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 is the main causative agent of post-weaning multisystemic wasting syndr... more Porcine circovirus type 2 is the main causative agent of post-weaning multisystemic wasting syndrome, which affects the immune system of swine and causes widespread epidemics in livestock farms resulting in signi cant piglet mortality and economic losses every year. Although several commercial vaccines were developed, the e ciency and safety need to be improved. Therefore, we have engineered the chimeric complex containing PCV2bCap protein based on virus like particles (VLPs) and the mouse polyomavirus (MPyV) as VLPs represent modern and safe alternative of classical vaccine with high B cells stimulating activity. The ability of this complex to induce an immune response in both mouse and pig models in vivo were evaluated. Firstly, experimental mice were divided into 4 groups and immunized with sterile buffer and VP1-PCV2bCap with different adjuvants, the immune response was monitored for 10 weeks. Robust immune response was detected after the rst immunization and gradually increased after the second and third dose, especially in mice immunized by recombinant protein with Emulsigen (10%) as an adjuvant. Subsequently, to con rm the vaccine e cacy in a target organism, 8-week-old piglets were immunized with VP1-PCV2bCap protein with Emulsigen (10%). The levels of anti-PCV2b speci c IgG antibodies were signi cantly increased in piglets after the second immunization. Finally, strong neutralizing activity of these antibodies was con rmed in PK-15 cells infected with PCV2 Stoon 1010. VP1-PCV2bCap protein complex appears as a promising candidate vaccine for preventing disease associated with PCV2 infection in pigs.
A combination of single-tube reverse transcription (RT)-PCR and nested PCR was used to identify s... more A combination of single-tube reverse transcription (RT)-PCR and nested PCR was used to identify spring viraemia of carp rhabdovirus (SVCV) in infected cell cultures and fish tissues. Two pairs of specific primers (external and internal) were selected from the glycoprotein gene sequence. A specific product of 470 bp was amplified from RNA derived from 34 SVCV isolates including the reference strain (Fijan), using RT-PCR with the external primers. The subsequent PCR using the internal primers yielded a specific product of 141 bp in all cases. No PCR product was obtained following attempts to amplify RNA derived from other fish viruses including pike fry rhabdovirus (PFRV), or from non-infected cells. The identity of the cDNA was confirmed by direct sequencing. PCR sensitivity in the cell-culture system was assessed as about 10-1 TCID 50 ml-1. PCR was further used for the detection of SVCV in 14 clinical samples. Nested PCR allowed us to diagnose the infection in all clinical samples in which SVCV infection was demonstrated by electron microscopy and ELISA. PCR amplification of the SVCV glycoprotein (G) gene is a potential method for rapid diagnosis of spring viraemia of carp; however, it is necessary to verify the method in a higher number of clinical tissue samples. PCR can now be added to current confirmatory diagnostic methods, for determination of SVCV in cell culture. Sequencing of RT-PCR products performed for 7 SVCV isolates (4 Czech, 2 Hungarian, and 1 isolate of unknown origin) revealed a high degree of homogeneity of the G gene region with that of the previously sequenced Fijan strain. The highest nucleotide variability (97.4 to 98.1% nucleotide similarity) was found between the Hungarian and the other isolates. Knowledge of genetic differences among SVCV isolates will be useful in the development of diagnostic methods and elaboration of vaccination programmes.
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...
Porcine circovirus causes the post-weaning multi-systemic wasting syndrome. Despite the existence... more Porcine circovirus causes the post-weaning multi-systemic wasting syndrome. Despite the existence of commercial vaccines, the development of more effective and cheaper vaccines is expected. The usage of chimeric antigens allows serological differentiation between naturally infected and vaccinated animals. In this work, recombinant pentameric vaccination protein particles spontaneously assembled from identical subunits-chimeric fusion proteins derived from circovirus capsid antigen Cap and a multimerizing subunit of mouse polyomavirus capsid protein VP1 were purified and characterized using asymmetric flow fieldflow fractionation (AF4) coupled with UV and MALS/DLS (multi-angle light scattering/dynamic light scattering) detectors. Various elution profiles were tested, including constant cross-flow and decreasing cross-flow (linearly and exponentially). The optimal sample retention, separation efficiency, and resolution were assessed by the comparison of the hydrodynamic radius (R h) measured by online DLS with the R h values calculated from the simplified retention equation according to the AF4 theory. The results show that the use of the combined elution profiles (exponential and constant cross-flow rates) reduces the time of the separation, prevents undesirable sample-membrane interaction, and yields better resolution. Besides, the results show no selfassociations of the individual pentameric particles into larger clusters and no sample degradation during the AF4 separation. The R g /R h ratios for different fractions are in good correlation with morphological analyses performed by transmission electron microscopy (TEM). Additionally to the online analysis, the individual fractions were subjected to offline analysis, including batch DLS, TEM, and SDS-PAGE, followed by Western blot.
