Genetic and antigenic characterization of Brazilian SRLV strains: Natural small ruminant interspecies transmission from mixed herds.

Cross-species transmission events and mixed infection of small ruminant lentiviruses (SRLVs) were studied in seven goats and two sheep from three small ruminant mixed flocks from Northeast and Southeast Brazil. Genetic and antigenic analyses with gag/env genes and ELISA multiepitope SU1/SU5 recombinant antigens were carried out, respectively. The genetic analysis of gag and env sequences showed high viral diversity in both species, MVV-like (subtype A1) and CAEV-like B1 in goats, and CAEV-like (subtype B1) in sheep, revealing SRLV interspecies transmission from sheep to goats and vice versa in Brazilian farms. Two Brazilian caprine lentiviruses were segregated in two new genetic clades based on gag analyses, which suggests a new classification into heterogenic genotype A. Furthermore, goat isolates were grouped into subtype A1 and B1 clusters. Cross-reactive antibodies were detected in goats using ELISA with a recombinant antigen carrying SU1 and SU5 immunodominant epitopes; the results showed anti-CAEV and MVV antibodies in goats and anti-CAEV antibodies in sheep. This result can be associated with the high divergence in the V4 region due to SRLV variability. All results confirm cross-species infection of SRLV in Brazilian mixed herds.

Molecular detection of bovine immunodeficiency virus (BIV) in bovines from the state of Minas Gerais, Brazil / Detecção molecular do vírus da imunodeficiência bovina (BIV) em bovinos do estado de Minas Gerais, Brasil

O vírus da imunodeficiência bovina é o agente causador da imunodeficiência viral bovina que é conhecido por infectar bovinos em todo o mundo. Como em outras infecções por retrovírus, os hospedeiros desenvolvem uma infecção de longo prazo e a maioria dos animais infectados permanece assintomática. O objetivo deste estudo foi detectar DNA proviral BIV em amostras de sangue de bovinos e estimar a ocorrência de infecção no estado de Minas Gerais, Brasil. Amostras de sangue de 391 bovinos foram coletadas de duas regiões do estado, Zona da Mata e Central. O DNA proviral foi detectado por reação em cadeia da polimerase semi-nested (SN-PCR). Os resultados de SN-PCR indicaram uma ocorrência de BIV de 12,5% no estado. Os produtos amplificados foram confirmados como BIV por sequenciamento e a similaridade da sequência de nucleotídeos com a estirpe de referência (R-29) foi de 99%. Este é o primeiro estudo que relata a presença do BIV em Minas Gerais, Brasil. Os resultados indicam a necessidade de realizar um estudo detalhado sobre a prevalência da infecção por BIV no Brasil.(AU)

Gold nanoparticle-carboxymethyl cellulose nanocolloids for detection of human immunodeficiency virus type-1 (HIV-1) using laser light scattering immunoassay.

It is estimated that over 100 million people have been infected with human immunodeficiency virus (HIV-1) resulting in approximately 30 million deaths globally. Herein, we designed and developed novel nano-immunoconjugates using gold nanoparticles (AuNPs) and carboxymethylcellulose (CMC) biopolymer, which performed simultaneously as an eco-friendly in situ reducing agent and surface stabilizing ligand for the aqueous colloidal process. These AuNPs-CMC nanocolloids were biofunctionalized with the gp41 glycoprotein receptor (AuNPs-CMC-gp41) or HIV monoclonal antibodies (AuNPs-CMC_PolyArg-abHIV) for detection using the laser light scattering immunoassay (LIA). These AuNPs-CMC bioengineered nanoconjugates were extensively characterized by morphological and physicochemical methods, which demonstrated the formation of spherical nanocrystalline colloidal AuNPs with the average size from 12 to 20 nm and surface plasmon resonance peak at 520 nm. Thus, stable nanocolloids were formed with core-shell nanostructures composed of AuNPs and biomacromolecules of CMC-gp41, which were cytocompatible based on in vitro cell viability results. The AuNPs-CMC-gp41 nanoconjugates were tested against HIV monoclonal antibodies conjugates (AuNPs-CMC_PolyArg-abHIV) using the light scattering immunoassay (LIA) where they behaved as active nanoprobes for the detection at nM level of HIV-1 antigenic proteins. This strategy offers a novel nanoplatform for creating bioprobes using green nanotechnology for the detection of HIV-1 and other virus-related diseases.

