BACKGROUNDTo detect quantitatively HCMV DNA in peripheral blood leukocytes to monitor the status of HCMV infection, evaluate the effectiveness of antiviral treatment with ganciclovir (GCV) combined with intravenous immunoglobulin (IVIG) and find out the relationship among the HCMV DNA levels, the state of infection and the clinical outcome.The long-term goal of the study was to establish a molecular diagnostic standard for HCMV infection in children.

METHODS45 cases of suspected HCMV-infected children were examined by PCR, ELISA and fluorescent quantitative (FQ)-PCR, respectively. Twenty five HCMV hepatitis cases of the 45 were randomly assigned to a treated group or a control group. Both groups were treated with prednisone, glucurone, Luminal and Xiaoyanlidanpian. But the treated group was given with GCV+IVIG in addition. Each infant of the two groups was checked with FQ-PCR at the five time points.

RESULTSThe positive rates of PCR, ELISA and FQ-PCR were 60.00%, 33.33% and 66.67%,their sensitivities were 84.38%, 46.88% and 93.75%, respectively. There was no significant difference in viral DNA copy numbers between the two groups before being treated (P>0.05), but there was significant difference between HCMV hepatitis and normal infants (P<0.001). Although viral load of both groups decreased in both groups, the viral load of the treated group decreased more significantly. The level of HCMV DNA fell to 103 copies/ml at second time point while that of the control group fell to the same level after third time point. The differences between the two groups at each time point were statistically significant (P<0.001). The results of 135 person times testing indicated that 103 copies/ml of FQ-PCR can be taken as a critical value for prediction of active HCMV infection.

CONCLUSIONSFQ-PCR may be one of the effective methods for diagnosis of HCMV disease; it can offer a key index in the diagnosis of HCMV active infection; dynamic detection of HCMV viral load can play a role not only in monitoring antiviral therapy, but also in evaluating the development and prognosis of HCMV disease.

Antiviral Agents ; therapeutic use ; Cytomegalovirus ; isolation & purification ; Cytomegalovirus Infections ; diagnosis ; drug therapy ; DNA, Viral ; blood ; Ganciclovir ; therapeutic use ; Humans ; Infant ; Infant, Newborn ; Polymerase Chain Reaction ; methods ; Sensitivity and Specificity ; Treatment Outcome

BACKGROUNDTo find a rapid and sensitive method for early diagnosis of nasopharyngeal carcinoma by using EBV TK kinase.

METHODSProkaryotic expression plasmid pRSETTK was constructed. EBV TK kinase was highly expressed in E.coil BL21 (DE3). The authors identified specificity of TK kinase by Western blot, then used purified TK kinase in ELISA to detect the IgG antibody in the serum of NPC patients.

RESULTSSpecific IgG antibody against TK kinase was found in the serum of NPC patients. The specificity and sensitivity of TK kinase were both 100% in Western blot and were 98.0% and 93.4% respectively in ELISA.

CONCLUSIONSThe EBV TK kinase showed high specificity and sensitivity in ELISA, therefore it can be used for early diagnosis of NPC

Antibodies, Viral ; blood ; Enzyme-Linked Immunosorbent Assay ; methods ; Herpesvirus 4, Human ; enzymology ; Humans ; Immunoglobulin G ; blood ; Nasopharyngeal Neoplasms ; diagnosis ; Recombinant Proteins ; biosynthesis ; immunology ; isolation & purification ; Sensitivity and Specificity ; Thymidine Kinase ; biosynthesis ; isolation & purification

BACKGROUNDTo identify the relationship of cervical cancer with pathogen infectious, cytokine and Se.

METHODSOn the one hand regarded tissues of the carcinoma of the uterine cervix with 195 cases as the experimental group and ordinary cervical tissues with 75 cases as the control group. Polymerase chain reaction (PCR) to detect them. On the other hand applied ELISA to detect cytokine IL-2R, TNF and fluorescent luminosity technique to detect element Se in the serums.

RESULTSIn the experimental group those infected pathogen were 166 cases (85.1%), In all of pathogen HPV 16,18,35 type were 60 cases (30.8%), HSV 2 were 60 cases (30.8%), While those ordinary tissues infected pathogen were 15 cases (20 0%),in the contrast group. HPV 16,18,35 type and HSV 2 mere 4.0% and 6.7% respectively,(P<0.001). In the serums of effective 62 experimental objects IL-2R (x=356.44 U/ml) and TNF (x=373.48 pg/ml) were much high than them in the serums of effective 36 contrast objects (P<0.001). But Se (x=0.058 mg/ml) was lower than it in the serums of contrast objects (P<0.05).