The aim of this study was to develop a suitable vaccine antigen against porcine circovirus 2 (PCV... more The aim of this study was to develop a suitable vaccine antigen against porcine circovirus 2 (PCV2), the causative agent of post-weaning multi-systemic wasting syndrome, which causes significant economic losses in swine breeding. Chimeric antigens containing PCV2b Cap protein sequences based on the mouse polyomavirus (MPyV) nanostructures were developed. First, universal vectors for baculovirus-directed production of chimeric MPyV VLPs or pentamers of the major capsid protein, VP1, were designed for their exploitation as vaccines against other pathogens. Various strategies were employed based on: A) exposure of selected immunogenic epitopes on the surface of MPyV VLPs by insertion into a surface loop of the VP1 protein, B) insertion of foreign protein molecules inside the VLPs, or C) fusion of a foreign protein or its part with the C-terminus of VP1 protein, to form giant pentamers of a chimeric protein. We evaluated these strategies by developing a recombinant vaccine against porcine circovirus 2. All candidate vaccines induced the production of antibodies against the capsid protein of porcine circovirus after immunization of mice. The candidate vaccine, Var C, based on fusion of mouse polyomavirus and porcine circovirus capsid proteins, could induce the production of antibodies with the highest PCV2 neutralizing capacity. Its ability to induce the production of neutralization antibodies was verified after immunization of pigs. The advantage of this vaccine, apart from its efficient production in insect cells and easy purification, is that it represents a DIVA (differentiating infected from vaccinated animals) vaccine, which also induces an immune response against the mouse polyoma VP1 protein and is thus able to distinguish between vaccinated and naturally infected animals.
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.
The objective of the present study was to carry out a small surveillance programme in Czech pig p... more The objective of the present study was to carry out a small surveillance programme in Czech pig production herds using the nested reverse transcriptase-polymerase chain reaction (nRT-PCR) technique to trace Hepatitis E virus (HEV) in different biological samples and to characterise the detected swine HEV isolates by phylogenetic analysis. A total of 32 piglets from 11 herds clinically suspected of postweaning multisystemic wasting syndrome (PMWS) were examined. Bile, liver tissue and serum samples were collected from each animal. Due to the high genetic variability of HEV, three sets of primers targeting each of the open reading frames (ORFs) of its genome were used. HEV RNA was most frequently detected in the bile samples (40.0%), followed by liver tissue (16.1%) and serum (3.2%). Seven (63.6%) of the 11 monitored farms were found to have at least one HEV RNA positive piglet. Specific 242 bp sequences within the ORF1 coding non-structural proteins were sequenced and phylogenetically analysed. Phylogenetic analysis using the neighbor-joining and maximum parsimony method confirmed that all detected Czech swine HEV isolates belonged to genotype III. Comparison of the Czech swine HEV isolates with corresponding sequences of swHEV available in GenBank failed to find any 100% homologous HEV isolate.
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 ...
HAL is a multidisciplinary open access archive for the deposit and dissemination of scientific re... more HAL is a multidisciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L'archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.
