Thesis (MSc)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: Potato Virus Y (PVY) and Potato Leafroll Virus (PLRV) are two of the most destructive potato viruses capable of drastically diminishing crop yields by up to 80%. The presence of these viruses in planting material namely seed potato stocks are routinely diagnosed by enzyme-linked immunosorbent assay (ELISA) kits. The kits currently used by Potatoes South Africa are obtained from Europe. These kits have produced false positive and false negative results in the past. Potatoes South Africa required an ELISA that was reliable, cheap and specific for the detection of South African strains of the two respective viruses. In this study the viral coat protein genes were amplified by RT-PCR from a South African source of infected plant material. The PVY and PLRV coat protein genes were subsequently cloned into pGEM-T Easy vector and sequenced. The sequences of the two viruses were aligned and compared to corresponding viral coat protein gene sequences obtained from Genbank. Subsequently the two amplified and cloned coat protein genes of PVY and PLRV were sub-cloned into an expression system (pET-14b) to induce and express the respective recombinant viral coat proteins. The induction of the cloned coat protein genes yielded successful production of the recombinant PVY coat protein but the induction and expression of the recombinant PLRV coat protein was unsuccessful. The isolated recombinant PVY CP was then used to immunize a rabbit to produce highly specific anti-PVY CP immunoglobulins. The antiserum obtained from the rabbit was used to develop an ELISA to detect the presence of PVY in seed potato stocks in South Africa. The ELISA kit was subsequently used in preliminary trials to determine if the kit could detect PVY infected plant material. The initial results of the ELISA trials using PVY infected material obtained from Potatoes South Africa yielded positive results. / AFRIKAANSE OPSOMMING: Aartappel Virus Y (PVY) en Aartappel Rolblad Virus (PLRV) is twee van die mees vernietigende aartappel virusse wat ‘n oes tot 80% kan verlaag. Virus infeksie van plant materiaal tewete aartappelmoere word deur “enzyme-linked immunosorbent assay” (ELISA) toetsstelle bevestig. Die toetsstelle wat op die oomblik gebruik word deur Aartappels Suid- Afrika word in Europa vervaardig. Hierdie toetsstelle het vals positiewe en vals negatiewe resultate in die verlede gegee. Aartappels Suid-Afrika benodig toetsstelle wat betroubaar, goedkoop en spesifiek vir Suid-Afrikaanse virus stamme is. In hierdie studie is besmette plantmateriaal vanuit Suid-Afrika gebruik vir die amplifisering van virale mantel proteïen gene met behulp van RT-PCR. Die PVY en PLRV mantel proteïen gene was daarna in die pGEM-T Easy vektor gekloneer en nukleotied volgordes is bepaal. Die nukleotied volgordes is met ander PVY en PLRV gene vanaf Genbank vergelyk. Die twee ge-amplifiseerde en gekloneerde mantel proteïen gene van PVY en PLRV is uitgesny en gekloneer in ‘n ekspressie sisteem (pET-14b) om die mantel proteïen te produseer. Induksie van die gekloneerde mantel proteïen gene het gelei tot die suksesvolle produksie van ‘n PVY mantel proteïen, maar produksie van die PLRV mantel proteïen was onsuksesvol. Die geïsoleerde PVY mantel proteïen is vervolgens gebruik vir die immunisering van ‘n konyn vir die produksie van konyn anti-PVY antiliggame. Die antiserum verkry vanaf die konyn is gebruik vir die ontwikkeling van ‘n ELISA vir die identifisering van PVY infeksies in aartappelmoere. Voorlopige proewe is deurgevoer om te bepaal of hierdie ELISA PVY infeksies in plantmateriaal sou kon opspoor. Aanvanklike resultate toon dat die ELISA suksesvol PVY infeksies in plantmateriaal verkry vanaf Aartappels Suid-Afrika kan opspoor.