CONCLUSIONSThe occurrence of carcinoma of the uterine cervix is closely connected with infection of HPV 16,18,35 and HSV2, high level of cellular factors IL-2R, TNF and low level of element Se.

Female ; Herpes Simplex ; virology ; Humans ; Papillomavirus Infections ; virology ; Polymerase Chain Reaction ; Receptors, Interleukin-2 ; blood ; Selenium ; blood ; Tumor Necrosis Factor-alpha ; metabolism ; Tumor Virus Infections ; virology ; Uterine Cervical Neoplasms ; blood ; virology

BACKGROUNDTo clone the type common antigen gD of human herpes simplex virus I, II (HSV-I, II), the authors constructed recombinant expression vector Pmal-c2/gD and induced to express the fusion protein MBP-gD.

METHODSThe authors extracted HSV DNA,amplified gD gene by PCR assay and directly cloned it into prokaryotic expression vector pMAL-c2, then transformed it into E.coli DH5alpha. After proved to be correct by PCR, double enzyme digestion and sequencing, the fusion protein is induced to express by IPTG and detected by both Western blot and ELISA.

RESULTSThe constructed expression vector pMAL-c2/gD can be expressed with high efficiency. The product expressed was about 35.5% of the total bacterium proteins by SDS?PAGE analysis and was found nearly 39% as soluble protein,61% as inclusion in cytoplasm.

CONCLUSIONSThe authors constructed recombinant expression vector pMAL-c2/gD, the Western blotting result showed that the recombinant protein could be identified with gD specific monoclonal antibody DL6. Therefore the protein was of natural antigenic structure of gD.

Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Humans ; Plasmids ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; Viral Envelope Proteins ; biosynthesis ; genetics

BACKGROUNDTo study the effects of dendritic cells (DC) transfected with recombinant adenovirus encoding Epstein-Barr virus(EBV)latent membrane protein 2A(LMP2A)gene, and to provide evidence for further investigation on the therapeutic vaccine against EBV-associated malignancies.

METHODSDCs were transfected with EBV-LMP2A recombinant adenovirus (Ad5-LMP2A), which was generated by homologous recombination. The expression of LMP2A protein on mature DC transfected with Ad5-LMP2A at different multiplicity of infection(MOI)was analyzed by fluorescence activated cell sorting(FACS), and the dead cells were counted by trypan blue staining. The alteration of surface markers on mature DC including CD1a, CD83, CD40, CD80, HLA?DR was detected by means of FACS before and after transfection. Meanwhile, the functions including stimulating allogenetic T cells reaction and expressing IL12 P40 mRNA on transfected DC were measured by methods of 3H-thymidine uptake and fluorescent semi?quantitative polymerase chain reaction (PCR), respectively.

RESULTSAbout 80% mature DC expressed LMP2A protein and >92% cells were viable after transfection at the MOI of 200 No. significant changes in the surface markers and the cytomorphology of mature DCs were detected during the transfection. Transfected DC still have strong potential to stimulate the proliferation of allogenetic T cells and to express IL-12 P40 mRNA.

CONCLUSIONEBV-LMP2A gene,which was carried by adenovirus vectors, could be transferred into DC with high efficiency. The function of mature DC was not affected significantly by the transfection of Ad5-LMP2A.

Adenoviridae ; genetics ; Cells, Cultured ; Dendritic Cells ; physiology ; Gene Expression ; Genetic Vectors ; Humans ; Recombination, Genetic ; Transfection ; Viral Matrix Proteins ; genetics

BACKGROUNDTo get early laboratory study of type specific antigenicity of herpes simplex virus II.

METHODSPCR and prokaryotic expression technique.

RESULTSHerpes simplex virus II type specific gene fragment was expressed in E.coli and the products can be used as specific antigen for the detection of anti\HSV in the recovery sera.

CONCLUSIONSCloning and express of HSVII type specific antigen found the basis for developing specific diagnosis methods and vaccine of HSV.