A combination of single-tube reverse transcription (RT)-PCR and nested PCR was used to identify s... more A combination of single-tube reverse transcription (RT)-PCR and nested PCR was used to identify spring viraemia of carp rhabdovirus (SVCV) in infected cell cultures and fish tissues. Two pairs of specific primers (external and internal) were selected from the glycoprotein gene sequence. A specific product of 470 bp was amplified from RNA derived from 34 SVCV isolates including the reference strain (Fijan), using RT-PCR with the external primers. The subsequent PCR using the internal primers yielded a specific product of 141 bp in all cases. No PCR product was obtained following attempts to amplify RNA derived from other fish viruses including pike fry rhabdovirus (PFRV), or from non-infected cells. The identity of the cDNA was confirmed by direct sequencing. PCR sensitivity in the cell-culture system was assessed as about 10-1 TCID 50 ml-1. PCR was further used for the detection of SVCV in 14 clinical samples. Nested PCR allowed us to diagnose the infection in all clinical samples in which SVCV infection was demonstrated by electron microscopy and ELISA. PCR amplification of the SVCV glycoprotein (G) gene is a potential method for rapid diagnosis of spring viraemia of carp; however, it is necessary to verify the method in a higher number of clinical tissue samples. PCR can now be added to current confirmatory diagnostic methods, for determination of SVCV in cell culture. Sequencing of RT-PCR products performed for 7 SVCV isolates (4 Czech, 2 Hungarian, and 1 isolate of unknown origin) revealed a high degree of homogeneity of the G gene region with that of the previously sequenced Fijan strain. The highest nucleotide variability (97.4 to 98.1% nucleotide similarity) was found between the Hungarian and the other isolates. Knowledge of genetic differences among SVCV isolates will be useful in the development of diagnostic methods and elaboration of vaccination programmes.
The hepatitis E virus (HEV), the causative agent of hepatitis E, is a non-enveloped RNA virus. Th... more The hepatitis E virus (HEV), the causative agent of hepatitis E, is a non-enveloped RNA virus. The HEV genome is formed by a non-segmented positive-sense RNA chain. The 3´end of the chain is polyadenylated and the 5´end is structurally characterised by the so called "capping". According to currently accepted taxonomy, HEV is classified in the genus Hepevirus, the only member of the Hepeviridae family. HE is usually transmitted via the faecal-oral route due to the fact that drinking water or water for industrial purposes is contaminated due to poor sanitation. This spread of HEV has been reported in developing countries of Asia, Africa, South and Central America. However, cases in countries with the sporadic occurrence of HEV have been associated with travelling to countries with an increased risk of infection (developing countries in Asia, Africa and America). HEV infections have subsequently been described in people who have not travelled to endemic countries. Further studies of the HEV suggested other routes of transmission and a zoonotic potential of the virus (pigs and deer as the potential source of human infection).
Horizontal electrophoresis of the rotavirus genome in aprose (EPA), indirect immunoenzymatic anal... more Horizontal electrophoresis of the rotavirus genome in aprose (EPA), indirect immunoenzymatic analysis (ELISA) and,counter-immunoelectroosJnOphoresis (CIEOP) were compared for the identification of .rotavirus in 94 feces .samples collected from scouring calves in 6 rearing premises. Positive results by all three methods were obtained in 48 animals (51 %), most effective being ELISA and EPA (both 43) and least effective CIEOP (29); The sensitivity of the rotavirus genome demonstration by EPA was equal to that of indirect ELISA (88 %), which demonstrates the group-specific. rotavirus antigen. The sensitivities of. EPA and BLISA were higher by 31 % than that of CIBOP. Identical results of BLlSAand EPA, CIBOP and EPA, and BLISA and CIBOP were. obtained in 89.3, 82.9 and 80.8 %, respectively. Compared with BLISA and CIBOP, EPA iSR straightforward procedure involving neither a complicated processing of samples, nor the preparation of a specific hyperimmune serum. The time required for BP.A equals to one half and one fifth of the time necessary for BLISA and CIBOP,respectively.