Thesis (MSc)--University of Stellenbosch, 2008. / ENGLISH ABSTRACT: Potato virus Y (PVY) is responsible for considerable yield losses in the South African potato industry. The incidence of this virus has greatly increased over the past few years. Even more worrying is the variation of symptoms observed during PVY infection and the recent appearance of the more virulent PVYNTN strain in local fields. This project aimed to investigate the possible genetic variation within the viral genome and to establish the origin of strains. The project also aimed to establish a dependable, area specific enzyme-linked immunosorbent assay (ELISA) to replace the currently used ELISAs. Currently seed potato certification is done using ELISA kits imported from Europe. These kits were developed for the detection of overseas variants of PVY and the use thereof in South Africa has in the past lead to false negatives. Finally, this project set out to develop, optimize and establish a sensitive and reliable real-time reverse transcriptase polymerase chain reaction (qRT-PCR) detection protocol for PVY. In the first part of the study the coat protein (CP) gene of PVY isolates from plant material obtained from various parts of South Africa was amplified using RT-PCR. The resulting cDNA was then sequenced directly or cloned into a vector and then sequenced. The resulting sequences were aligned in a data matrix with international reference sequences, analyzed and grouped according to strain. Examination of the CP gene within this matrix as well as phylogenetic analysis revealed six main groups of PVY. These six groups included the traditional PVYN and PVYO groups and a recombinant group. Furthermore it also revealed variants of PVYN and PVYO. These mutants and recombinants pose a threat as they may lead to South African strains of PVY expressing coat proteins which vary from those found overseas. This may render the currently used European ELISA method of detection less effective and subsequently result in an increase in viral prevalence. This reinforced the need for a detection method based on local viral strains. Phylogenetic and Simplot analysis also confirmed that a recombinant strain between PVYN and PVYO had evolved and that PVYNTN was such a recombinant. The second part of the study aimed to develop and establish detection methods based on local variants of PVY. This included the development of ELISA and qRT-PCR detection methods of PVY. Previously amplified cDNA of the PVY CP gene was cloned into an expression vector and successfully expressed. Antibodies produced against the recombinant protein, when used in ELISA, however, failed to achieve the required levels of sensitivity. This prompted the development of qRT-PCR detection methods for PVY. Primer combinations for PVY were designed using the previously established CP gene data matrix. A reliable and sensitive SYBR® Green I based qRT-PCR assay was developed for the detection of PVY. The assay effectively detected all known South African variants of PVY. Furthermore, a Taqman® assay was developed for the detection of all variants of PVY. The Taqman® assay was 10 fold less sensitive and does not allow for amplicon verification through melting curve analysis, but it does add more specificity due to the addition of the probe. Although these qRT-PCR detection methods are still too expensive to replace the routine diagnostics done with ELISA, they do offer the opportunity to screen valuable mother material and confirm borderline cases in seed certification. / AFRIKAANSE OPSOMMING: Aartappel virus Y (PVY) is verantwoordelik vir aansienlike opbrengsverliese in die Suid-Afrikaanse aartappelindustrie. Die insidensie van infeksie deur die virus het drasties toegeneem oor die afgelope jare. Wat egter meer kommerwekkend is, is die groter variasie in simptome van PVY infeksie en die onlangse voorkoms ‘n meer virulente ras, PVYNTN. Hierdie projek poog om moontlike genetiese variasie van PVY te ondersoek en om die oorsprong van rasse op te spoor. Die projek het ook gepoog ook om ‘n bruikbare, betroubare en area spesifieke “enzyme-linked immunosorbent assay” (ELISA) toets te ontwikkel om die huidige ingevoerde ELISA te vervang. Hierdie toetse is ontwikkel om oorsese variante van PVY op te spoor en die gebruik daarvan het in die verlede gelei tot vals negatiewes. Verder is daar ook ondersoek ingestel na die ontwikkeling van ‘n sensitiewe en betroubare “real-time reverse transcriptase polymerase chain reaction” (qRT-PCR) protokol vir die opsporing van PVY. In die eerste deel van die studie is die mantelproteïen geen van PVY isolate vanuit plant materiaal geamplifiseer deur die gebruik van