Genetic variability and phylogeny of the 5' long terminal repeat from Brazilian bovine leukemia virus.

We conducted a phylogenetic analysis of 22 strains of bovine leukemia virus obtained by polymerase chain reaction to amplify a 582-base pair fragment of the transcriptional regulatory region 5' long terminal repeat (LTR). Twenty-two samples of proviral DNA from peripheral blood mononuclear cells containing bovine leukemia virus from naturally infected bovine from 4 distinct geographic regions in Brazil were investigated. The products obtained by polymerase chain reaction were subjected to direct sequencing and sequence alignment. Fragments of 422 nucleotides were obtained, located between positions -118 and +303 base pairs of the 5'LTR. These fragments corresponded to 80% of the LTR region and included 56% of sub-region U3, 100% of R, and 82.5% of U5. Phylogenetic analysis of these sequences showed a high conservation degree in the 5'LTR region, with 5 well defined groups. However, a hotspot occurrence in the R-U5 region was also observed, which contained 40% of all nucleotide variability observed.

Indirect ELISA for diagnosis of Brucella ovis infection in rams / ELISA indireto para diagnóstico da infecção por Brucella ovis em carneiros

Brucella ovis is a major cause of epididymitis in sexually mature rams, resulting in subfertility, infertility, and economic losses for the sheep industry worldwide. The aim of this study was to develop an indirect ELISA (iELISA) using recombinant proteins, namely rBoP59 and rBP26, as antigens for serological diagnosis of B. ovis infection. The BoP59 and BP26 recombinant proteins were expressed in E. coli and purified by affinity chromatography. Antigenicity was tested by Western blot and iELISA. Standardization of iELISA was performed with 500ng and 1µg BoP59 and rBP26 per well, testing serum from uninfected and experimentally infected rams. rBP26 was effective in distinguishing positive from negative rams. The rBP26 iELISA developed in this study is the first to use a completely purified rBP26 as antigen resulting in high sensitivity (100%) and specificity (90.2%), and an overall accuracy equal to 1.0.(AU)

Brucella ovis é uma das principais causas de epididimite em carneiros sexualmente maduros, resultando em subfertilidade e infertilidade e consequentes perdas econômicas para a ovinocultura em todo o mundo. O objetivo deste estudo foi desenvolver ELISA indireto (ELISAi), utilizando como antígeno proteínas recombinantes BoP59r e BP26r para diagnóstico da infecção por B. ovis. BoP59r e BP26r foram expressas em E. coli e purificadas por cromatografia de afinidade e a antigenicidade testada por Western blot e ELISAi. A padronização do ELISAi foi realizada testando 500 ng e 1 µg de BoP59r e BP26r por poço e soros de carneiros infectados e não infectados. A BP26r foi eficiente em diferenciar ovinos negativos de positivos. O ELISAi com BP26 desenvolvido neste estudo foi o primeiro a utilizar BP26 completamente purificada como antígeno, resultando em elevada sensibilidade (100%) e sensibilidade (90,2%), com acurácia igual a 1,0.(AU)

Indirect ELISA for diagnosis of Brucella ovis infection in rams / ELISA indireto para diagnóstico da infecção por Brucella ovis em carneiros

Brucella ovis is a major cause of epididymitis in sexually mature rams, resulting in subfertility, infertility, and economic losses for the sheep industry worldwide. The aim of this study was to develop an indirect ELISA (iELISA) using recombinant proteins, namely rBoP59 and rBP26, as antigens for serological diagnosis of B. ovis infection. The BoP59 and BP26 recombinant proteins were expressed in E. coli and purified by affinity chromatography. Antigenicity was tested by Western blot and iELISA. Standardization of iELISA was performed with 500ng and 1µg BoP59 and rBP26 per well, testing serum from uninfected and experimentally infected rams. rBP26 was effective in distinguishing positive from negative rams. The rBP26 iELISA developed in this study is the first to use a completely purified rBP26 as antigen resulting in high sensitivity (100%) and specificity (90.2%), and an overall accuracy equal to 1.0...