Five bovine rotavh-us strains were isolated in monolayers of idA-I04 cell culture from faeces of ... more Five bovine rotavh-us strains were isolated in monolayers of idA-I04 cell culture from faeces of 3-to ll-day-old calves suffering from gastroenteritis. Cytophatic effect, accompanied by the release of cells from the glass surface of cultivation flasks was ()bs~ed,from the 5th to the 6th passage. After the stabilization of the cytopathic effect, the highest virus concentration was observed in cell cultures that had been, frozen 18-36 hrs after inoculation. Electron microscopy revealed particles with typical rotavirus morphology. Antigenic relationship of the isolates with the reference strain Lincolil was' confirmed by ELIS:A. The isolates were classified as members of the serological group A by ELISA and immunofluorescence assay. The identity of the rotavirus isolates was also confirmed by electrophoresis in agarose gel. The assay confirmed that their genome consists of segmented viral RNA, which produced in the electric field migration patterns, electrophorogrammes, typical of rotaviruses. In 4 isolates the migration speed of the segments in the electric field was identical with that ()f the reference strain. A different electropherotype was identified in the remaining isolate. Bovine rotavirus, diarrhoea, cell culture, isolation Rotaviruses are at present ~nsidered as important Causal agents of diarrheic infection~ i~ many domestic and wild ruminants, swine, monkey, horse, cat, dog, mouse, rabbit, poultry,
The objective of our study was to diagnose the postweaning multisystemic wasting syndrome (PMWS) ... more The objective of our study was to diagnose the postweaning multisystemic wasting syndrome (PMWS) and to determine the prevalence of the disease in 33 swine herds in the Czech Republic using the results of laboratory examinations of 100 pigs expressing the signs of wasting at the end of 2007. Microscopic lesions associated with the presence of porcine circovirus 2 (PCV2) antigen were detected in the lymph nodes from 39 of 100 diseased pigs (39%). Based on individual assessment of severity of microscopic lymphoid lesions associated with high amounts of PCV2 antigen, PMWS was confirmed in 4 out of 39 pigs originating from 3 of 33 herds (9%). The epidemiological study indicates that PCV2 infections associated with PMWS disease are only sporadically present in the Czech Republic. Subsequently used real time PCR technique confirmed the relation between PMWS status at the individual pig level and PCV2 DNA concentration. PCV2 DNA load in lymph nodes of PMWS-affected pigs were about 3 logs higher than the levels detected in the PMWS-nonaffected group (P < 0.05). Other parallel viral infections (PRRSV, PPV) were detected by real time PCR techniques in 21 out of 39 PCV2 infected pigs (54%). The results of serological examination of blood samples collected during the necropsy of 100 pigs are suggestive of great prevalence of PCV2 infections in pig herds; nevertheless serum samples collected from individual pigs at a single point in time had a low diagnostic value.
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 is the main causative agent of post-weaning multisystemic wasting syndr... more Porcine circovirus type 2 is the main causative agent of post-weaning multisystemic wasting syndrome, which affects the immune system of swine and causes widespread epidemics in livestock farms resulting in signi cant piglet mortality and economic losses every year. Although several commercial vaccines were developed, the e ciency and safety need to be improved. Therefore, we have engineered the chimeric complex containing PCV2bCap protein based on virus like particles (VLPs) and the mouse polyomavirus (MPyV) as VLPs represent modern and safe alternative of classical vaccine with high B cells stimulating activity. The ability of this complex to induce an immune response in both mouse and pig models in vivo were evaluated. Firstly, experimental mice were divided into 4 groups and immunized with sterile buffer and VP1-PCV2bCap with different adjuvants, the immune response was monitored for 10 weeks. Robust immune response was detected after the rst immunization and gradually increased after the second and third dose, especially in mice immunized by recombinant protein with Emulsigen (10%) as an adjuvant. Subsequently, to con rm the vaccine e cacy in a target organism, 8-week-old piglets were immunized with VP1-PCV2bCap protein with Emulsigen (10%). The levels of anti-PCV2b speci c IgG antibodies were signi cantly increased in piglets after the second immunization. Finally, strong neutralizing activity of these antibodies was con rmed in PK-15 cells infected with PCV2 Stoon 1010. VP1-PCV2bCap protein complex appears as a promising candidate vaccine for preventing disease associated with PCV2 infection in pigs.
