Hepatitis A

Two positive clones inserted DNA fragment of HBV coded for intracellular HBsAg synthesis from 2 strains of P.pastoris: GS115-47-4 (Mut+ His 4) and KM71-47-1 (Muts; His4 arg4 aox1: ARG4) were selected for developing a DNA recombinant hepatitis B vaccine in laboratory scale. Different types of growth media and expression media were used for the study on HBsAg expression levels from those two clones. The highest level of HBsAg - 1318mcg/l with MGYH/MMH medium after 96h and 2220 mcg/l with BMGY/BMMY medium after 72h of induction was obtained in clone GS115-47-4 and 241 mcg/l after 48h and 1680 mcg/l after 48h in clone KM71-47-1 observed were respectively.
Hepatitis B Surface Antigens ; DNA

Most hepatitis A virus replication in cell cutter has been reported to be nonlytic and relatively slow. A rapidly replicating isolate of strain HM-175 from persistently infected, serially passed cell cultures (pHM-175) was found to induce a cytopathic effect. This observation allowed the development of a classic plaque assay for pHM-175 in FRhK-4 cells (fetal rhesus kidney) which were used for the preparation of some lytic virus stocks and for all plaque assays. All cells were cultured at 37oC in DMEM + 10% FCS + 5mcg/ml gentamicin sulfate (GIBCO, Grand, NY). The pHM-175 stock was made by decanting supernatant medium and freezing infected cells in DMEM at -70oC.
Hepatitis A virus ; Titrimetry

50 patients with 50 teeth with dental stem defect due to the endodontis (74%) and other causes received the reconstruction by false cast and post. The primary results found that the rate of good reconstruction was 88%. The shortcomings of technique comprised longer duration of reconstruction and higher cost
Dental Care ; Delivery of Health Care

Monolayer primary monkey kidney cell culture was infected with vaccine production hepatitis A virus strain HM-175 at a multiplicity of infection (MOI) of 0.05 PFU cell and incubation period was 21 days. Then the culture was stored at 70oC. Freeze and thaw 3 times for cell breakage and virus release. Centrifugation for separation of cell debris, then cell debris was sonicated with 3 cycles of 30 second and 30 seconds intervals, centrifugation separation of cell debris. Cell culture supernatant and supernatant after centrifugation were mixed for further ultrafitration and concentration using filtration system pellicon following one rate-zonal ultracetrifugation in sucrose gradient solution were conducted for HAV separation and purification. A purified solution of HAV antigen including empty and full particles was obtained by using this method, which was further, treated with 1/2000 formalin for HAV inactivation at 35oC - 96h. Hepatitis A vaccine prepared by this method meets the WHO minimum requirement for this vaccine.
Hepatitis A virus ; kidney ; cells ; Hepatitis A Vaccines

The hepatitis A virus strain HM-175 was adapted to primary monkey kidney cell culture Maccaca mulatta for the study of development of an inactivated purified hepatitis A vaccine derived from cell culture. Using immuno-fluorescent method, it was detected that the hepatitis A viruses replicated very well inside cytoplasma and induced a typical cytophatic effect. After 10 days of inoculation, about 50% of cells were infected. Hepatitis A viruses replicated very slowly. This study found that the time for complet inducing CPE was 21 days and titers of released viruses and cell associated viruses are similar. Therefore, the procedure for preparation of hepatitis A vaccine should include the step of cell sonication for separation of all cells associated viruses. 10 days after inoculation was the best time to have high titer. A serial passage of HAV HM-175 on primary cell culture of monkey kidney was not successful in getting higher titer and shorter time of inoculation, so it was not necessary to conduct serial passages. The optimal infectious dose was determinated as 0.03 pfu/cell
Hepatitis A virus ; kidney ; cells ; Hepatitis A Vaccines

The hepatitis A virus strain HM-175 was adapted to primary monkey kidney cell culture Maccaca mulatta for the study of development of an inactivated purified hepatitis A vaccine derived from cell culture. Using immuno-fluorescent method, it was detected that the hepatitis A viruses replicated very well inside cytoplasma and induced a typical cytophatic effect. After 10 days of inoculation, about 50% of cells were infected. Hepatitis A viruses replicated very slowly. This study found that the time for complet inducing CPE was 21 days and titers of released viruses and cell associated viruses are similar. Therefore, the procedure for preparation of hepatitis A vaccine should include the step of cell sonication for separation of all cells associated viruses. 10 days after inoculation was the best time to have high titer. A serial passage of HAV HM-175 on primary cell culture of monkey kidney was not successful in getting higher titer and shorter time of inoculation, so it was not necessary to conduct serial passages. The optimal infectious dose was determinated as 0.03 pfu/cell
Hepatitis A virus ; kidney ; cells ; Hepatitis A Vaccines

Positive clones from Pichia pastoris strain GS115-47-4 and KM71-47-1 were grown in 1200 ml BMGY/BMMY medium into 2 liters fermentor and 50 ml innoculum with high density was added, temperature was 30oC, agitation speed was 300 rpm and pressure was 0.5 kg/cm2. The biomass was harvested after 72 h of induction in case of GS115-47-4 strain and 48 h for KM71-47-1 strain. The density was 3,9 x 109 cells/ml. After cell lysis, the total HBsAg contents were obtained approximately 30 mg in both strains. 2 consecutive isopycnic KBr gradient ultracentifugations following one rate-zonal sucrose were performed.
hepatitis B vaccines ; isolation & purification ; Hepatitis B Surface Antigens

After integrating hepatitis B vaccine produced by National Institute of Hygiene and Epidemiology into the Expanded Program on Immunization (EPI), seropositive rate and geometric mean titre of anti-HBs of 12-24 month old children were 69% and 157 mIU/ml in urban group, respectively. The results were higher than rural group (48.6% and 107.2 mIU/ml, respectively). Seroprotective rate increased from 48.7% to 80.2% after booster dose. This rate was highest (93.3%) in children received the booster dose 8-10 months after primary immunization. In the different time of booster dose, level of anti-HBs of 12-24 month old was higher than before
Hepatitis B ; Vaccination ; child

Background: As avian influenza A/H5N1 epidemic spreads rapidly, many corporations, vaccine manufacturers in cooperation with laboratories around the world has conducted research and developed H5N1 vaccine for both humans and poultry. Objective: Evaluate the adaptation and cloning of rgh5n1 vaccine reference strain to vero cells in order to produce master seed virus and working seed virus. Subject and Method: The reverse genetics derived A/H5N1 virus strain (rgH5N1) was studied for adaptation to Vero cells by serial passage. It has been shown that the rgH5N1 strain can be propagated in Vero cells and cause CPE with the highest virus titer 1010,9 PFU/ml at Vero passage 15. The rgH5N1 strain was cloned by using plaque purification method and passaged to obtain a high stable virus titer. Conclusion: The reverse genetics derived A/H5N1 virus strain was propagated in Vero cells. Master seed virus and working seed virus were obtained at passage 6.
rgh5n1 ; avian influenza ; vero cells