Schmallenberg virus (SBV), discovered in continental Europe in late 2011, causes mild clinical si... more Schmallenberg virus (SBV), discovered in continental Europe in late 2011, causes mild clinical signs in adult ruminants, including diarrhoea and reduced milk yield. However, fetal infection can lead to severe malformation in newborn offspring. To develop improved reagents for SBV serology, a high-level yeast expression system was employed to produce recombinant SBV nucleocapsid (N) protein. Recombinant SBV N protein was investigated as an antigen in SBV-specific IgG enzyme immunoassay and used for generation of monoclonal antibodies (MAbs). Yeast-expressed SBV N protein was reactive with anti-SBV IgG-positive cow serum specimens collected from different farms of Lithuania. After immunization of mice with recombinant SBV N protein, four MAbs were generated. The MAbs raised against recombinant SBV N protein reacted with native viral nucleocapsids in SBV-infected BHK cells by immunofluorescence assay. The reactivity of recombinant N protein with SBV-positive cow serum specimens and the ability of the MAbs to recognize virus-infected cells confirm the antigenic similarity between yeast-expressed SBV N protein and native viral nucleocapsids. Our study demonstrates that yeast expression system is suitable for high-level production of recombinant SBV N protein and provides the first evidence on the presence of SBV-specific antibodies in cow serum specimens collected in Lithuania.
ABSTRACT Sereika V., R. Lelesius, D. Zienius: Seroprevalence of Antibodies against Porcine Tescho... more ABSTRACT Sereika V., R. Lelesius, D. Zienius: Seroprevalence of Antibodies against Porcine Teschovirus 1 in Lithuania. Acta Vet Brno 2007, 76: 231-236. The objective of the present work was to study the serological status and epidemiological situation of porcine teschovirus 1 (PTV-1) on swine farms in different Lithuanian regions by using virus neutralization test and virus isolation. Clinical, epidemiological, serological and virological examinations were performed for the diagnosis of Teschen disease (porcine enterovirus encephalomyelitis, TE). Swine blood serum samples (n = 1680) were collected on 28 farms (60 samples per farm) of 8 regions in 2003. For virus isolation 29 samples of pathological material were taken from the brains of pigs (2 - 4 months of age) that died suddenly on seven swine farms in five regions. Epidemiological and clinical examination revealed no signs of porcine enterovirus encephalomyelitis on 32 Lithuanian swine farms. PTV-1 was not isolated and identified in the PK-15 cell culture. Antibodies against PTV-1, detected by using VNT, were found on all investigated farms and in all age groups. Negative serum (2.3%, 39 from 1680) samples were found in 15.5% (16 from 103) of 2-4-month-old pigs and in 4.7% (23 from 494) of 4-6-month-old ones. The positive serological and negative epidemiological, clinical and virological results suggest that less virulent or avirulent PTV-1 strains were spread on Lithuanian swine farms.
Porcine parvovirus (PPV) is a widespread infectious virus that causes serious reproductive diseas... more Porcine parvovirus (PPV) is a widespread infectious virus that causes serious reproductive diseases of swine and death of piglets. The gene coding for the major capsid protein VP2 of PPV was amplified using viral nucleic acid extract from swine serum and inserted into yeast Saccharomyces cerevisiae expression plasmid. Recombinant PPV VP2 protein was efficiently expressed in yeast and purified using density gradient centrifugation. Electron microscopy analysis of purified PPV VP2 protein revealed the self-assembly of virus-like particles (VLPs). Nine monoclonal antibodies (MAbs) against the recombinant PPV VP2 protein were generated. The specificity of the newly generated MAbs was proven by immunofluorescence analysis of PPV-infected cells. Indirect IgG ELISA based on the recombinant VLPs for detection of PPV-specific antibodies in swine sera was developed and evaluated. The sensitivity and specificity of the new assay were found to be 93.4% and 97.4%, respectively. In conclusion, yeast S. cerevisiae represents a promising expression system for generating recombinant PPV VP2 protein VLPs of diagnostic relevance.
