Equine infectious anemia virus (EIAV) is a macrophage-tropic retrovirus that causes persistent disease in horses and ponies. In addition to its structural proteins, EIAV encodes four regulatory/accessory genes, tat, rev, ttm, and S2. It has been documented EIAV S2 gene expression is essential for disease expression of EIAV. Using a yeast two-hybrid assay, it was shown that S2 protein interacts with human NF-KB2. NF-KB2 plays a key role in the alternative or non-canonical NF-KB pathway. In order to determine if the interaction of S2 with NF-KB2 might be relevant to equine disease, a cDNA representing full length equine NF-KB2 was generated in our laboratory using PCR and rapid amplification of cDNA ends. To our knowledge this is the first time that equine NF-KB2 cDNAs have been recovered and characterized. The sequence of equine NF-KB2 was 95% homologous to human overall, however a major difference was found in the ankyrin repeat region where protein-protein interactions occur. Two splice variants of equine NF-KB2 were found that correspond to splice variants of human NF- KB2. We tested the interaction of EIAV S2 and equine NF-KB2 using the yeast two hybrid system (Y2H) and co-immunoprecipitation. Unfortunately we were not able to detect an interaction between EIAV S2 and equine NF-KB2 in either system. Despite this result, NF-KB2 is an important component in the immune response so we examined its expression in equine macrophages. Moreover we were interested to know if EIAV might affect expression levels of equine NF-KB2, as NF-KB2 is a target of other viruses. Hence, the expression level of equine NF-KB2 was measured in uninfected and infected primary equine monocyte- derived macrophage (eMDM). Using quantitative PCR we determined that equine NF-KB2 gene expression is decreased in eMDM after 3 days post plating, about the time that monocytes start to differentiate into mature macrophages. However EIAV infection of eMDM upregulated the expression level of NF-KB2.

Submitted by Renorbio (renorbioba@ufba.br) on 2017-08-15T16:15:18Z No. of bitstreams: 1 tese Dellane Martins Tigre 75folhas 2017.pdf: 1616856 bytes, checksum: a34a008544e31d63c94fa0cf795cc2ff (MD5) / Approved for entry into archive by Delba Rosa (delba@ufba.br) on 2017-08-28T13:03:56Z (GMT) No. of bitstreams: 1 tese Dellane Martins Tigre 75folhas 2017.pdf: 1616856 bytes, checksum: a34a008544e31d63c94fa0cf795cc2ff (MD5) / Made available in DSpace on 2017-08-28T13:03:56Z (GMT). No. of bitstreams: 1 tese Dellane Martins Tigre 75folhas 2017.pdf: 1616856 bytes, checksum: a34a008544e31d63c94fa0cf795cc2ff (MD5) / O vírus da Anemia Infecciosa Equina (EIAV), membro da família Retroviridae, gênero Lentivirus, causa uma doença de curso crônico e latente em equídeos. A infecção é limitada a equinos, asininos e muares e caracteriza-se por episódios febris, perda de peso, debilidade progressiva, mucosas ictéricas, edemas subcutâneos e anemia. A AIE não tem tratamento nem vacina eficaz. O diagnóstico clínico é difícil pelo fato que os sinais da doença não são específicos, além disso, após a fase aguda da infecção a maioria dos animais se torna portador assintomático do vírus. Neste estudo nós detectamos e caracterizamos filogeneticamente o vírus isolado na Bahia, Brasil. A partir de amostras de sangue e soro de animais de diferentes municípios do estado utilizamos a técnica de referência pela Organização Mundial para Sanidade Animal (OIE), a prova sorológica de IDGA, e as técnicas moleculares de nested-PCR e nested-RT-PCR. No total 82 animais foram examinados neste estudo por IDGA e PCR. Primers para o gene gag foram utilizados para amplificar o DNA proviral/RNA do EIAV, nos ensaios de nested-PCR e nested-RT-PCR respectivamente. Amplicons de 15 amostras positivas por nested-PCR foram submetidas ao sequenciamento e análise filogenética. As sequencias de EIAV analisadas neste estudo formam um clado com as cepas WSU5, EIAVUK e EIAVwyoming, todas dos EUA. 51 amostras (62,2%) foram positivas por nested-PCR, enquanto apenas 31 amostras (37,8%) foram positivas por IDGA. No presente estudo utilizando técnicas moleculares foi possível demonstrar que animais portadores assintomáticos do EIAV, com diferentes status sorológico apresentam vírus e/ou DNA proviral detectável por PCR, em PBMC e plasma, demonstrando que o vírus se replica mesmo na presença de