Brucella ovis é uma das principais causas de epididimite em carneiros sexualmente maduros, resultando em subfertilidade e infertilidade e consequentes perdas econômicas para a ovinocultura em todo o mundo. O objetivo deste estudo foi desenvolver ELISA indireto (ELISAi), utilizando como antígeno proteínas recombinantes BoP59r e BP26r para diagnóstico da infecção por B. ovis. BoP59r e BP26r foram expressas em E. coli e purificadas por cromatografia de afinidade e a antigenicidade testada por Western blot e ELISAi. A padronização do ELISAi foi realizada testando 500 ng e 1 µg de BoP59r e BP26r por poço e soros de carneiros infectados e não infectados. A BP26r foi eficiente em diferenciar ovinos negativos de positivos. O ELISAi com BP26 desenvolvido neste estudo foi o primeiro a utilizar BP26 completamente purificada como antígeno, resultando em elevada sensibilidade (100%) e sensibilidade (90,2%), com acurácia igual a 1,0...

Genetic variation of bovine leukemia virus (BLV) after replication in cell culture and experimental animals.

This article reports the selection of bovine leukemia virus (BLV) variants after continuous passage in cell lines or experimental animals. Two wild BLV strains isolated from 2 naturally infected Holstein dairy cows in Brazil (cow codes: 485 and 141) were used for the experimental infection of 1 sheep and FLK cells, and 1 rabbit and CC81 cells. Viral DNA was isolated several months after infection, and env gene nucleotide and amino acid sequences of the "passaged" variants were compared against the 2 original infecting wild strains. The sequences of the original infecting wild strains were not recovered after their replication in the cell lines or experimental animals. These results indicate that genetic variation occurred after BLV replication in vivo and in vitro, with new variants being selected.

PCR em tempo real para detecção do vírus da doença de Aujeszky / Real time PCR for detection of Aujeszky's disease virus

O objetivo deste trabalho foi desenvolver uma PCR em tempo real (qPCR) para o diagnóstico rápido e sensível da doença de Aujeszky. Os iniciadores amplificaram um fragmento de 123 pares de base do gene codificante da glicoproteína D. A qPCR foi testada em 25 amostras de cérebro de suíno positivas e 85 amostras negativas para DA no isolamento viral e na soroneutralização. A sensibilidade analítica foi calculada com acréscimo de um isolado brasileiro do SuHV-1 titulado em amostras de cérebro de suíno negativas na soroneutralização e na PCR. A técnica apresentou sensibilidade analítica de 10-0,5 TCID50/50µL. A qPCR foi capaz de distinguir reações inespecíficas devido a dímero de oligonucleotídeos iniciadores ou amplificações, além do alvo designado (evitando, assim, os falso-positivos), e de obter resultados rápidos.(AU)

The aim of this study was to validate a low-cost real-time PCR for a quick and sensitive diagnosis of the disease. The fluorofore used was a DNA intercalating agent, one of the cheaper detection systems. Primers amplified a 123 base pairs fragment of the gene coding for glycoprotein D. PCR was tested on 25 samples of pig brain positive for AD and 85 samples negative in viral isolation and serum neutralization. The detection limit was calculated on samples of pig brain contaminated with a Brazilian isolate of SuHV-1. The technique had a detection limit of 10-0,5 TCID50/50µL. PCR was able to distinguish nonspecific reactions due to primer dimers (thus avoiding false positives) and get faster results.(AU)

PCR em tempo real para detecção do vírus da doença de Aujeszky / Real time PCR for detection of Aujeszky's disease virus

O objetivo deste trabalho foi desenvolver uma PCR em tempo real (qPCR) para o diagnóstico rápido e sensível da doença de Aujeszky. Os iniciadores amplificaram um fragmento de 123 pares de base do gene codificante da glicoproteína D. A qPCR foi testada em 25 amostras de cérebro de suíno positivas e 85 amostras negativas para DA no isolamento viral e na soroneutralização. A sensibilidade analítica foi calculada com acréscimo de um isolado brasileiro do SuHV-1 titulado em amostras de cérebro de suíno negativas na soroneutralização e na PCR. A técnica apresentou sensibilidade analítica de 10-0,5 TCID50/50µL. A qPCR foi capaz de distinguir reações inespecíficas devido a dímero de oligonucleotídeos iniciadores ou amplificações, além do alvo designado (evitando, assim, os falso-positivos), e de obter resultados rápidos.

The aim of this study was to validate a low-cost real-time PCR for a quick and sensitive diagnosis of the disease. The fluorofore used was a DNA intercalating agent, one of the cheaper detection systems. Primers amplified a 123 base pairs fragment of the gene coding for glycoprotein D. PCR was tested on 25 samples of pig brain positive for AD and 85 samples negative in viral isolation and serum neutralization. The detection limit was calculated on samples of pig brain contaminated with a Brazilian isolate of SuHV-1. The technique had a detection limit of 10-0,5 TCID50/50µL. PCR was able to distinguish nonspecific reactions due to primer dimers (thus avoiding false positives) and get faster results.

