Padronização de um ELISA indireto a partir da utilização de uma proteína de superfície do vírus da anemia infecciosa equina como antígeno para o diagnóstico dessa enfermidade / Standardization of an indirect ELISA using a surface protein of the equine infectious anemia virus as an antigen for the diagnosis of this disease

A anemia infecciosa equina é uma importante enfermidade que acomete os equídeos em todo o mundo, se apresentando de forma aguda, crônica e assintomática causando grandes prejuízos para a economia tanto para criadores que vivem do trabalho desses animais quantos aos criadores que investem no melhoramento das raças, impedindo o acesso ao mercado tanto nacional quanto internacional. O Ministério da Agricultura, Pecuária e Abastecimento considera o IDGA como teste oficial para diagnóstico dessa enfermidade, porém essa técnica é demorada e muita vez acaba sendo subjetiva, dependendo da experiencia particular de cada Laboratorista. Além de não conseguir detectar animais no início da infecção. Logo, a necessidade de se buscar novas técnicas como o ELISA indireto que aperfeiçoem o tempo de análise dos resultados, facilita a automação e obtém resultados confiáveis. O estudo realizado teve como objetivo padronizar uma técnica de ELISA indireto utilizando uma proteína de envelope viral GP90 como antígeno para diagnóstico da anemia infecciosa equina. Avaliando o desempenho do teste a partir da sensibilidade, especificidade e valores preditivos positivo e negativo. Os valores obtidos foram: 91,11%, 93,33%, 91,11% e 93,33% respectivamente. Concluiu-se que o teste apresenta bom desempenho, além da possibilidade de detectar amimais positivos no início da infecção.

Equine infectious anemia is an important disease that affects horses all over the world, presenting in an acute, chronic and asymptomatic way, causing great damage to the economy, both for breeders who live off the work of these animals and for breeders who invest in the improvement of breeds, preventing access to both national and international markets. The Ministry of Agriculture, Livestock and Food Supply considers AGID to be the official test for diagnosing this disease, but this technique takes time and often ends up being subjective, depending on the particular experience of each laboratory worker. In addition to not being able to detect animals at the beginning of the infection. Therefore, the need to seek new techniques such as indirect ELISA that improve the time of analysis of results, facilitate automation and obtain reliable results. The aim of this study was to standardize an indirect ELISA technique using a GP90 viral envelope protein as an antigen for the diagnosis of equine infectious anemia. Evaluating test performance based on sensitivity, specificity and positive and negative predictive values. The values obtained were 91.11%, 93.33%, 91.11 and 93.33 respectively. It was concluded that the test performs well, in addition to the possibility of detecting positive animals at the beginning of the infection.

Equine infectious anaemia in equids of Southern Pantanal, Brazil: seroprevalence and evaluation of the adoption of a control programme / Anemia infecciosa equina em equídeos do Pantanal Sul, Brasil: soroprevalência e avaliação da adoção de um programa de controle

The working equid population in Corumbá, Southern Pantanal, is very large and has a crucial role in the main economic activity of the State of Mato Grosso do Sul, the beef cattle industry. The aim of the present study was to estimate the prevalence of equine infectious anaemia (EIA) in working equids of ranches in the municipality of Corumbá, by the official agar gel immunodiffusion (AGID) test, and evaluate the adoption of the Programme for the Prevention and Control of Equine Infectious Anaemia proposed by Embrapa Pantanal and official entities in the 1990s. From September to November 2009, forty ranches distributed through the area of the municipality were visited, and serum samples were obtained from 721 equines and 232 mules. According to previous publications and the present data, it was concluded that the prevalence of EIA in this population has increased from 18.17% to 38.60%, which represents at this time approximately 13,000 infected animals. There was no significant difference between the apparent prevalence of equines and mules. It was also verified that the control programme was not known by the greater part of the interviewed ranch owners, managers and foremen and, in their perception, EIA is not a primary threat to address. Among the studied variables, the serologic testing practice significantly reduced the risk for the presence of EIA seropositivity, as well as the separation of riding equipment and segregation of seropositives.(AU)

