Ultraviolet irradiation is an effective method of virus and bacteria inactivation. The dose of UV... more Ultraviolet irradiation is an effective method of virus and bacteria inactivation. The dose of UV-C light necessary for baculovirus inactivation by measurement of fluorescent GFP protein produced by baculovirus expression system after the irradiation of baculovirus culture in doses ranging from 3.5 to 42 J/m2 was determined. At a dose of 36.8 J/m2, only 0.5% of GFP-expressing cells were detected by flow cytometry and confocal microscopy. The stability of purified VP1-PCV2bCap protein produced by baculovirus expression system was analyzed after the irradiation at doses ranging from 3.5 to 19.3 J/m2. Up to the dose of 11 J/m2, no significant effect of UV-C light on the stability of VP1-PCV2bCap was detected. We observed a dose-dependent increase in VP1-PCV2bCap-specific immune response in BALB/c mice immunized by recombinant protein sterilized by irradiation in dose 11 J/m2 with no significant difference between vaccines sterilized by UV-C light and filtration. A substantial differenc...
Porcine circovirus type 2 (PCV2) is the main causative agents of post-weaning multi-systemic wast... more Porcine circovirus type 2 (PCV2) is the main causative agents of post-weaning multi-systemic wasting syndrome leading to significant economic losses in swine population. Although vaccination is the most effective strategy to decrease morbidity and mortality rate, the virus persist in immunized populations and causes subclinical infections with symptoms similar to porcine circovirus diseases. To develop new effective vaccine against PCV2 infection, we prepared chimeric complex VP1-PCV2bCap containing PCV2bCap protein fused with mouse polyomavirus (MPyV). Complex was expressed in baculovirus expression system and purified by nickel agarose. From eluate containing large aggregates of purified proteins, fraction containing pantameres of VP1-PCV2bCap protein was separated by Asymmetrical flow field-flow fractionation and fractions were analyzed by electron microscopy with nano-gold labeling of PCV2Cap portion of the fused protein. 6 weeks old BALB/c mice were subcutaneously vaccinated by...
Rotaviruses are major cause of acute diarrhea in animals and humans which can result in huge eco-... more Rotaviruses are major cause of acute diarrhea in animals and humans which can result in huge eco- nomic losses in farm animals including pigs. We collected 195 samples of feces of diarrhoeic animals. Rotavirus was demonstrated by electron microscopy using the method of negative staining in 27 samples and by ELISA test using monoclonal antibodies to the group antigen VP6
Besides group A rotaviruses, group B and C rotaviruses have been detected as the cause of diarrhe... more Besides group A rotaviruses, group B and C rotaviruses have been detected as the cause of diarrheal diseases in pigs. Of a set of 329 faecal samples from pigs, 16 samples were selected in which rotavirus was detected by electron microscopy and at the same time group A rotavirus was excluded by ELISA method. Rotaviruses were assayed using specific primers
Atypical form of myxomatosis, which caused non-lethal and clinically mild disease in domestic rab... more Atypical form of myxomatosis, which caused non-lethal and clinically mild disease in domestic rabbits 1 month after immunization with a commercially available vaccine MXT, is described. The isolated myxoma virus designated as Litovel 2 (Li-2) did not induce systemic disease following subcutaneous and intradermal applications in susceptible experimental rabbits but led to the immune response demonstrated by ELISA. No severe disease was induced in those Li-2 inoculated rabbits by challenge with the virulent strains Lausanne (Lu) or Sanar (SA), while the control animals showed nodular form of myxomatosis with lethal course of the illness. Restriction fragment length polymorphism (RFLP) of genomic DNA with KpnI and BamHI endonucleases was used for genetic characterization of the Li-2 isolate, the vaccine strain MXT and both virulent strains Lu and SA, respectively. In general, RFLP analysis has shown to be informative for inferring genetic relatedness between myxoma viruses. Based on restriction endonuclease DNA fragment size distribution, it was evident that the pathogenic strain SA is genetically related to the reference strain Lu and the isolate Li-2 is more related, but not identical, to the vaccination strain MXT.
Journal of Virological Methods - J VIROL METH, 2009
A capsid protein of porcine circovirus 2 (PCV 2) serves as a diagnostic antigen for the detection... more A capsid protein of porcine circovirus 2 (PCV 2) serves as a diagnostic antigen for the detection of PCV 2-associated disease known as a postweaning multisystemic wasting syndrome (PMWS). In this report, a bacterial expression system was developed for the expression and purification of the full-length PCV 2 capsid (Cap) protein from a codon-optimized cap gene. Replacement of rare arginine codons located at the 5′ end of the cap reading frame with codons optimal for E. coli was found to overcome the poor expression of the viral protein in the prokaryotic system. The Cap protein was purified to greater than 95% homogeneity by using a single cation-exchange chromatography at a yield of 10mg per litre of bacterial culture. Despite the failure of the E. coli-expressed Cap protein to self-assemble into virus-like particles (VLPs), the immunization of mice with recombinant Cap yielded antibodies with the same specificity as those raised against native PCV 2 virions. In addition, the antige...
... caliciviruses. A higher degree of heterogeneity was observed between the isolates of EBHSV an... more ... caliciviruses. A higher degree of heterogeneity was observed between the isolates of EBHSV and RHDV. Author ... caliciviruses. A higher degree of heterogeneity was observed between the isolates of EBHSV and RHDV. 1997 ...