Pseudorabies virus can be classified into five genotypes using partial sequences of UL44

Suid herpesvirus 1 (SuHV-1) is the causative agent of pseudorabies (PR), a disease of great importance due to the huge losses it causes in the swine industry. The aim of this study was to determine a method for genotyping SuHV-1 based on partial sequences of the gene coding for glycoprotein C (gC) and to elucidate the possible reasons for the variability of this region. A total of 109 gCsequences collected from GenBank were divided into five major groups after reconstruction of a phylogenetic tree by Bayesian inference. The analysis showed that a portion of gC (approximately 671 bp) is under selective pressure at various points that coincide with regions of protein disorder. It was also possible to divide SuHV-1 into five genotypes that evolved under different selective pressures. These genotypes are not specific to countries or continents, perhaps due to multiple introduction events related to the importation of swine.(AU)

Pseudorabies virus can be classified into five genotypes using partial sequences of UL44

Suid herpesvirus 1 (SuHV-1) is the causative agent of pseudorabies (PR), a disease of great importance due to the huge losses it causes in the swine industry. The aim of this study was to determine a method for genotyping SuHV-1 based on partial sequences of the gene coding for glycoprotein C (gC) and to elucidate the possible reasons for the variability of this region. A total of 109 gCsequences collected from GenBank were divided into five major groups after reconstruction of a phylogenetic tree by Bayesian inference. The analysis showed that a portion of gC (approximately 671 bp) is under selective pressure at various points that coincide with regions of protein disorder. It was also possible to divide SuHV-1 into five genotypes that evolved under different selective pressures. These genotypes are not specific to countries or continents, perhaps due to multiple introduction events related to the importation of swine.

Selection of peptides for serological detection of equine infectious anemia.

Equine infectious anemia caused by equine infectious anemia virus is an important disease due to its high severity and incidence in animals. We used a phage display library to isolate peptides that can be considered potential markers for equine infectious anemia diagnosis. We selected peptides using IgG purified from a pool comprised of 20 sera from animals naturally infected with equine infectious anemia virus. The diagnostic potential of these peptides was investigated by ELISA, Western blot and dot blot with purified IgG and serum samples. Based on the results, we chose a peptide mimetic for glycoprotein gp45 epitopes of equine infectious anemia virus, with potential for use as an antigen in indirect diagnostic assays. Synthesis of this peptide has possible applications for the development of new diagnostic tools for this disease.

Pseudorabies virus can be classified into five genotypes using partial sequences of UL44.

Suid herpesvirus 1 (SuHV-1) is the causative agent of pseudorabies (PR), a disease of great importance due to the huge losses it causes in the swine industry. The aim of this study was to determine a method for genotyping SuHV-1 based on partial sequences of the gene coding for glycoprotein C (gC) and to elucidate the possible reasons for the variability of this region. A total of 109 gCsequences collected from GenBank were divided into five major groups after reconstruction of a phylogenetic tree by Bayesian inference. The analysis showed that a portion of gC (approximately 671 bp) is under selective pressure at various points that coincide with regions of protein disorder. It was also possible to divide SuHV-1 into five genotypes that evolved under different selective pressures. These genotypes are not specific to countries or continents, perhaps due to multiple introduction events related to the importation of swine.

Multiplex PCR for detection of Suid herpesvirus 1, Bovine herpesvirus 1, Bovine herpesvirus 5, Ovine herpesvirus 2 / PCR Multiplex para detecção dos principais herpesvírus neurológicos de ruminantes

Desenvolveu-se uma PCR multiplex (mPCR) para diagnóstico diferencial de encefalite bovina causada por herpesvírus suíno 1 (SuHV-1), herpesvírus bovino 1 (BoHV-1), herpesvírus bovino 5 (BoHV-5) e herpesvírus ovino 2 (OvHV-2). Os iniciadores foram projetados após alinhamento de sequências disponíveis no banco de genomas (GenBank) e a reação foi padronizada levando-se em consideração a concentração dos reagentes e os tipos diferentes de DNA polimerase. Após determinação da especificidade e sensibilidade, 65 amostras de encéfalo de bovinos com síndrome neurológica foram submetidas à análise. A sensibilidade analítica para detecção de BoHV-1, BoHV-5 e SuHV-1 foi, respectivamente, 10(1,2) TCID50/50µL, 10(1,0) TCID50/50µL, 10(1,3) TCID50/50µL na reação multiplex. Das 65 amostras analisadas, 10 foram positivas para BoHV-5, uma para BoHV-1 e cinco para OvHV-2. A mPCR descrita neste trabalho mostrou-se uma técnica útil para o diagnóstico diferencial de enfermidades relacionadas ao sistema nervoso central de bovinos.(AU)

