Research on adaptation and cloning of rgh5n1 vaccine reference strain to vero cells

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.

Knowledge and practice on HBV prevention among pregnant women in Hanoi (2005-2006) and some related factors of HBsAg positive rate

Background: hepatitis due to virus B is now one of the leading concerns in the community health care throughout the world. Vietnam was a country located in high risk areas for hepatitis B virus (HBV) infection with rate of HBV infection in the community ranged from 11.3 to 25.5%, the rate of HBsAg positive in pregnant women from 12-16%. Objectives: to evaluate knowledge and practice on HBV prevention among pregnant women in Hanoi in 2005-2006 period; to study on some related factors of HBsAg positive rate. Subjectives and Method: a cross sectional study (with analysis on knowledge and practice about HBV prevention) was carried out on 1.300 pregnant women above 28 gestational weeks at Hanoi Obstetrics/Gynecology Hospital in 2005. Results: the rates of women with knowledge about HBV prevention were 38.5% at good level, 24.4% at acceptable level, and 37.7% at unacceptable level. The rates of practice on HBV prevention were 58.5% at satisfied level and 41.5% at notsatified level. Knowledge was likely related to practice, age, educational level, and HBV positive rate. Practice on HBV prevention was also related to HBV infection. Conclusions: the rates of women with knowledge about HBV prevention at good level were relatively high. The rates of practice on HBV prevention at satisfied level were relatively low. Related factors of HBsAg positive rate: knowledge and practice about HBV prevention.

Study on adaptation of hepatitis A virus strain HM-175 to primary monkey kidney cell culture for preparation of an inactivated hepatitis A vaccine

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

Using plaque method for titration of hepatitis A virus (HAV)

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.

A study on expression level of DNA recombinant intracellular HBsAg in Pichia pastoris

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.

Purification of yeast derived HBsAg from Pichia pastoris for preparation of a DNA recombinant hepatitis B vaccine

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.

Study on adaptation of hepatitis A virus strain HM-175 to primary monkey kidney cell culture for preparation of an inactivated hepatitis A vaccine

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

Study on purification of hepatitis A virus strain HM-175 from primary monkey kidney cell culture of Maccaca mulatta for preparation of an inactivated hepatitis A vaccine

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.