A população de equídeos de serviço em Corumbá, Pantanal Sul, é muito numerosa e tem um papel crucial na principal atividade econômica do estado de Mato Grosso do Sul, a pecuária de corte extensiva. O objetivo deste trabalho foi estimar a prevalência atual da anemia infecciosa equina (AIE) em equídeos de serviço em fazendas do município de Corumbá, pelo teste oficial de imunodifusão em gel de ágar (IDGA), e avaliar a adoção do Programa de Prevenção e Controle da Anemia Infecciosa Equina proposto pela Embrapa Pantanal e entidades oficiais nos anos 1990. De setembro a novembro de 2009, quarenta fazendas distribuídas na área do município foram visitadas, e amostras de soro obtidas de 721 equinos e 232 muares. De acordo com publicações anteriores e os dados obtidos neste trabalho, concluiu-se que a prevalência da AIE nesta população aumentou de 18.17% para 38,60%, o que representa atualmente cerca de 13.000 animais infectados. Não houve diferença significativa entre as prevalências aparentes de equinos e muares. Verificou-se, também, que o programa de controle era desconhecido pela maior parte dos produtores, gerentes e capatazes entrevistados e, na percepção dos mesmos, a AIE não é uma ameaça importante a ser enfrentada. Dentre as variáveis estudadas, a prática da realização de testes sorológicos reduziu significantemente o risco para a presença de soropositividade para AIE, assim como a separação dos equipamentos de montaria e a segregação dos soropositivos.(AU)

Anemia infecciosa equina: Una revisión / Equine infectious anemia: A review

La Anemia Infecciosa Equina (en ingles Equine Infectious Anemia EIA), es una enfermedad viral que afecta a los équidos a nivel mundial. El agente causal, pertenece al género Lentivirus, de la familia Retroviridae, subfamilia Orthoretrovirinae. La enfermedad se caracteriza por episodios febriles recurrentes, trombocitopenia, anemia, pérdida de peso y edema de las partes bajas del cuerpo; si no se produce la muerte en el curso de los ataques clínicos agudos, se produce una fase crónica y la enfermedad tiende a convertirse en latente. La AIE debe notificarse ante la OIE: Organización Mundial de Sanidad Animal. En 2015 la OIE mediante Código Sanitario para los Animales Terrestres se estableció a la Anemia Infecciosa Equina en la lista única de enfermedades e infecciones de los équidos. Los veterinarios que detecten un caso de anemia infecciosa equina deben seguir las pautas nacionales y/o locales para la notificación y las pruebas de diagnóstico correspondientes. Este artículo describe los aspectos más relevantes de la Anemia Infecciosa Equina.

Equine Infectious Anemia (EIA) is a viral disease that affects horses worldwide. The causative agent belongs to the genus Lentivirus, the family Retroviridae, subfamily Orthoretrovirinae. The disease is characterized by recurrent episodes of fever, thrombocytopenia, anemia, weight loss and edema of the lower parts of the body; if death does not occur in the course of the acute clinical attacks, a chronic stage occurs and the disease tends to become dormant. The IEA must be reported to the OIE: World Organization for Animal Health. In 2015 by Health Code OIE Terrestrial Animal it was established Equine Infectious Anemia in the single list of diseases and infections of horses. Veterinarians detected a case of equine infectious anemia should follow national and / or local to the notification and guidelines appropriate diagnostic tests. This article describes the most relevant aspects of the Equine Infectious Anemia.

The efficacy of ELISA commercial kits for the screening of equine infectious anemia virus infection / Eficacia de ensayos de ELISA comerciales para el screening de infección por el virus de la anemia infecciosa equina

The most used and reliable indicator of equine infectious anemia virus (EIAV) infection is the detection of its specific antibodies in horse serum. In the present study, the performance of two commercial ELISA tests for the detection of EIAV antibodies as well as the potential advantages of their use as an EIAV infection screening tool were evaluated in 302 horse serum samples. Both ELISA assays showed 100% diagnostic sensitivity, and 92.3-94.3% diagnostic specificity. Discordant results were analyzed by immunoblot. The results showed that both ELISA tests are very efficient at detecting EIAV infected animals, allowing to identify a higher number of positive horse cases. Thus, ELISA assays can be useful tools in EIA control and eradication.

