Vaccinia virus is the prototype orthopoxvirus that has been used as a vaccine strain for small pox. This virus has been used to express a variety of cellular and viral genes in mammalian cells at high levels. Interleukin-4 (IL-4) has been found to stimulate the proliferation of T cells and enhance the cytolytic activity of cytotoxic T lymphocytes. To test the immunotherapeutic potential of IL-4 delivered in vivo by poxvirus, a recombinant vaccinia virus expressing the murine IL-4 gene (RVVmIL-4) was constructed. A high level of IL-4 production was confirmed by infecting HeLa cells and measuring IL-4 in cell culture supernatant by ELISA. As a tumor model, two cell lines were used: the murine T leukemic line P388 and the murine breast cancer line TS/A. CDF1 mice were intraperitoneally inoculated with 1 x 105 cells of P388. Mice were injected at the same site with 5 x 105 PFU of recombinant vaccinia virus; first, 3 days after the injection of tumor cells and thereafter once every week for 3 weeks. Intraperitoneal injections of RVVmIL-4 significantly prolonged the survival time of mice inoculated with tumor cells. All mice injected with RVVmIL-4 remained alive for 30 days after the postinoculation of tumor cells, while 100% and 70% of the animals injected with saline or wild type vaccinia virus died, respectively. In another tumor model using TS/A, tumor was established by subcutaneously inoculating 2 x 105 tumor cells to BALB/c mice. After tumor formation was confirmed on day 4 in all mice, 5 x 106 PFU of RVVmIL-4 was inoculated subcutaneously three times, once every week for 3 weeks. The TS/A tumor was eradicated in two of the nine mice. Seven of the nine mice treated with RVVmIL-4 developed a tumor, but tumor growth was significantly delayed compared to those treated with saline or wild type vaccinia virus. These results indicate that recombinant vaccinia viruses may be used as a convenient tool for delivering immunomodulator genes to a variety of tumors.
Animals ; Breast Neoplasms ; Cell Culture Techniques ; Cell Line ; Enzyme-Linked Immunosorbent Assay ; Genes, Viral ; HeLa Cells ; Humans ; Injections, Intraperitoneal ; Interleukin-4* ; Interleukins* ; Mice ; Orthopoxvirus ; T-Lymphocytes ; T-Lymphocytes, Cytotoxic ; Vaccinia virus* ; Vaccinia*

To establish in vivo antiviral evaluation system by using murine herpesvirus intracerebral infection model, 5-6 female BALB/c mice per group aged 5 weeks were inoculated i.c. into cerebrum with different inocular HSV-1 F. Signs of clinical disease noted everyday for one month. Observed were body weight decrease, neurological signs and death caused by encephalitis. Mice discontinued body weight decrease were recovered from the disease, and keratitis was often observed during recovery. The groups inoculated with higher than 1,000 PFU showed 100% mortaltiy and LD50 was <100 PFU/mouse. To study the effect of virus inoculum sizes on antiviral effect of acyclovir (ACV), mice inoculated with different inocula were administered i.p. with different doses of ACV immediately after infection, and twice a day for 5 days. The higher inculum size, the less protective. ED50 of ACV was >25, >25, 18.4 and 8.0mg/kg b.i.d. in the group infected with 1,000,000, 100,000, 10,000 and 1,000 PFU/mouse, respectively. LD50 of ACV was 62.5 mg/kg b.i.d. Therapeutic index of ACV was <2.5, <2.5, 3.0 and 7.0 in the groups with inocula 1,000,000, 100,000, 10,000 and 1,000 PFU/mouse, respectively. Inoculum size 1,000 PFU/mouse showing 100% mortaltiy and 5-6 days mean time to death, 5 days drug administration and 14 days observation will be future exeperimental conditions.
Acyclovir* ; Animals ; Body Weight ; Cerebrum ; Encephalitis ; Female ; Herpesvirus 1, Human ; Humans ; Keratitis ; Lethal Dose 50 ; Mice*

Human papillomavirus (HPV) 16, E7 proteins derived from the prototype (Bac73) and natural variant (Bac101) E7 open reading frame were produced in Sf9 insect cells. The variant E7 gene occurred naturally by substitution mutation at the position of 88 nucleotide, resulting serine instead of asparagine. Using E7 specific monoclonal antibody (VD6), both E7 proteins were identified in recombinant baculovirus infected SF9 cells. Radiolabelling and immunoprecipitation analysis revealed that both E7 proteins were phosphoproteins. Immunostaining result showed that E7 proteins were mainly localized in the cytoplasm. Nuclear form of E7 proteins was also detected after a sequential fractionation procedure for removing chromatin structure. Considering that the VD6 recognition site in E7 protein is located within 10 amino acid at the N-terminus, this region appears to be blocked by the nuclear component. Western blot analysis revealed that nuclear form was more abundant than cytoplasmic E7 proteins. Time course immunostaining showed that the primary location of E7 protein was the nucleus and exported to the cytoplasm as proteins were accumulated. These events occurred similarly in both Bac73 and Bac101 infected Sf9 cells, suggesting that these two proteins may have similar biological functions.
Asparagine ; Baculoviridae* ; Blotting, Western ; Chromatin ; Cytoplasm ; Humans* ; Immunoprecipitation ; Insects ; Open Reading Frames ; Phosphoproteins ; Serine ; Sf9 Cells

No abstract available.