RT-PCR. Hierdie materiaal is vanaf verskeie streke in Suid-Afrika ontvang. ‘n Volgordebepalingsreaksie is uitgevoer op gekloneerde of ongekloneerde cDNA verkry uit die RT-PCR. DNA volgordes is in ‘n data matriks geplaas en vergelyk met internationale volgordes om die plaaslike isolate te analiseer en te groepeer. Deur vergelyking en filogenetiese ontleding kon ses hoofgroepe van PVY geïdentifiseer word, wat tradisionele PVYN en PVYO, sowel as ‘n rekombinante ras en variante binne die tradisionele PVYN en PVYO groepe ingesluit het. Rekombinante en mutante kan veroorsaak dat Suid-Afrikanse rasse van PVY mantelproteïene uitdruk wat afwyk van die oorsese rasse wat tot gevolg mag hê dat die ELISAs van oorsee minder effektief kan wees en kan lei tot verhoogde virus voorkoms. Die realiteit en gevaar versterk die gedagte dat ‘n deteksie metode gebaseer op plaaslike virusse absoluut krities is. Filogenetiese sowel as Simplot analise het bevestig dat ’n mutante ras tussen PVYN en PVYO ontstaan het en dat PVYNTN ’n rekombinante ras is. Die tweede deel van die studie was daarop gemik om deteksie metodes te ontwikkel wat gebaseer was op plaaslike variante van PVY. Dit sluit die ontwikkeling van ELISA sowel as qRT-PCR deteksie van PVY in. Voorheen geamplifiseerde cDNA is in ‘n ekspressievektor gekloneer en suksesvol uitgedruk. Teenliggaampies teen die rekombinante proteïen, indien in ELISA aangewend, kon egter nie die nodige sensitiwiteit oplewer nie. Dit het aanleiding gegee tot ontwikkeling van qRT-PCR deteksie metodes. Inleier kombinasies vir PVY was ontwikkel deur die gebruik van die bestaande mantelproteïen geen data matrikse. ‘n Betroubare en sensitiewe SYBR® Green I qRT-PCR deteksie protokol was ontwikkel vir die effektiewe deteksie van alle bekende Suid-Afrikanse rasse van PVY. Verder is ‘n sogenaamde “Taqman®” protokol ook ontwikkel vir deteksie van alle rasse. Die “Taqman®” protokol was 10 voudiglik minder gevoelig and laat nie bevestiging deur smeltkurwe analise toe nie, maar verleen meer spesifisiteit deur die toevoeging van die “Taqman® probe”. Hierdie qRT-PCR deteksie metodes is tans te duur om as roetine diagnostiese toetse te gebruik en kan dus nie ELISA vervang nie, maar hulle bied wel die geleentheid om waardevolle moeder materiaal te toets en grensgevalle in aartappelsaad sertifisering te bevestig.

published_or_final_version / abstract / Zoology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Microbiology / Master / Master of Philosophy

As hepatites B e C constituem ainda importante problema de saúde pública no mundo, com cifras de portadores crônicos estimados em cerca de 350 milhões e de 170 milhões, respectivamente, fazendo com que número elevado de pessoas se encontrem sob risco de cronificação e evolução para cirrose hepática e hepatocarcinoma. Entre os fatores de risco mais conhecidos para hepatite B estão às transfusões de sangue e derivados, o contato com sangue e/ou com secreções, por relações sexuais desprotegidas, compartilhamento de seringas e/ou agulhas para uso de drogas injetáveis, tatuagem e piercing com material contaminado. A hepatite C é transmitida primordialmente por via parenteral e a sua prevenção faz-se de modo semelhante à hepatite B. Diversos fatores de risco para ambas parecem ser fazer presentes na população surda, a qual é historicamente marginalizada em termos de acesso a informações e serviços de saúde e sobre a qual não existem referências de investigações sobre hepatites virais. Esse trabalho teve como objetivos estimar a prevalência de sorologia positiva para hepatite B e C e investigar possíveis fatores de risco entre a população surda de Ribeirão Preto. Foram estudados 88 surdos, aos quais foi apresentado DVD explicativo sinalizado em Libras sobre características e riscos das hepatites B e C. Aos que concordaram em participar foi aplicado questionário padronizado e coletada uma amostra de sangue para realização de testes imunoenzimáticos para detecção dos marcadores HBsAg, anti-HBs, anti-HBc e anti-HCV, realizados no Laboratório de Sorologia do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto. A análise sorológica revelou presença de infecção atual ou pregressa por hepatite B em sete participantes, correspondendo a 8% de prevalência total de marcadores (IC95%: 2,3 13,7). Ao se analisar as possíveis variáveis de risco, encontrou-se associação entre a infecção e as variáveis: ser nascido em outra Unidade Federada que não São Paulo e antecedente de encarceramento. Os participantes mostraram grande desconhecimento sobre aspectos básicos relacionados à transmissão das hepatites virais, indicando necessidade de políticas de saúde pública voltadas para esta população, e