El mejor indicador de la infección por el virus de la anemia infecciosa equina (Equine infectious anemia virus, EIAV) es la detección de anticuerpos específicos en el suero del caballo. En el presente trabajo se evaluó la capacidad de detección de anticuerpos contra EIAV de dos equipos de ELISA comerciales utilizando 302 muestras de suero equino, así como las ventajas potenciales de su uso como herramientas de screening. Ambos ensayos de ELISA presentaron 100 % de sensibilidad diagnóstica y una especificidad diagnóstica del orden de 92,3 a 94,3 %. Las muestras discordantes fueron analizadas por inmunoblot. Los resultados mostraron que las dos pruebas ELISA son muy eficientes para detectar animales infectados por EIAV, al permitir identificar un mayor número de animales positivos que la prueba de inmunodifusión en gel de agar, oficialmente aprobada en la República Argentina para la certificación de los animales. Las pruebas de ELISA constituyen herramientas muy útiles en los programas de control y de erradicación de la infección por EIAV.

The application of single-genome amplification and sequencing in genomic analysis of an attenuated EIAV vaccine / 病毒学报

Our previous studies found that the Chinese attenuated EIAV vaccine was composed of a pool of quasispecies, which showed a complicated diversity called "multi-species". Further determining the viral composition of these species in the vaccine should improve the identification of predominant viruses in the vaccine and facilitate the analysis of in vivo evolution of EIAV and the vaccine. In this study, the comparison of fidelities in amplifying and sequencing the V3 to V5 fragment of EIAV envelope gp90 gene by either a single-genome amplification (SGA) approach or the traditional RT-PCR (bulk PCR) was performed. Results revealed that the diversities were 1.84% and 1.88% for SGA- and bulk PCR-derived sequences, respectively. Futher analysis revealed that beside the sequences highly homologous to those derived by the bulk PCR, nine of 73 sequences derived by SGA contained a deduced amino acid domain that was identical to the corresponding domain in the virulent strain LN40. In addition, sequences with deletion of one predicted amino acid residual was detected by using SGA The presence of these less populated sequences provided additional evidence for the "multi-species" hypothesis for the action mechanism of the EIAV vaccine. Furthermore, based on the analysis of sampling bias, Our results that the difference in copy number of each viral specie in the pool of quasispecies resulted in the inefficiency to amplify viral sequences that were in low population by bulk PCR. Therefore, the sequences amplified by bulk PCR could not correctly represent the composition of quasispecies. As an approach based on the amplification and sequencing single isolated genome, SGA significantly improved the weakness of bulk PCR and appeared its advantage in analysis of EIAV genome composition with high variety.

Study of the correlation between the plasma viral load and protective immunity induced by the equine infectious anemia attenuated vaccine and its parental virulent strain / 病毒学报

The threshold hypothesis of attenuated lentiviral vaccine considers that the type of host response to infections of lentiviruses depends on the viral load. To evaluate the correlation between viral loads of the attenuated vaccine strain of equine infectious anemia virus (EIAV) and their effects to induce protective immunity, longitudinal plasma viral loads in groups of horses inoculated with either an attenuated EIAV vaccine strain (EIAV(DLV125)) or sub-lethal dose of an EIAV virulent strain (EIAV(LN40)) were compared. Similar levels of plasma viral loads ranging from 10(3)-10(5) copies/mL were detected from samples of these two groups of animals (P > 0.05) during 23 weeks post the inoculation. However, different responses to the challenge performed thereafter with lethal dose of the EIAV virulent strain were observed from the groups of horses inoculated with either EIAV(DLV125) or sub-lethal dose of EIAV(LN40). The protective efficiency was 67% (3 of 4 cases) and 0 (none of 2 cases), respectively. Our results implicate that the viral load of EIAV attenuated vaccine is not the primary factor, or at least not the solo primary factor, to determine the establishment of immune protection.