Ultraviolet irradiation is an effective method of virus and bacteria inactivation. The dose of UV... more Ultraviolet irradiation is an effective method of virus and bacteria inactivation. The dose of UV-C light necessary for baculovirus inactivation by measurement of fluorescent GFP protein produced by baculovirus expression system after the irradiation of baculovirus culture in doses ranging from 3.5 to 42 J/m2 was determined. At a dose of 36.8 J/m2, only 0.5% of GFP-expressing cells were detected by flow cytometry and confocal microscopy. The stability of purified VP1-PCV2bCap protein produced by baculovirus expression system was analyzed after the irradiation at doses ranging from 3.5 to 19.3 J/m2. Up to the dose of 11 J/m2, no significant effect of UV-C light on the stability of VP1-PCV2bCap was detected. We observed a dose-dependent increase in VP1-PCV2bCap-specific immune response in BALB/c mice immunized by recombinant protein sterilized by irradiation in dose 11 J/m2 with no significant difference between vaccines sterilized by UV-C light and filtration. A substantial differenc...
Porcine circovirus type 2 (PCV2) is the main causative agents of post-weaning multi-systemic wast... more Porcine circovirus type 2 (PCV2) is the main causative agents of post-weaning multi-systemic wasting syndrome leading to significant economic losses in swine population. Although vaccination is the most effective strategy to decrease morbidity and mortality rate, the virus persist in immunized populations and causes subclinical infections with symptoms similar to porcine circovirus diseases. To develop new effective vaccine against PCV2 infection, we prepared chimeric complex VP1-PCV2bCap containing PCV2bCap protein fused with mouse polyomavirus (MPyV). Complex was expressed in baculovirus expression system and purified by nickel agarose. From eluate containing large aggregates of purified proteins, fraction containing pantameres of VP1-PCV2bCap protein was separated by Asymmetrical flow field-flow fractionation and fractions were analyzed by electron microscopy with nano-gold labeling of PCV2Cap portion of the fused protein. 6 weeks old BALB/c mice were subcutaneously vaccinated by...
Rotaviruses are major cause of acute diarrhea in animals and humans which can result in huge eco-... more Rotaviruses are major cause of acute diarrhea in animals and humans which can result in huge eco- nomic losses in farm animals including pigs. We collected 195 samples of feces of diarrhoeic animals. Rotavirus was demonstrated by electron microscopy using the method of negative staining in 27 samples and by ELISA test using monoclonal antibodies to the group antigen VP6
Besides group A rotaviruses, group B and C rotaviruses have been detected as the cause of diarrhe... more Besides group A rotaviruses, group B and C rotaviruses have been detected as the cause of diarrheal diseases in pigs. Of a set of 329 faecal samples from pigs, 16 samples were selected in which rotavirus was detected by electron microscopy and at the same time group A rotavirus was excluded by ELISA method. Rotaviruses were assayed using specific primers
Atypical form of myxomatosis, which caused non-lethal and clinically mild disease in domestic rab... more Atypical form of myxomatosis, which caused non-lethal and clinically mild disease in domestic rabbits 1 month after immunization with a commercially available vaccine MXT, is described. The isolated myxoma virus designated as Litovel 2 (Li-2) did not induce systemic disease following subcutaneous and intradermal applications in susceptible experimental rabbits but led to the immune response demonstrated by ELISA. No severe disease was induced in those Li-2 inoculated rabbits by challenge with the virulent strains Lausanne (Lu) or Sanar (SA), while the control animals showed nodular form of myxomatosis with lethal course of the illness. Restriction fragment length polymorphism (RFLP) of genomic DNA with KpnI and BamHI endonucleases was used for genetic characterization of the Li-2 isolate, the vaccine strain MXT and both virulent strains Lu and SA, respectively. In general, RFLP analysis has shown to be informative for inferring genetic relatedness between myxoma viruses. Based on restriction endonuclease DNA fragment size distribution, it was evident that the pathogenic strain SA is genetically related to the reference strain Lu and the isolate Li-2 is more related, but not identical, to the vaccination strain MXT.
Journal of Virological Methods - J VIROL METH, 2009
A capsid protein of porcine circovirus 2 (PCV 2) serves as a diagnostic antigen for the detection... more A capsid protein of porcine circovirus 2 (PCV 2) serves as a diagnostic antigen for the detection of PCV 2-associated disease known as a postweaning multisystemic wasting syndrome (PMWS). In this report, a bacterial expression system was developed for the expression and purification of the full-length PCV 2 capsid (Cap) protein from a codon-optimized cap gene. Replacement of rare arginine codons located at the 5′ end of the cap reading frame with codons optimal for E. coli was found to overcome the poor expression of the viral protein in the prokaryotic system. The Cap protein was purified to greater than 95% homogeneity by using a single cation-exchange chromatography at a yield of 10mg per litre of bacterial culture. Despite the failure of the E. coli-expressed Cap protein to self-assemble into virus-like particles (VLPs), the immunization of mice with recombinant Cap yielded antibodies with the same specificity as those raised against native PCV 2 virions. In addition, the antige...
... caliciviruses. A higher degree of heterogeneity was observed between the isolates of EBHSV an... more ... caliciviruses. A higher degree of heterogeneity was observed between the isolates of EBHSV and RHDV. Author ... caliciviruses. A higher degree of heterogeneity was observed between the isolates of EBHSV and RHDV. 1997 ...
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