% of patients (4/16) negative FISH results could not be obtained, but it was much lower than that of before imatinib-treatment. When FISH became negative, RQ-PCR showed positive results. Conclusions RQ-PCR is a more sensitive technique in the detection of bcr/abl fusion gene than the FISH. It is an important way to monitor the tumor cell during the treatment with imatinib mesylate in chronic myeloid leukemia patients.

Objective To establish a clinical test assay for detecting common respiratory viruses and enteroviruses (EV) by using mixed cultured Madin-Darby canine kidney cells (MDCK), human epidermoid cancer cells (Hep-2) and African green monkey kidney cells (Vero) to isolate common respiratory viruses and enteroviruses. Methods Throat swabs with influenza A and B viruses,adenovirus and EV71 were incubated with mixed cultured MDCK, Hep-2 and Vero in a single vial to observe the presence of cytopathic effects. Polymerase chain reaction (PCR), reverse transcription (RT)-PCR and monoclonal antibody-based immunofluorescene assay were also used for confirmatory test. Results The sensitive cell lines developed obvious cytopathic effects to the corresponding viruses, which were confirmed by the specific green particles observed by immunofluorescence assay and specific target PCR segments. Conclusions The shell-vial of mixed cells can simultaneously isolate different common respiratory viruses and EV. The isolated pathogens can be further confirmed by antigen test and PCR. This assay may improve the diagnosis of clinical viral diseases.

Objective To study the gene characteristics of VP1 region of Enterovirus 71 (EV71) strains isolated from clinical specimens of children with hand-foot-and-mouth disease(HFMD) in Shanghai in 2010.MethodsEighteen EV71 isolates were selected from different periods of year 2010,including strains isolated from fatal cases and non-fatal cases.Complete VP1 gene (891nucleotides) of the eighteen EV71 isolates were amplified and sequenced,and then compared with that of genotype A,B,C reference EV71 strains in GeneBank by homogeneity and phylogenetic tree analyses.ResultsThe nucleotide homogeneities between these 18 Shanghai strains and the representative isolates of genotype A and B were 81.5 ％ -82.6 ％ and 83.4 ％- 84.2 ％,respectively,while the amino acid homogeneities were 94.3 ％- 95.0％ and 96.6％ -97.0％,respectively.The nucleotide and amino acid homogeneities between the 18 Shanghai strains and the representative isolates of genotype C were 87.4％- 99.2％ and 98.7％ -100.0％,respectively.Of note,the nucleotide and amino acid homogeneities between Shanghai strains and Fuyang EV71strains (representative strain of C4 subtype) appeared to be 97.8％- 99.2％ and 99.3％- 100.0％,respectively.The eighteen EV71 Shanghai strains were classified as genotype C,subgenogroup C4 in the phylogenetic tree.There was no remarkable difference in VP1 gene between the strains isolated from fatal cases and non-fatal cases.ConclusionThe EV71 strains isolated from Shanghai belong to subgenogroup C4.

Objective To evaluate the survivability of hand foot mouth disease(HFMD)virus,in tap water for daily use.Methods HFMD viruses were isolated from cases of HFMD in Shanghai and Zhejiang from in 2008.Six isolated strains (five subtype of enterovirus 71 and one coxackie virus)were selected in this study.These viruses were mixed with chloride 1.0 mg/L tap-water and then inoculated into Vero cells.The cytopathic effect (CPE)was checked everyday in order to survey the survivability of each virus strain.The decline of virus survivability was analyzed by scatter diagram.Results These six strains of HMFD virus could survive longer than one month in tap water with initial chloride concentration of 1.0 mg/L and still had celluar infectivity.The survivabilities were varied between viruses isolated from different HFMD cases.Conclusions The survivabilities of enterovirus 71 and coxackie virus stains are quite strong in water.Therefore,the transmission route of water-borne pathogens should be monitored in regions using tap water during HFMD epidemic period.