The aim of this study was to develop a multiplex PCR (mPCR) for the differential diagnosis of bovine encephalitis caused by the suid herpesvirus 1 (SuHV-1), bovine herpesvirus 1 (BoHV-1), bovine herpesvirus 5 (BoHV-5) and ovine herpesvirus 2 (OvHV -2). The primers were designed after alignment of sequences available in GenBank and the reaction was developed by taking into account the concentration of reagents and different types of DNA polymerase. After determining the specificity and sensitivity to PCR, 65 brain samples from cattle with neurological syndrome were submitted to the reaction. The analytical sensitivity for detection of BoHV-1, BoHV-5 and SuHV-1 was, respectively, 10(1,2) TCID50/50µL, 10(1,0) TCID50/50µL, 10(1,3) TCID50/50µL. Ten samples were positive for BoHV-5, one for BoHV-1, one for SuHV-1 and five for OvHV-2. The mPCR described here is a useful technique for the differential diagnosis of diseases related to the central nervous system of cattle.(AU)

PCR Multiplex para detecção dos principais herpesvírus neurológicos de ruminantes / Multiplex PCR for detection of Suid herpesvirus 1, Bovine herpesvirus 1, Bovine herpesvirus 5, Ovine herpesvirus 2

Desenvolveu-se uma PCR multiplex (mPCR) para diagnóstico diferencial de encefalite bovina causada por herpesvírus suíno 1 (SuHV-1), herpesvírus bovino 1 (BoHV-1), herpesvírus bovino 5 (BoHV-5) e herpesvírus ovino 2 (OvHV-2). Os iniciadores foram projetados após alinhamento de sequências disponíveis no banco de genomas (GenBank) e a reação foi padronizada levando-se em consideração a concentração dos reagentes e os tipos diferentes de DNA polimerase. Após determinação da especificidade e sensibilidade, 65 amostras de encéfalo de bovinos com síndrome neurológica foram submetidas à análise. A sensibilidade analítica para detecção de BoHV-1, BoHV-5 e SuHV-1 foi, respectivamente, 10(1,2) TCID50/50µL, 10(1,0) TCID50/50µL, 10(1,3) TCID50/50µL na reação multiplex. Das 65 amostras analisadas, 10 foram positivas para BoHV-5, uma para BoHV-1 e cinco para OvHV-2. A mPCR descrita neste trabalho mostrou-se uma técnica útil para o diagnóstico diferencial de enfermidades relacionadas ao sistema nervoso central de bovinos.

The aim of this study was to develop a multiplex PCR (mPCR) for the differential diagnosis of bovine encephalitis caused by the suid herpesvirus 1 (SuHV-1), bovine herpesvirus 1 (BoHV-1), bovine herpesvirus 5 (BoHV-5) and ovine herpesvirus 2 (OvHV -2). The primers were designed after alignment of sequences available in GenBank and the reaction was developed by taking into account the concentration of reagents and different types of DNA polymerase. After determining the specificity and sensitivity to PCR, 65 brain samples from cattle with neurological syndrome were submitted to the reaction. The analytical sensitivity for detection of BoHV-1, BoHV-5 and SuHV-1 was, respectively, 10(1,2) TCID50/50µL, 10(1,0) TCID50/50µL, 10(1,3) TCID50/50µL. Ten samples were positive for BoHV-5, one for BoHV-1, one for SuHV-1 and five for OvHV-2. The mPCR described here is a useful technique for the differential diagnosis of diseases related to the central nervous system of cattle.