A combination of single-tube reverse transcription (RT)-PCR and nested PCR was used to identify s... more A combination of single-tube reverse transcription (RT)-PCR and nested PCR was used to identify spring viraemia of carp rhabdovirus (SVCV) in infected cell cultures and fish tissues. Two pairs of specific primers (external and internal) were selected from the glycoprotein gene sequence. A specific product of 470 bp was amplified from RNA derived from 34 SVCV isolates including the reference strain (Fijan), using RT-PCR with the external primers. The subsequent PCR using the internal primers yielded a specific product of 141 bp in all cases. No PCR product was obtained following attempts to amplify RNA derived from other fish viruses including pike fry rhabdovirus (PFRV), or from non-infected cells. The identity of the cDNA was confirmed by direct sequencing. PCR sensitivity in the cell-culture system was assessed as about 10-1 TCID 50 ml-1. PCR was further used for the detection of SVCV in 14 clinical samples. Nested PCR allowed us to diagnose the infection in all clinical samples in which SVCV infection was demonstrated by electron microscopy and ELISA. PCR amplification of the SVCV glycoprotein (G) gene is a potential method for rapid diagnosis of spring viraemia of carp; however, it is necessary to verify the method in a higher number of clinical tissue samples. PCR can now be added to current confirmatory diagnostic methods, for determination of SVCV in cell culture. Sequencing of RT-PCR products performed for 7 SVCV isolates (4 Czech, 2 Hungarian, and 1 isolate of unknown origin) revealed a high degree of homogeneity of the G gene region with that of the previously sequenced Fijan strain. The highest nucleotide variability (97.4 to 98.1% nucleotide similarity) was found between the Hungarian and the other isolates. Knowledge of genetic differences among SVCV isolates will be useful in the development of diagnostic methods and elaboration of vaccination programmes.
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...
Porcine circovirus causes the post-weaning multi-systemic wasting syndrome. Despite the existence... more Porcine circovirus causes the post-weaning multi-systemic wasting syndrome. Despite the existence of commercial vaccines, the development of more effective and cheaper vaccines is expected. The usage of chimeric antigens allows serological differentiation between naturally infected and vaccinated animals. In this work, recombinant pentameric vaccination protein particles spontaneously assembled from identical subunits-chimeric fusion proteins derived from circovirus capsid antigen Cap and a multimerizing subunit of mouse polyomavirus capsid protein VP1 were purified and characterized using asymmetric flow fieldflow fractionation (AF4) coupled with UV and MALS/DLS (multi-angle light scattering/dynamic light scattering) detectors. Various elution profiles were tested, including constant cross-flow and decreasing cross-flow (linearly and exponentially). The optimal sample retention, separation efficiency, and resolution were assessed by the comparison of the hydrodynamic radius (R h) measured by online DLS with the R h values calculated from the simplified retention equation according to the AF4 theory. The results show that the use of the combined elution profiles (exponential and constant cross-flow rates) reduces the time of the separation, prevents undesirable sample-membrane interaction, and yields better resolution. Besides, the results show no selfassociations of the individual pentameric particles into larger clusters and no sample degradation during the AF4 separation. The R g /R h ratios for different fractions are in good correlation with morphological analyses performed by transmission electron microscopy (TEM). Additionally to the online analysis, the individual fractions were subjected to offline analysis, including batch DLS, TEM, and SDS-PAGE, followed by Western blot.
The aim of this study was to develop a suitable vaccine antigen against porcine circovirus 2 (PCV... more The aim of this study was to develop a suitable vaccine antigen against porcine circovirus 2 (PCV2), the causative agent of post-weaning multi-systemic wasting syndrome, which causes significant economic losses in swine breeding. Chimeric antigens containing PCV2b Cap protein sequences based on the mouse polyomavirus (MPyV) nanostructures were developed. First, universal vectors for baculovirus-directed production of chimeric MPyV VLPs or pentamers of the major capsid protein, VP1, were designed for their exploitation as vaccines against other pathogens. Various strategies were employed based on: A) exposure of selected immunogenic epitopes on the surface of MPyV VLPs by insertion into a surface loop of the VP1 protein, B) insertion of foreign protein molecules inside the VLPs, or C) fusion of a foreign protein or its part with the C-terminus of VP1 protein, to form giant pentamers of a chimeric protein. We evaluated these strategies by developing a recombinant vaccine against porcine circovirus 2. All candidate vaccines induced the production of antibodies against the capsid protein of porcine circovirus after immunization of mice. The candidate vaccine, Var C, based on fusion of mouse polyomavirus and porcine circovirus capsid proteins, could induce the production of antibodies with the highest PCV2 neutralizing capacity. Its ability to induce the production of neutralization antibodies was verified after immunization of pigs. The advantage of this vaccine, apart from its efficient production in insect cells and easy purification, is that it represents a DIVA (differentiating infected from vaccinated animals) vaccine, which also induces an immune response against the mouse polyoma VP1 protein and is thus able to distinguish between vaccinated and naturally infected animals.