Schmallenberg virus (SBV), discovered in continental Europe in late 2011, causes mild clinical si... more Schmallenberg virus (SBV), discovered in continental Europe in late 2011, causes mild clinical signs in adult ruminants, including diarrhoea and reduced milk yield. However, fetal infection can lead to severe malformation in newborn offspring. To develop improved reagents for SBV serology, a high-level yeast expression system was employed to produce recombinant SBV nucleocapsid (N) protein. Recombinant SBV N protein was investigated as an antigen in SBV-specific IgG enzyme immunoassay and used for generation of monoclonal antibodies (MAbs). Yeast-expressed SBV N protein was reactive with anti-SBV IgG-positive cow serum specimens collected from different farms of Lithuania. After immunization of mice with recombinant SBV N protein, four MAbs were generated. The MAbs raised against recombinant SBV N protein reacted with native viral nucleocapsids in SBV-infected BHK cells by immunofluorescence assay. The reactivity of recombinant N protein with SBV-positive cow serum specimens and the ability of the MAbs to recognize virus-infected cells confirm the antigenic similarity between yeast-expressed SBV N protein and native viral nucleocapsids. Our study demonstrates that yeast expression system is suitable for high-level production of recombinant SBV N protein and provides the first evidence on the presence of SBV-specific antibodies in cow serum specimens collected in Lithuania.
ABSTRACT Sereika V., R. Lelesius, D. Zienius: Seroprevalence of Antibodies against Porcine Tescho... more ABSTRACT Sereika V., R. Lelesius, D. Zienius: Seroprevalence of Antibodies against Porcine Teschovirus 1 in Lithuania. Acta Vet Brno 2007, 76: 231-236. The objective of the present work was to study the serological status and epidemiological situation of porcine teschovirus 1 (PTV-1) on swine farms in different Lithuanian regions by using virus neutralization test and virus isolation. Clinical, epidemiological, serological and virological examinations were performed for the diagnosis of Teschen disease (porcine enterovirus encephalomyelitis, TE). Swine blood serum samples (n = 1680) were collected on 28 farms (60 samples per farm) of 8 regions in 2003. For virus isolation 29 samples of pathological material were taken from the brains of pigs (2 - 4 months of age) that died suddenly on seven swine farms in five regions. Epidemiological and clinical examination revealed no signs of porcine enterovirus encephalomyelitis on 32 Lithuanian swine farms. PTV-1 was not isolated and identified in the PK-15 cell culture. Antibodies against PTV-1, detected by using VNT, were found on all investigated farms and in all age groups. Negative serum (2.3%, 39 from 1680) samples were found in 15.5% (16 from 103) of 2-4-month-old pigs and in 4.7% (23 from 494) of 4-6-month-old ones. The positive serological and negative epidemiological, clinical and virological results suggest that less virulent or avirulent PTV-1 strains were spread on Lithuanian swine farms.
Porcine parvovirus (PPV) is a widespread infectious virus that causes serious reproductive diseas... more Porcine parvovirus (PPV) is a widespread infectious virus that causes serious reproductive diseases of swine and death of piglets. The gene coding for the major capsid protein VP2 of PPV was amplified using viral nucleic acid extract from swine serum and inserted into yeast Saccharomyces cerevisiae expression plasmid. Recombinant PPV VP2 protein was efficiently expressed in yeast and purified using density gradient centrifugation. Electron microscopy analysis of purified PPV VP2 protein revealed the self-assembly of virus-like particles (VLPs). Nine monoclonal antibodies (MAbs) against the recombinant PPV VP2 protein were generated. The specificity of the newly generated MAbs was proven by immunofluorescence analysis of PPV-infected cells. Indirect IgG ELISA based on the recombinant VLPs for detection of PPV-specific antibodies in swine sera was developed and evaluated. The sensitivity and specificity of the new assay were found to be 93.4% and 97.4%, respectively. In conclusion, yeast S. cerevisiae represents a promising expression system for generating recombinant PPV VP2 protein VLPs of diagnostic relevance.
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