Avaliação da nested PCR em comparação aos testes sorológicos IDGA e ELISA para o diagnóstico da anemia infecciosa equina / Evaluation of nested PCR compared with AGID and ELISA serological tests for equine infectious anemia diagnosis

Comparou-se a técnica nested PCR (nPCR) com os testes sorológicos IDGA e ELISA para o diagnóstico da anemia infecciosa equina. Amostras do DNA provenientes das células mononucleares do sangue periférico foram submetidas à amplificação do gene gag pela nPCR, que apresentou valores de sensibilidade e especificidade relativas de 90 por cento e 52,9 por cento, respectivamente, em relação à IDGA, e valores de 85,7 por cento e 49 por cento, respectivamente, em relação ao ELISA. Considerando-se os fatores referentes às limitações de cada técnica, pode ser sugerido o uso da nPCR como teste de diagnóstico complementar para AIE em amostras brasileiras.(AU)

The nested polymerase chain reaction (nPCR) technique was compared to AGID and ELISA serological tests for the diagnosis of Equine Infectious Anemia. DNA samples from the peripheral blood mononuclear cells were subjected to the amplification of the gag gene by nPCR, which showed relative sensibility and specificity values of 90.0 percent and 52.9 percent respectively, compared to the AGID and values of 85.7 percent and 49.0 percent, respectively, as compared to ELISA. Considering the factors concerning the limitations of each technique, the use of nPCR can be suggested as a complementary diagnostic test for EIA in Brazilian samples.(AU)

Avaliação da nested PCR em comparação aos testes sorológicos IDGA e ELISA para o diagnóstico da anemia infecciosa equina / Evaluation of nested PCR compared with AGID and ELISA serological tests for equine infectious anemia diagnosis

Comparou-se a técnica nested PCR (nPCR) com os testes sorológicos IDGA e ELISA para o diagnóstico da anemia infecciosa equina. Amostras do DNA provenientes das células mononucleares do sangue periférico foram submetidas à amplificação do gene gag pela nPCR, que apresentou valores de sensibilidade e especificidade relativas de 90 por cento e 52,9 por cento, respectivamente, em relação à IDGA, e valores de 85,7 por cento e 49 por cento, respectivamente, em relação ao ELISA. Considerando-se os fatores referentes às limitações de cada técnica, pode ser sugerido o uso da nPCR como teste de diagnóstico complementar para AIE em amostras brasileiras.

The nested polymerase chain reaction (nPCR) technique was compared to AGID and ELISA serological tests for the diagnosis of Equine Infectious Anemia. DNA samples from the peripheral blood mononuclear cells were subjected to the amplification of the gag gene by nPCR, which showed relative sensibility and specificity values of 90.0 percent and 52.9 percent respectively, compared to the AGID and values of 85.7 percent and 49.0 percent, respectively, as compared to ELISA. Considering the factors concerning the limitations of each technique, the use of nPCR can be suggested as a complementary diagnostic test for EIA in Brazilian samples.

PCR duplex para diferenciação de amostras vacinais e selvagens do vírus da doença de Aujeszky / A duplex PCR for differentiation of wild and vaccine virus of Aujeszkyïs disease

A duplex PCR was developed to differentiate the wild-type virus from the attenuated virus used in vaccinations. The PCR was able to amplify fragments of 493bp for glycoprotein E (gE) gene and 207bp for glycoprotein B (gB) gene. The analytical sensitivity was determined by addition of a virus field sample titled in the brain samples of pigs. The standard virus strain Shope, the vaccine strain Bartha, and ten other field isolates were subjected to PCR. The PCR was able to amplify fragments of gE and gB in all field samples and only fragments of gB were amplified in the attenuated virus, as expected. The technique was able to detect up to 100.5 TCID50/50mL virus in samples of brain. Duplex PCR proved to be an important tool for differentiation of naturally-infected animals and animals vaccinated with the virus deleted for gE.(AU)

PCR duplex para diferenciação de amostras vacinais e selvagens do vírus da doença de Aujeszky / A duplex PCR for differentiation of wild and vaccine virus of Aujeszkyïs disease

A duplex PCR was developed to differentiate the wild-type virus from the attenuated virus used in vaccinations. The PCR was able to amplify fragments of 493bp for glycoprotein E (gE) gene and 207bp for glycoprotein B (gB) gene. The analytical sensitivity was determined by addition of a virus field sample titled in the brain samples of pigs. The standard virus strain Shope, the vaccine strain Bartha, and ten other field isolates were subjected to PCR. The PCR was able to amplify fragments of gE and gB in all field samples and only fragments of gB were amplified in the attenuated virus, as expected. The technique was able to detect up to 100.5 TCID50/50mL virus in samples of brain. Duplex PCR proved to be an important tool for differentiation of naturally-infected animals and animals vaccinated with the virus deleted for gE.