Objective To evaluate the survival ability of enterovirus 71 (EV71) on different surface and under different climate.Methods Each 1 × 105 tissue culture infective dose 50 (TCID50)EV71 was added on different aseptic surface of plastic,rubber,cloth and wood,respectively.Then these materials were put into biotron (artificial climatic chamber) which could simulate different temperature and moisture.The viruses were recovered after a definite time and then inoculated into Vero cell.The cytopathic effect (CPE) was observed everyday to survey the survival ability of EV71 on different medium surface.Results The recovery rates of EV71 on medium surface ranged from 89 ％-93 ％.The survival time of EV71 on medium surface varied under different climatic conditions.The longest survival time of the virus was observed under the condition of 20 ℃ as the temperature and 80％ as the humidity.After 24 hours of incubation,the infectious titer on plastic surface reduced about 4 lg.After 72 hours of incubation,the infectious titer reduced at least 3.89 lg on cloth and wood surface.Conclusions Temperature and humidity can affect the survival time of EV71 on medium surface,which is longer in the condition of low temperature and high humidity.The survival ability of EV71 on natural cloth and wood surface is better than that on synthetic plastic surface.

Objective To test the levels of enterovirus 71 (EV71) antibody among children of different ages in Shanghai in 2011,and to investigate the relationship between antibody levels and virus infection.Methods EV71 antibody was detected by microneutralization assay from the serum specimens of healthy children of different ages collected during July to August,2011.The results were analyzed by t test for quantitative data with normal distribution,and by x2 test for count data.Results The positive rate of EV71 antibody among the 93 serum specimens was 58.1％ (54/93).The geometric mean titer (GMT) of EV71-specific neutralizing antibody was 1 ∶ 14.48.The positive rate of EV71 antibody in infants less than 6 months old was 87.5％ (21/24),and the GMT was 1∶29.56.In children aged 2 to 3 years,the positive rate of EV71 antibody decreased to 3.7％ (1/27),and GMT decreased to 1∶4.21,which were both statistically significantly lower than those less than 6 months old (x2 =36.37,t=7.58; both P＜0.01).The positive rate of EV71 antibody increased to 83.3％ (20/24) in children aged 5 to 6 years,with GMT reaching 1∶21.74.Whereas in children aged 7 to 8 years,the positive rate was 66.7％ (12/18) and GMT was 1∶20.76,without statistically significant difference compared with those aged 5 to 6 years (x2 =1.58,t=0.597; both P＞0.05).No statistically significant difference was found between boys and girls in positive rate of EV71 antibody [62.7 ％ (32/51) vs 52.4 ％ (22/42),x2 =1.02,P＞0.05] or GMT (1 ∶ 16.23 vs 1 ∶ 12.61,t=0.881,P＞0.05).Conclusions Children aged 2 to 3 years were at higher risk for EV71 infection,with EV71 antibody level significantly lower than other age groups.

Objective To understand the genetic and antigenic variations of influenza A (H1N1) isolates in Shanghai area in winter of 2010.Methods A total of 137 throat swabs were collected from patients with influenza-like illness in the sentinel hospital in Shanghai area from December 2010 to January 2011,then inoculated into Madin-Darby canine kidney (MDCK) cells.The types of influenza were identified by direct immunofluorescence assay (DIF) and influenza A (H1N1) subtype was determined by reverse transcriptase-polymerase chain reaction (RT-PCR).The mutations of gene and amino acid locus were analyzed through the whole genome sequencing of hemagglutinin (HA),neuraminidase (NA) and polymerase (PB2) segments from some influenza A (H1N1) isolates.Results Total of 53 human influenza virus strains were isolated including 48 influenza A (H1N1) virus strains.Nineteen strains were selected for sequencing by simple random sampling.The phylogenetic tree of HA gene revealed that the latest isolates and most of influenza A (H1N1) viruses isolated before June 2010 were not in the same stem.Analysis of amino acid residues in HA protein showed that mutations were found in antigenic determinant region in some strains.Residues at the enzyme active sites of NA protein were strictly conservative,no change was observed in amino acid residues which were related to drug resistance against oseltamivir and zanamivir.The 627 and 701 residues in PB2 protein were glutamic acid and aspartic acid,respectively,which was still the feature of avian influenza virus,but E677G mutation was detected.Conclusion Compared to influenza A (H1N1) strains isolated in spring and summer,some variations have been detected in the strains isolated in Shanghai area in winter of 2010,some antigen drift and adaptive evolution in mammalian hosts have appeared.