Orientador: Clarice Weis Arns / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-05T13:06:28Z (GMT). No. of bitstreams: 1 Carvalho_RodrigoFranco_D.pdf: 1262824 bytes, checksum: 9c044e92e7146ea5bb090e025e656061 (MD5) Previous issue date: 2005 / Resumo: O herpesvírus eqüino do tipo 1 (EHV-1) é um membro da subfamília Alfaherpesvirinae, implicado no surgimento de distúrbios respiratórios, reprodutivos e nervosos em cavalos. A principal forma de contaminação dos animais é através do contato direto com secreções contaminadas pelo vírus. No eqüino, a disseminação do vírus ocorre pela transposição da infecção respiratória a outros órgãos e sistemas através da corrente sanguínea. Pouco se sabe sobre a ocorrência do EHV-1 no Brasil. Dessa forma, este estudo teve por objetivo o isolamento do EHV-1 a partir de material biológico e produção e análise de dados moleculares de isolados brasileiros de EHV-1. Durante este estudo, foi realizado o isolamento de uma amostra de EHV-1 a partir da inoculação de material clínico em células de derme eqüina (ED). Este isolado foi diagnosticado como EHV-1 através da reação em cadeia da polimerase (PCR) para o gene da timidina quinase (tk). Neste trabalho, foram também realizados os seqüenciamentos de fragmentos de PCR derivados do isolado aqui descrito, de uma outra amostra brasileira de EHV-1 e de duas amostras estrangeiras do vírus para análise filogenética. A análise comparativa entre seqüências permitiu inferências sobre o nível de divergência entre os vírus estudados, além da listagem de seqüências regulatórias para atividade gênica em um sítio do genoma localizado próximo ao gene tk. Na região genômica reportada foram contextualizadas ao menos três genes (ORF 38, ORF 37 e ORF 36). Os dados levantados com o seqüenciamento de amostras de EHV-1 de origens geográficas distintas (Brasil, Europa e América do Norte) não mostraram divergências, o que pode estar associado a um processo seletivo constritivo, que impediria a fixação de novas mutações naquela região. A ausência de divergências também pode estar associada à importância dessa região na regulação gênica do EHV-1. Também é um indicativo para a fidelidade dos mecanismos de replicação envolvidos na síntese do DNA viral, o que sugere a importância da região estudada na regulação da expressão gênica do EHV-1 / Abstract: Equine Herpesvirus Type 1 (EHV-1) is a member of Alphaherpesvirinae subfamily implicated with abortions, respiratory and neurological disturbs in horses. The principal mode of viral transmission is through close contact virus-containing secretions of infected horses. Systemic pathogenesis in which this virus is implicated combines primary respiratory infection and spread of viral particles through the circulatory/lymphatic system. Until today, there are only few studies involving the isolation of this virus in Brazil. Thus, the main goal of this study was the isolation of EHV-1 from biological material and the production and analysis of molecular data derived from Brazilian EHV-1 isolates. Clinical samples were screened by inoculation into Equine Dermis (ED) cells monolayers, searching for the characteristic citopathic effect produced by EHV-1. Inoculation of one tissue sample has presented a suggestive citopathic effect. Re-inoculation of the original tissue homogenate in a second, independent experiment reproduced the same positive result. Following these observations, infection agent diagnostic was done by PCR for thymidine kinase (tk) gene. The results demonstrated that sample was EHV-1 positive. In this work, it was done either the sequencing of PCR fragments derived from two Brazilian and two foreign samples of EHV-1 for filogenetic and genomic analyses purposes. It was assigned at least three Open Reading Frames contexts (ORF 38, ORF 37, ORF 36). The data do not show genetic variation between sequences. The high level of genetic conservation for this region, despite the distinct geographic origins (Brazil, Europe and North America) of EHV-1 samples studied, indicates a strong selection process against the fixation of new mutations. It also highlights a high level of fidelity for DNA replication and strongly suggests the importance of the studied region for EHV-1 gene regulation / Doutorado / Microbiologia / Doutor em Genetica e Biologia Molecular

The project was undertaken to monitor a group of 30 foals on a farm both clinically and microbiologically from birth until one year of age, to determine the aetiology of upper respiratory tract infections and to establish immune profiles of some of the known respiratory viral pathogens. One to two months prior to the birth of their foals, blood for serology was collected from the mares. The same specimens were collected from the foals just after birth, prior to suckling and a day after suckling. Thereafter the foals were examined monthly for the presence of respiratory disease and specimens taken. The following specimens were collected from each foal: three nasopharyngeal swabs, (one for virus isolation, one for bacteria and fungus isolation, and one for mycoplasma isolation) and blood that was allowed to clot. Blood was collected in heparin from sick foals with elevated rectal temperatures. Virus isolation was done on roller tube cultures of equine embryonic lung (EEL), Vero cells and rabbit kidney 13 (RK13) cells. The bacteria (including mycoplasmas) and fungi were cultured from the swabs and identified using a variety of traditional methods. The serum neutralization test (SNT) was used to detect antibodies to equid herpesvirus 1 (EHV-1), equid herpesvirus 4 (EHV-4), equine rhinovirus 1 (ERV-1), equine rhinovirus 2 (ERV-2) and equine adenovirus 1 (EAdV-1). The complement fixation test (CFT) was used to detect antibodies to EHV-1 and EHV-4 and the haemagglutination inhibition test (HIT) antibodies to equine influenzavirus (EIV). Only EHV-4 was cultured from the nasopharyngeal swabs of nine foals when they were 5 to 6 months of age and from one foal two months later. A wide variety of bacteria and fungi were cultured and it was established that coagulase-negative staphylococci, viridans streptococci, Moraxella spp. and Flavobacterium spp. predominated in most of the samples. Several potential bacterial pathogens were isolated but the most common were Streptococcus equi subsp. zooepidemicus, Actinobacillus equuland Staphylococcus aureus. Colostrum-derived antibodies were detected for all the viruses in all but two of the foals. It was found that the foals had similar or slightly higher titres than their mothers. The levels declined in direct proportion to what they initially were and were depleted by the time the foals were 2 to 7 months of age. Antibodies to natural infection was detected to EHV-4, ERV-2 and EAdV-1. A rise in antibody titres occurred when the foals were 5 to 6 months of age, two months later and when they were one year of age. Antibodies resulting from immunization was detected to EHV-1, EHV-4 and EIV. It was established that the most important virus causing upper respiratory tract disease of the foals from 5 to 12 months of age was EHV-1 with EAdV-1 playing a minor role. These viruses caused repeated bouts of infection with a two to five months interval. Streptococcus equi subsp. zooepidemicus was considered to be the most important secondary pathogen. Prior to this period most of the foals were healthy with only a few suffering from upper respiratory disease. The aetiology was not determined in these cases, but based on the bacteriology results, it was suspected that some of them were suffering from bacterial infections. / Dissertation (MSc (Veterinary Science))--University of Pretoria, 2005. / Veterinary Tropical Diseases / unrestricted