mecanismos de defesa imunológicos do hospedeiro, sendo o animal portador assintomático e sorologicamente negativo, importante reservatório do vírus no plantel, e sugerindo que o controle da AIE baseado apenas no IDGA precisa ser revisto pelos órgãos fiscalizadores no Brasil. / The Equine Infectious Anemia Virus (EIAV), a member of the family Retroviridae, genus Lentivirus, causes a disease of chronic and latent course in equidae. Infection is limited to horses, asinines and mules and is characterized by febrile episodes, weight loss, progressive weakness, icteric mucous, subcutaneous edema and anemia. The equine infectious anemia (EIA) has no effective treatment or vaccine. The clinical diagnosis is difficult due to the fact that the signs of the disease are not specific; moreover, after the acute phase of infection, most animals become asymptomatic carriers of the virus. In this study we detected and characterized phylogenetically the virus isolated in Bahia, Brazil. We used the reference technique from the World Organization for Animal Health (OIE), the serological test of AGID, and the molecular techniques of nested-PCR and nested-RT-PCR from blood and serum samples from different municipalities of the state. In total, 82 animals were examined in this study by AGID and PCR. Primers for the gag gene were used to amplify the EIAV proviral DNA/RNA in the nested-PCR and nested-RT-PCR assays, respectively. Amplicons of 15 samples positive by nested-PCR were submitted to sequencing and phylogenetic analysis. The EIAV sequences analyzed in this study form a clade with strains WSU5, EIAVUK and EIAVwyoming, all from the USA. 51 samples (62.2%) were positive by nested-PCR, whereas only 31 samples (37.8%) were positive by AGID. In the present study using molecular techniques, it was possible to demonstrate that asymptomatic carriers of EIAV, with different serological status, have virus and/or DNA proviral detectable by PCR, in PBMC and plasma, demonstrating that the virus replicates even in the presence of immunological defense mechanisms of the host, being the asymptomatic and serologically negative carrier animal, an important reservoir of the virus in the establishment, and suggesting that the control of the EIA based only on the AGID test needs to be reviewed by the inspection agencies in Brazil.

The lentiviral capsid core, which encapsulates the viral RNA genome, is delivered into the target cell cytoplasm during the viral entry process. In the cytoplasm, the conical core undergoes morphological changes, which are termed uncoating. Proper uncoating has been shown to be critical for the infectivity of the lentivirus HIV-1. In addition, the HIV-1 capsid protein is critical for the process of nuclear import of the preintegration complex (PIC). The lentivirus equine infectious anemia virus (EIAV) shares many similarities to HIV-1, including similarities in the capsid protein. In particular, both HIV-1 and EIAV capsid contain a proline-rich loop region in the amino terminal domain of capsid between helices 4 and 5. The host cellular factor cyclophilin A binds this loop in HIV-1 and is critical for proper uncoating. We hypothesized that this helix 4-5 loop was also critical for EIAV infectivity at some early step in the viral infection cycle. We created a panel of amino acid substitution mutations in this loop region. Some of the mutations resulted in severely deleterious effects on EIAV infectivity. Some mutations caused a slight increase in infectivity. The deleterious mutations did not affect uncoating or reverse transcription but appeared to block nuclear import of the PIC. Those mutations in which infectivity was slightly increased did not exhibit significantly different phenotypes from wild-type EIAV at any of the stages examined. The results of this study lend further support to the role of capsid as a determinant of nuclear import and suggest that viral and cellular factors critical to HIV-1 import may also be applicable to EIAV. Future research should focus on identifying the causes of the defects in nuclear import observed for some mutants, as well as attempt to identify the reason for the infectivity increase in others. In addition, inclusion of EIAV in future studies of nuclear import involving HIV-1 can broaden the scope of the data to lentiviruses in general rather than HIV-1 in particular.