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.
The objective of the present study was to carry out a small surveillance programme in Czech pig p... more The objective of the present study was to carry out a small surveillance programme in Czech pig production herds using the nested reverse transcriptase-polymerase chain reaction (nRT-PCR) technique to trace Hepatitis E virus (HEV) in different biological samples and to characterise the detected swine HEV isolates by phylogenetic analysis. A total of 32 piglets from 11 herds clinically suspected of postweaning multisystemic wasting syndrome (PMWS) were examined. Bile, liver tissue and serum samples were collected from each animal. Due to the high genetic variability of HEV, three sets of primers targeting each of the open reading frames (ORFs) of its genome were used. HEV RNA was most frequently detected in the bile samples (40.0%), followed by liver tissue (16.1%) and serum (3.2%). Seven (63.6%) of the 11 monitored farms were found to have at least one HEV RNA positive piglet. Specific 242 bp sequences within the ORF1 coding non-structural proteins were sequenced and phylogenetically analysed. Phylogenetic analysis using the neighbor-joining and maximum parsimony method confirmed that all detected Czech swine HEV isolates belonged to genotype III. Comparison of the Czech swine HEV isolates with corresponding sequences of swHEV available in GenBank failed to find any 100% homologous HEV isolate.
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 ...
HAL is a multidisciplinary open access archive for the deposit and dissemination of scientific re... more HAL is a multidisciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L'archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.
A combination of single-tube reverse transcription (RT)-PCR and nested PCR was used to identify s... more A combination of single-tube reverse transcription (RT)-PCR and nested PCR was used to identify spring viraemia of carp rhabdovirus (SVCV) in infected cell cultures and fish tissues. Two pairs of specific primers (external and internal) were selected from the glycoprotein gene sequence. A specific product of 470 bp was amplified from RNA derived from 34 SVCV isolates including the reference strain (Fijan), using RT-PCR with the external primers. The subsequent PCR using the internal primers yielded a specific product of 141 bp in all cases. No PCR product was obtained following attempts to amplify RNA derived from other fish viruses including pike fry rhabdovirus (PFRV), or from non-infected cells. The identity of the cDNA was confirmed by direct sequencing. PCR sensitivity in the cell-culture system was assessed as about 10-1 TCID 50 ml-1. PCR was further used for the detection of SVCV in 14 clinical samples. Nested PCR allowed us to diagnose the infection in all clinical samples in which SVCV infection was demonstrated by electron microscopy and ELISA. PCR amplification of the SVCV glycoprotein (G) gene is a potential method for rapid diagnosis of spring viraemia of carp; however, it is necessary to verify the method in a higher number of clinical tissue samples. PCR can now be added to current confirmatory diagnostic methods, for determination of SVCV in cell culture. Sequencing of RT-PCR products performed for 7 SVCV isolates (4 Czech, 2 Hungarian, and 1 isolate of unknown origin) revealed a high degree of homogeneity of the G gene region with that of the previously sequenced Fijan strain. The highest nucleotide variability (97.4 to 98.1% nucleotide similarity) was found between the Hungarian and the other isolates. Knowledge of genetic differences among SVCV isolates will be useful in the development of diagnostic methods and elaboration of vaccination programmes.
The hepatitis E virus (HEV), the causative agent of hepatitis E, is a non-enveloped RNA virus. Th... more The hepatitis E virus (HEV), the causative agent of hepatitis E, is a non-enveloped RNA virus. The HEV genome is formed by a non-segmented positive-sense RNA chain. The 3´end of the chain is polyadenylated and the 5´end is structurally characterised by the so called "capping". According to currently accepted taxonomy, HEV is classified in the genus Hepevirus, the only member of the Hepeviridae family. HE is usually transmitted via the faecal-oral route due to the fact that drinking water or water for industrial purposes is contaminated due to poor sanitation. This spread of HEV has been reported in developing countries of Asia, Africa, South and Central America. However, cases in countries with the sporadic occurrence of HEV have been associated with travelling to countries with an increased risk of infection (developing countries in Asia, Africa and America). HEV infections have subsequently been described in people who have not travelled to endemic countries. Further studies of the HEV suggested other routes of transmission and a zoonotic potential of the virus (pigs and deer as the potential source of human infection).