Construction and in vitro evaluation of an infectious clone of the equine infectious anemia virus vaccine strain EIAV(FDDV) with four reverse-mutated vaccine-specific sites in the S2 gene / 病毒学报

To elucidate the function of the S2 gene in equine infectious anemia virus (EIAV) and its role in the attenuation of the Chinese attenuated EIAV vaccine strains, the S2 in the EIAV vaccine strain EIAV (FDDV) was reverse-mutated and the in vitro replication character of the resultant virus was evaluated. Based on the sequence variation of the S2 gene between the EIAV virulent strains and vaccine strains, all the four vaccine-specific sites in the S2 of an EIAV(FDDV) infectious clone, pFDDV3-8, were reverse-mutated to the sequences of the virulent strain EIAV(DV115). The reverse-mutated molecular clone pFDDVS2r1-3-4-5 was used to transfect fetal donkey dermal (FDD) cells for rescuing the derived virus vpFDDVS2r1-3-4-5. The production and replication of vpFDDVS2r1-3-4-5 in FDD cells were proved by RT-PCR, immune fluorescence assay and reverse transcriptase activity assay. Typical virons of EIAV were clearly observed under the electron microscopy. The parallel analysis of the dynamic replication of the reverse-mutated viral clone vpFDDVS2r1-3-4-5 and its parental virus vpFDDV3-8 showed that the virus with four reverse mutations in the S2 replicated only slightly slower than its parental vaccine strain in FDD cells. This result implicates that the mutations in the S2 of the EIAV vaccine strains did not significantly alter the viral replication in vitro. Further studies on the in vivo replication of the reverse-mutated viral clone are required for understanding the relationship between the S2 and the attenuated pathogenesis of EIAV attenuated vaccines.

Comparison of proviral genomes between the Chinese EIAV donkey leukocyte-attenuated vaccine and its parental virulent strain / 病毒学报

The donkey leukocyte-attenuated vaccine of equine infectious anemia virus (EIAV) was the first lentiviral vaccine that induced solid protection from the infection of virulent strains. To elucidate the mechanism of increased immunogenicity and attenuated virulence of the vaccine, the proviral genomic DNA of an EIAV vaccine strain, EIAV(DLV121) was analyzed and compared with the genome of a parental virulent strain EIAV(DV117). Full length viral genomic DNAs were amplified as two segments by LA-PCR and were cloned. Because of the genomic diversity of retroviral quasispecies, 10 full-length sequences of EIAV(DLV121) and 4 full-length sequences of EIAV(DV117) from randomly picked clones were analyzed. Results showed that the average length of the complete nucleotide sequence of EIAV(DLV121) was 8,236bp and EIAV(DV117) was 8,249bp, with the inter-strain diversity of 2.8%. As for individual genes between the vaccine and virulent strains, the differences in nucleotide sequence of S2, LTR and env were significantly higher than the other genes with the diversity of 4.1%, 3.7% and 3.1%, respectively. Considerable variations in deduced amino acid sequences were found in S2, S3 and env. The diversities were 10.4%, 5.6% and 4.8%, respectively. Furthermore, the LTR of EIAV(DLV121) consisted of at least 5 subtypes grouped by their nucleotide sequences. There were two additional N-linked glycosylation sites in the deduced amino acid sequence of EIAV(DV117) gp90 than that of EIAV(DLV121). Among glycosylation sites in the gp90 of virulent strain, 3 were found unique only in EIAV(DV117), of which 2 were located in the principle neutralizing domain (PND). In addition, there was one EIAV(DLV121) -specific glycosylation site, which was positioned in the PND, too. Taken together, it is clear that greatly increased genomic diversity exists in the EIAV vaccine strain, which provides important information for the further study on biological characters of the Chinese EIAV attenuated vaccine.