Submitted by Socorro Pontes (socorrop@ufersa.edu.br) on 2017-06-29T13:21:31Z No. of bitstreams: 1 RebecaJFC_DISSERT.pdf: 1231711 bytes, checksum: 845448c874cc7680ba5ba551b9029235 (MD5) / Made available in DSpace on 2017-06-29T13:21:31Z (GMT). No. of bitstreams: 1 RebecaJFC_DISSERT.pdf: 1231711 bytes, checksum: 845448c874cc7680ba5ba551b9029235 (MD5) Previous issue date: 2017-02-14 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Equine infectious anemia (EIA) is the most important viral disease among horses. It is an endemic disease in populations of Equidae throughout the world. All equids are considered to be susceptible although most of the published work are focused in horses, with a lack of information on EIA in donkeys. In the State of Rio Grande do Norte, Brazil thousands of donkeys live free, establishing a problem for the control of diseases like the EIA, since they may play a role as reservoirs. In this work, blood samples taken from 409 animals (asinine and equine) were submitted to IDGA, ELISA pgp45 and nested-PCR tests, using gag and LTR gene as molecular markers. Four samples (0.98%) were positive in at least one serological test, and of these, three (0.73%) were positive to nested-PCR. We did not get enough material for sequencing of LTR samples from asinine, then only the amplified referring to the positive equine sample was sequenced. The alignment (BLAST) allowed the identification of the sequence as EIAV with 95% of similarity with european strains. Three product from nested-PCR products for the gag gene were sequenced and submitted to phylogenetic analysis. The result of this analysis suggested that the samples obtained from the horse had the same origin as strains from North America, and that the sequences from donkey samples had no homology with any other already published available EIAV sequence. In the light of the results from this work, additional studies are required. So we could confirm that we found a new strain of EIAV or endogenous retrovirus that are capable of expressing genes that are homologous to EIAV or even a new species of lentivirus Although the presented data are incomplete, they reveal an interesting scenario for the study of EIAV infection in equids ther than the horses / A anemia infecciosa equina (AIE) é a doença infecciosa de etiologia viral mais importante entre os equinos. É uma doença endêmica em populações de equídeos por todo o mundo. Considera-se que todos os equídeos são susceptíveis embora os trabalhos publicados se concentrem em equinos, sendo escassas as informações sobre AIE em asininos. No Rio Grande do Norte (RN) milhares de asininos vivem livres, constituindo um problema para o controle de enfermidades como a AIE, pois podem ser prováveis reservatórios e fontes de transmissão delas. Neste trabalho, amostras de 409 animais (asininos e equinos) foram submetidas aos testes de IDGA, ELISA pgp45 e nested-PCR, utilizando iniciadores para o gene gag e LTR. Quatro amostras (0,98%) foram positivas em pelo menos um teste sorológico, e dessas, três amostras (0,73%) foram positivas na nested-PCR. Não obtivemos material suficiente para sequenciamento das amostras de LTR dos asininos sendo sequenciado apenas o amplificado de LTR referente à amostra do equino positivo. O alinhamento (BLAST) permitiu a identificação da sequência como vírus da AIE (EIAV) com 95% de similaridade de nucleotídeos com estirpes europeias. Os três produtos obtidos da nested-PCR para o gene gag foram sequenciados e submetidos à análise filogenética. O resultado da análise sugeriu que a sequência obtida do cavalo confirmaram a identificação como EIAV, com a mesma origem de isolados da América do Norte, e que as sequencias de asininos não possuem identidade com nenhuma outra deo EIAV até o momento publicada. Desta forma, estudos adicionais são necessários para confirmar se, com estes resultados, foram identificados um lentivirus ainda não descrito que infecta Equus asinus (e qual papel ele teria para outros equídeos) ou se trata de retrovírus endógenos que expressa proteínas usadas como marcadores de diagnóstico da AIE. Embora os dados apresentados sejam incipientes, revelam um cenário interessante para o estudo das lentiviroses em asininos / 2017-06-29

Equine infectious anemia virus (EIAV) is an important animal model to study the contribution of macrophages in viral persistence during lentiviral infections. EIAV is unique amongst the lentiviruses in that it causes a rapid, rather than the very slow disease progression, characteristic of other lentiviral infections. The accessory gene, S2, unique to EIAV, is an important determinant in viral pathogenesis. A functional S2 gene is required to achieve high-titer viremia and the development of disease in infected horses. Despite its essential role, the mechanisms by which S2 influences EIAV pathogenesis remain elusive. The goal of this research was to gain insight into the role of S2 in pathogenesis. To accomplish this goal we: (i) Examined the effects of EIAV and its S2 protein in the regulation of the cytokine and chemokine responses in macrophages, (ii) Assessed the influence of EIAV infection and the effect of S2 on global gene expression in macrophages and (iii) Identified host cellular proteins that interact with S2 as a starting point for the identification of host factors implicated in S2 function. The results from this study provide evidence for a role of S2 in enhancing a proinflammatory cytokine and chemokine response in infected macrophages. Specifically, S2 enhances the expression of IL-1 alpha, IL-1 beta IL-8, MCP-2, MIP-1 beta, IP-10 and a newly discovered cytokine, IL-34. Involvement of S2 in cytokine and chemokine dysregulation may contribute to disease development by optimizing the host cell environment to promote viral dissemination and replication. Microarray analyses revealed an interesting set of differentially expressed genes upon EIAV infection. Genes affected by EIAV were involved in the immune response, transcription, translation, cell cycle and cell survival. Finally, we used the yeast two-hybrid system to identify S2 host cellular interacting proteins. We identified osteosarcoma amplified 9 (OS-9) and proteasome 26S ATPase subunit 3 (PSMC3) proteins as interacting partners of S2. Additional evidence is needed to demonstrate the physiological relevance of these interactions in vivo. In summary, the results from this study contribute towards our understanding of the role S2 in disease expression and allow the formulation of new hypotheses as to the potential mechanisms of action of S2 during EIAV infection.