Horizontal electrophoresis of the rotavirus genome in aprose (EPA), indirect immunoenzymatic anal... more Horizontal electrophoresis of the rotavirus genome in aprose (EPA), indirect immunoenzymatic analysis (ELISA) and,counter-immunoelectroosJnOphoresis (CIEOP) were compared for the identification of .rotavirus in 94 feces .samples collected from scouring calves in 6 rearing premises. Positive results by all three methods were obtained in 48 animals (51 %), most effective being ELISA and EPA (both 43) and least effective CIEOP (29); The sensitivity of the rotavirus genome demonstration by EPA was equal to that of indirect ELISA (88 %), which demonstrates the group-specific. rotavirus antigen. The sensitivities of. EPA and BLISA were higher by 31 % than that of CIBOP. Identical results of BLlSAand EPA, CIBOP and EPA, and BLISA and CIBOP were. obtained in 89.3, 82.9 and 80.8 %, respectively. Compared with BLISA and CIBOP, EPA iSR straightforward procedure involving neither a complicated processing of samples, nor the preparation of a specific hyperimmune serum. The time required for BP.A equals to one half and one fifth of the time necessary for BLISA and CIBOP,respectively.
Five bovine rotavh-us strains were isolated in monolayers of idA-I04 cell culture from faeces of ... more Five bovine rotavh-us strains were isolated in monolayers of idA-I04 cell culture from faeces of 3-to ll-day-old calves suffering from gastroenteritis. Cytophatic effect, accompanied by the release of cells from the glass surface of cultivation flasks was ()bs~ed,from the 5th to the 6th passage. After the stabilization of the cytopathic effect, the highest virus concentration was observed in cell cultures that had been, frozen 18-36 hrs after inoculation. Electron microscopy revealed particles with typical rotavirus morphology. Antigenic relationship of the isolates with the reference strain Lincolil was' confirmed by ELIS:A. The isolates were classified as members of the serological group A by ELISA and immunofluorescence assay. The identity of the rotavirus isolates was also confirmed by electrophoresis in agarose gel. The assay confirmed that their genome consists of segmented viral RNA, which produced in the electric field migration patterns, electrophorogrammes, typical of rotaviruses. In 4 isolates the migration speed of the segments in the electric field was identical with that ()f the reference strain. A different electropherotype was identified in the remaining isolate. Bovine rotavirus, diarrhoea, cell culture, isolation Rotaviruses are at present ~nsidered as important Causal agents of diarrheic infection~ i~ many domestic and wild ruminants, swine, monkey, horse, cat, dog, mouse, rabbit, poultry,
The objective of our study was to diagnose the postweaning multisystemic wasting syndrome (PMWS) ... more The objective of our study was to diagnose the postweaning multisystemic wasting syndrome (PMWS) and to determine the prevalence of the disease in 33 swine herds in the Czech Republic using the results of laboratory examinations of 100 pigs expressing the signs of wasting at the end of 2007. Microscopic lesions associated with the presence of porcine circovirus 2 (PCV2) antigen were detected in the lymph nodes from 39 of 100 diseased pigs (39%). Based on individual assessment of severity of microscopic lymphoid lesions associated with high amounts of PCV2 antigen, PMWS was confirmed in 4 out of 39 pigs originating from 3 of 33 herds (9%). The epidemiological study indicates that PCV2 infections associated with PMWS disease are only sporadically present in the Czech Republic. Subsequently used real time PCR technique confirmed the relation between PMWS status at the individual pig level and PCV2 DNA concentration. PCV2 DNA load in lymph nodes of PMWS-affected pigs were about 3 logs higher than the levels detected in the PMWS-nonaffected group (P < 0.05). Other parallel viral infections (PRRSV, PPV) were detected by real time PCR techniques in 21 out of 39 PCV2 infected pigs (54%). The results of serological examination of blood samples collected during the necropsy of 100 pigs are suggestive of great prevalence of PCV2 infections in pig herds; nevertheless serum samples collected from individual pigs at a single point in time had a low diagnostic value.
Uploads
Papers by Ivan Pšikal