Construction of recombinant lentivirus vaccine with single round replication / 中华流行病学杂志

<p><b>OBJECTIVE</b>To develop a safe and effective lentivirus vaccine model and provide insights into the development of other lentivirus vaccines.</p><p><b>METHODS</b>In this study, a construct of pGPT was made by deleting env gene in the infectious Equine infectious anemia virus (EIAV) molecular clone of WU57. Since the overlaping of EIAV Rev gene with env gene, there was no Rev gene in the construct of pGPT. For compensation of Rev function, the construct of pGPTC was made by inserting 4 copies of constitutive RNA transport elements (CTEs) from Mason-Pfizer monkey virus into the construct of pGPT. In addition, a construct designated pTEB expressing EIAV Env protein was made while env gene-minus viruses were made by co-transfection of pGPT/pTEB or pGPTC/pTEB into 293 cells. Western blot was used to identify the development of recombinant virus particles. Then immunofluorescence assay was used to evaluate the infectivity of recombinant virus particles in vitro.</p><p><b>RESULTS</b>EIAV proteins expression was detected in the supernatant of transfected 293 cells by Western blot within pGPTC/pTEB transfected cells. However, no evidence of EIAV proteins expression was observed within pGPT/pTEB transfected cells. EIAV proteins expression was detected in the first round but not in the second round infected EK cells with EIAV(GPTC) by immunofluorescence assay.</p><p><b>CONCLUSION</b>Rev/RRE was necessary for expression of viral structural proteins; CTEs from Mason-Pfizer monkey virus was functionally interchangeable with EIAV Rev/RRE to help RNAs transportation out of nucleus to express structural proteins and EIAV particles were produced in the transfected 293 cells. A live EIAV recombinant virus with single round infection had been developed.</p>

Levantamento soro-epidemiológico da infecção pelo vírus da Anemia Infecciosa Eqüina, da Influenza Eqüina-2 e do Herpesvírus Eqüino-1 em rebanhos do sul do Estado do Pará, Brasil / Seroepidemiological survey of infection of equine infectious anemia, equine influenza type 2 and equine herpesvirus type 1 in herds of south Pará State, Brazil

Os vírus da anemia infecciosa eqüina (EIAV), da influenza eqüina tipo 2 (EIV-2) e o herpesvírus eqüino tipo 1 (EHV-1) são agentes causadores de enfermidades que podem causar graves prejuízos econômicos. O objetivo deste presente estudo foi estimar a freqüência de anticorpos contra o EIAV, EIV-2 e o EHV-1 em rebanhos do sul do Estado do Pará, Brasil. Os anticorpos contra EIAV, EIV-2 e EHV- 1 foram detectados pelo teste de IDGA, pelo método de inibição da hemaglutinação e pela técnica de soroneutralização (TCID50 =100), respectivamente. Amostras de sangue de 672, 514 e de 506 equídeos saudáveis e sem histórico de vacinação contra nenhum dos três vírus foram testadas, respectivamente, para EIAV, EIV-2, EHV-1. A seguinte freqüência de soro reativos foi observada: 1,34% para o EIAV; 35,79% para o EIV-2; 45,45% para o EHV-1. Estes resultados indicam que estes agentes estão presentes no rebanho paraense e a adoção de medidas de manejo e profilaxia devem ser priorizadas, garantindo deste modo, a prosperidade da eqüideocultura brasileira.

Equine infectious anemia virus (EIA V), equine influenza virus type 2 (EIV-2) and equine herpesvirus type 1 (EHV-1) are the causal agents of diseases that may bring economical Iosses. The aim of this present study was to estimate the frequency of antibodies against EIAV, EIV-2 and EHV-1 in herds of south Pará State, Brazil. Antibodies against EIAV, EIV-2 and EHV-1 were detected by AGID, hemagglutination inhibition method and serum neutralization technique (TCID50 =100), respectively. Blood samples of 572, 514, and 506 healthy equine unvaccinated against any of the three viruses were tested, respectively, for EIAV, EIV-2 and EHV-1. The following frequencies of serum reactors animals were observed: EIAV,1,34%; EIV-2, 35,79%; EHV-1, 45,45%. These results show that the agents are present in herds from Pará herds and the adoption of measures of management and prophylaxis should be prioritized, ensuring, thereby, the prosperity of brazilian's breeding equine.