No abstract available.
Glucose* ; Hantaan virus* ; Lactose* ; Trehalose*

Noabstract available.
Hantaan virus*

In view of the potential of replicase protein as a diagnostic reagent for human caliciviruses (HuCVs), we have cloned and over-expressed this gene from the Snow Mountain-like Korean strains in Escherichia coli as a fusion protein with glutathione S-transferase (GST, and described the preliminary antigenic characterization of the recombinant products. Each 470bp fragment corresponding to highly conserved region of RNA-dependent RNA polymerase was generated by RT-PCR from stools of two diarrheal children, cloned in pMOSBlue T-vector, and subcloned between the EcoRI and SalI restriction sites of pGEX-47-3, a GST gene fusion vector, yielding pGCVpol. This construct expressed a Snow Mountain-like HuCV replicate under the control of the IPTG-inducible pac promoter. An extract prepared by sonication of the E. coli cell inclusion bodies bearing pGCVpol products was purified and analyzed by SDS-PAGE. After Coomassie blue staining, it was shown that the recombinant replicase migrated on the gels with an approximate molecular mass of 46.5 kDa, that was subsequently cleaved into a 26 kDa GST fragment and a 20.5 kDa replicase protein upon digestion with thrombin protease. The replicase was recognized on immunoblotting with the sera from symptomatic children with the HuCV-associated diarrhea but not by asymptomatic sera from adults. The results presented the first biological activity of individually expressed HuCV replicase subunit and provided important reagents for diagnosis of HuCV infection.
Adult ; Child ; Clone Cells ; Diagnosis ; Diarrhea ; Digestion ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; Gels ; Gene Fusion ; Glutathione Transferase ; Humans ; Immunoblotting ; Inclusion Bodies ; Indicators and Reagents ; RNA Replicase ; Snow* ; Sonication ; Staphylococcal Protein A ; Thrombin

Most of the current licenced hepatitis B vaccines are being produced by recombinant DNA technology in large fermentation cultures of Saccharomyces cerevisiae of yeast cells which carry the gene coded for hepatitis B virus surface antigen. These vaccines are proved very effective clinically and the immunogenicity of vaccines could be maintained for a long time under refrigeration. To develope the stabilizer that could increase the stability of hepatitis B virus vaccine which could be stored for a long period at room temperature or higher conditions, glucose, lactose and sucrose solutions in phosphate buffered saline were added into hepatitis B vaccine respectively to make 2.5%, 5%, 7.5% and 10% final concentration in vaccines. These sugar-vaccine mixtures were stored at room temperature for one month, two months and three months respectively and then inoculated into ICR mice intramuscularly. On the fourteenth day after inoculation, mice were bled and sera were tested for the evaluation of efficacies of vaccines. The results showed that 5% glucose, 7.5% lactose and sucrose increased the stability of vaccines in some degree and this method could be applied for the production of other viral vaccines and bacterial vaccines.
Animals ; Antigens, Surface ; Bacterial Vaccines ; Carbohydrates* ; DNA, Recombinant ; Fermentation ; Glucose ; Hepatitis B Vaccines ; Hepatitis B virus* ; Hepatitis B* ; Hepatitis* ; Lactose ; Mice ; Mice, Inbred ICR ; Refrigeration ; Saccharomyces cerevisiae ; Sucrose ; Vaccines ; Viral Vaccines ; Yeasts

We evaluated Immunochromatographic assay kit to screen HBsAg in human serum. When the reference HBsAg was applyed to ICA, HA and EIA kits, the limit of detection for HBsAg were found out to be 4, 2 and 0.25 ng/ml respectively. But ICA kit required 5 minutes to read the result whereas HA and EIA kit more than one hour. The sensitivity was 97% (29 of 30 samples) and the specificity 100% (45 samples) compared with conventional EIA. The ICA kit needs no instrument or machine to perform the test contrary to the conventional methods. Therefore, this rapid and sensitive ICA kit can be used for HBsAg-screening, especially in the emergency room and in the scene of the accident.
Emergency Service, Hospital ; Hepatitis B Surface Antigens* ; Hepatitis B* ; Hepatitis* ; Humans ; Immunochromatography* ; Limit of Detection ; Sensitivity and Specificity

Serum samples from 123 males and 123 females collected by age in 1996 were analyzed for antibodies against surface antigen of Hepatitis B virus and C22-3, C200 antigens of Hepatitis C virus. Sera from the children under the age of 10 showed 30% seropositivity to the surface antigen of Hepatitis B virus, 33.3% in 10~19 year group, 20% in 20~29 year group, 17.6% in 30~39 year group, 3.3% in 40~49 year group, 5.9% in 50~59 year group, 8,3% in 60~69 year group, 2.9% in 70~79 year group, but antibody could not found in 80~86 year group. 12 out of 123 male sera were positive, 19 out of 123 female sera were positive and overall rate of positivity of antibody against surface antigen of Hepatitis B virus was 12.6%. Serum samples from peoples under the age of 30 had not antibody against C22-3, C200 antigens of Hepatitis C virus. The positivity rate was 2.9% in 30~39 year group. 5 out of 30 sera from 40~49 year age group were positive, and 3 positive sera showed extremely high titer (1:524,288) but the titers of two remaining sera were 1:32, 1:8,192 respectively. 5.9% was positive in 50~59 year group, 8.3% in 60~69 year group, 11.8% in 70~79 year group but all negative in 80~86 yea. group. 6 out of 123 male sera were positive (4.9%), 9 out of 123 female sera were positive (7.3%). Overall .ate of positivity of antibody against C22-3, C200 antigen of Hepatitis C virus was 6.1%. None out of 246 sera had both antibodies against Hepatitis B virus and Hepatitis C virus.
Antibodies* ; Antigens, Surface ; Child ; Female ; Hepacivirus* ; Hepatitis B virus* ; Hepatitis B* ; Hepatitis C* ; Hepatitis* ; Humans* ; Male