Antigens, Viral ; immunology ; Cloning, Molecular ; Gene Expression ; Herpesvirus 2, Human ; genetics ; Humans ; Immunoglobulin G ; blood ; Polymerase Chain Reaction ; Recombinant Proteins ; immunology

BACKGROUNDTo evaluate the influence of assays with primer labeled with fluorochrome (Cy5) and dUTP labeled with Cy5 on the signal intensity of the chip for detection of hepatitis B virus (HBV) gene polymorphism.

METHODSThe P-region and pre-C/C-region of HBV gene were amplified by polymerase chain reaction (PCR) with Cy5 labeled primer or Cy5 labeled dUTP. The amplicons of the two assays were hybridized with chips, scanned and analyzed by computer software for the detection of HBV gene polymorphism.

RESULTSThe signal intensity of assay with Cy5 labeled dUTP was slightly higher than that of assay with Cy5 labeled primer, but non?specific signal intensity of the assay with Cy5 labeled dUTP was higher. The result of 42 samples showed that there was no significant difference between the two assays, and that both had a good repeatability and CV value (15%-20%).

CONCLUSIONSThe assay with Cy5 labeled primer may replace the assay with Cy5 labeled dUTP as a routine method to detect HBV gene polymorphism, and it is simpler and cheaper.

DNA, Viral ; isolation & purification ; Fluorescent Dyes ; Genome, Viral ; Hepatitis B ; virology ; Hepatitis B virus ; genetics ; Humans ; Polymerase Chain Reaction ; methods ; Polymorphism, Genetic

BACKGROUNDTo explore the possibility of using retrovirus vector to carry HBV vector, and to prove that replication defective HBV could be normally packaged.

METHODSTwo kinds of full length of mutant HBV gene, which express dominant negative mutants, were inserted into retrovirus vector. After recombinant retroviruses were harvested, they were used to infect Hep G2 and 2.2.15 cell line. Then the expression of HBV core antigen in the Hep G2 cell was examined by immune fluorescence, and the existence of recombinant HB virion in the culture medium was examined by PCR.

RESULTSHigh titer of recombinant retroviruses were obtained in the culture medium of transfected PA317 cell line. Core antigen was detectable in the recombinant retrovirus infected Hep G2 cell. Recombinant HB virion was detectable in the culture medium of recombinant retrovirus infected 2.2.15 cell.

CONCLUSIONSThe results suggested that recombinant retrovirus could carry HBV vector and express HBV products. When structural protein is offered by wt-HBV, the recombinant retrovirus may function as HBV vector, therefore it could be used in anti?HBV gene therapy.

Genetic Therapy ; Genetic Vectors ; Hepatitis B Core Antigens ; biosynthesis ; Hepatitis B virus ; genetics ; Humans ; Recombination, Genetic ; Retroviridae ; genetics ; Tumor Cells, Cultured ; Virus Replication

BACKGROUNDTo search a novel sensitive, specific and lower cost method applicable for quantitative analysis of the hepatitis B virus DNA extensively.

METHODSQuantitative analysis of the DNA from 100 sera by real-time PCR with Sybr green 1. The results of Sybr's assay were compared with the results obtained with Taqman's fluorescent quantitative assay.

RESULTSTaqman real-time PCR could help evaluate the level of virus reliably. The results of Sybr's assay were in agreement with the Taqman's assay, but detection rate was lower.

CONCLUSIONSSybr green 1 real-time PCR appeared to be convenient and cheap, but detection rate was lower.

DNA, Viral ; blood ; Fluorescent Dyes ; Hepatitis B virus ; genetics ; Humans ; Organic Chemicals ; Polymerase Chain Reaction ; methods ; Sensitivity and Specificity

BACKGROUNDTo investigate the method and therapeutic efficacy of artificial liver support system (ALSS) in treatment of severe viral hepatitis.

METHODSA total of 83 patients including 66 with severe viral hepatitis were treated with ALSS using Baxter-550 artificial kidney and Biologic-DT system.

RESULTSThe levels of mean bilirubin, ALT, AST, BUN, Cr and endotoxin was significantly decreased after the treatment. Of the 66 patients?with severe viral hepatitis, 31(47.0%) had improvement in symptoms and 35 (53.0%) died or left hospital. In the control group,50(27.6%) out of the 181 had improvement in symptoms and 131(72.4%) died or left hospital.

CONCLUSIONSALSS could exert certain therapeutic effects on severe viral hepatitis.

Adult ; Female ; Hepatitis, Viral, Human ; therapy ; Humans ; Liver, Artificial ; Male ; Middle Aged ; Treatment Outcome