Expression and immunogenicity of equine infectious anemia virus membrane protein GP90 / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>Membrane protein GP90 of China equine infectious anemia virus (EIAV) vaccine strain (DLV) and its parental wild type LN strain were expressed with Bac-to-Bac baculovirus expression system and BALB/c mice were inoculated with purified protein, thereby to explore the availability of protein for differential diagnosis and potential for preparing genetically engineered vaccine.</p><p><b>METHODS</b>The authors infected donkey PBMC culture with China EIAV vaccine strain (DLV) and its parental wild type LN strain, extracted its proviral DNA as template, amplified the GP90 of DLV and LN, respectively, and expressed with Bac-to-Bac baculovirus expression system. The sf9 insect cells were infected with the recombinant baculovirus and the expressed proteins were purified by IMAC. BALB/c mice were inoculated with purified protein. The specific binding Abs generated in the immunized mice were determined by ELISA method. The neutralizing assay was set up to determine the neutralizing capability of the antigens generated in immunized animals.</p><p><b>RESULTS</b>The recombinant virus expressing viral antigens was determined by Western blot. The expressed proteins were purified by IMAC resulting in the protein purity of 87%(DLV) and 82%(LN), respectively. The antibody titer of the groups with and without adjuvant was 1 600 and 800, respectively. Serial 2 fold dilutions of the immunized mice sera were reacted with 100 TCID50 of EIAV. The end point of immunization assay was to protect 50% cells form CPE caused by EIAV in donkey skin cells. The neutralizing antibody titer was in the range 40 to 80 from animal immunized with and without adjuvant.</p><p><b>CONCLUSIONS</b>Membrane proteins of EIAV vaccine strain and wild type strain were successfully expressed in eukaryotic cell expression system according to the scheduled plan. The proteins showed strong immunogenicity and could activate animals to produce anti-EIAV specific antibody including neutralizing antibody to EIAV.</p>

Soroprevalência da anemia infecciosa eqüina, da arterite viral dos eqüinos e do aborto viral eqüino no município de Uruará, PA, Brasil / Seroprevalence of equine infection anemia, equine viral arteritis and equine viral abortion in Uruará municipality, Pará state, Brazil

Os vírus da anemia infecciosa eqüina (VAIE), da arterite viral dos eqüinos (VAVE) e do aborto viral eqüino (Herpesvírus eqüino tipo 1, HVE-1) säo agentes causadores de enfermidades nos eqüídeos que podem causar graves prejuízos econômicos. O objetivo do presente trabalho foi estimar a soroprevalência de anticorpos contra os vírus VAIE, VAVE e HVE-1, utilizando como unidades de análise os eqüídeos e as propriedades rurais do tipo familiar do município de Uruará, PA. Os anticorpos contra o VAIE foram pesquisados pela prova de imunodifusäo em gel de ágar e os anticorpos contra o VAVE e o HVE-1 pela prova de soroneutralizaçäo. O tamanho da amostra foi estimado a partir de um total de 2069 propriedades, caracterizadas por agricultura familiar e ausência de vacinações contra o VAVE e o HVE-1. Foi adotado um nível de confiança de 90 por cento, com uma precisäo de 15 por cento e prevalência estimada de 50 por cento. As seguintes prevalências de animais soro reatores para os diferentes vírus foram observadas: VAIE: 17,71 por cento (IC 10,67 - 26,83 por cento); HVE-1: 17,71 por cento (IC 10,67 - 26,83 por cento) e VAVE: 0,00 por cento (IC 0,00 - 3,77 por cento). As seguintes prevalências de propriedades com pelo menos um animal soro reator para os diferentes vírus foram observadas: VAIE: 53 por cento (IC 38,12 - 68,12 por cento); HVE-1: 40.62 por cento (IC 25.96 - 56.65 por cento) e VAVE: 0 por cento (IC 0 - 6.94 por cento)