que levem em consideração suas particularidades linguística e cultural. / Hepatitis B and C are still an important public health problem in the world, with chronic carriers estimated at around 350 million and 170 million, respectively, causing a large number of people to be at risk of chronic forms and progression to liver cirrhosis and hepatocellular carcinoma. Among the most well-known risk factors for hepatitis B are blood and derivatives transfusions, contact with blood and/or secretions by unprotected sex, sharing needles and/or syringes in injectable drug use, tattooing and piercing with contaminated material. Hepatitis C is transmitted primarily by parenteral way and its prevention is similar to hepatitis B. Several risk factors for both seem to be present in the deaf population, which is historically marginalized in terms of access to health information and services and on which there are no references to research on viral hepatitis. This work aimed to estimate the prevalence of positive serology for hepatitis B and C and to investigate possible risk factors among the deaf population of Ribeirão Preto. An explanatory DVD about the features and risks of hepatitis B and C was presented to them in Brazilian Sign Language. Eighty eight deaf agreed to participate, signed a free and informed consent form and were included in the investigation. A standardized questionnaire was applied and a sample of blood was collected. Immunoenzymatic tests for detection of HBsAg, anti-HBS, anti-HBC and anti-HCV markers were carried out at the Serum Laboratory of the University Hospital of the Ribeirão Preto Medical School, University of São Paulo. The serological analysis revealed the presence of current or previous infection by hepatitis B in seven participants, representing 8% of total prevalence of markers (CI95%: 2,3 13,7). When analyzing the possible risk factors, it was found association between infection and the variables being born in another State other than São Paulo and past history of imprisonment. No positive samples for hepatitis C were found. The participants showed great ignorance about basic aspects related to the transmission of viral hepatitis, indicating need for public health policies directed to this population that takes into account their linguistic and cultural singularities.

A morte súbita dos citros (MSC) foi identificada em 2001, no município de Comendador Gomes, Minas Gerais, e desde então foi responsável pela perda de 4 milhões de plantas na região sul do Triângulo Mineiro, e no norte e noroeste do estado de São Paulo. É uma doença de combinação copa/porta-enxerto, afetando principalmente laranjeira doce enxertada em limoeiro Cravo, e que culmina na morte das plantas. Passados dez anos do seu relato, até hoje não se tem conhecimento exato do agente causal e dos possíveis vetores. Sabe-se, todavia, que em todas as plantas com morte súbita encontram-se o vírus da tristeza dos citros (Citrus tristeza virus - CTV) e um vírus do Gênero Marafivirus, Família Tymoviridae, denominado Citrus sudden death associated virus (CSDaV). Devido esse fato, há a necessidade de separá-los para testar os postulados de Koch para o CSDaV. O principal objetivo deste trabalho foi tentar isolar o CSDaV para verificar o seu real envolvimento com a MSC. Também se procurou purificar esse vírus para a produção de antissoro policlonal para trabalhos de diagnose da doença. Para a purificação do CSDaV foi utilizado o protocolo de purificação do Potato leaf roll virus, porém os resultados não foram satisfatórios devido a constante presença do CTV. Tentativas de remoção do CTV por meio de imunoprecipitação com antissoro homólogo não foram satisfatórias. O antissoro produzido reagiu indistintamente com extratos de plantas infectadas com o CSDaV e o CTV. O CSDaV foi transmitido mecanicamente para plantas de Nicotiana sp., N. clevelandii, Chenopodium amaranticolor e C. quinoa, causando principalmente infecção localizada. A presença do vírus foi confirmada por RT-PCR e a sua identidade por meio do sequenciamento de nucleotídeos do produto da amplificação. Tentativas de transmissão do CSDaV usando inóculo de extrato de folhas de plantas do campo , por meio da Cuscuta sp., através de cortes no tronco das plantas com lâmina embebida no inóculo, com pulgões Toxoptera citricida aparentemente virulíferos somente para o tymovirus e por meio da inoculação de sementes de citros deram resultados negativos. / Citrus sudden death (CSD) disease was identified in 2001, at Comendador Comes County, State of Minas Gerais, Brazil. Since then the disease has caused the death of 4 million trees in Southwestern Minas Gerais State and Northern São Paulo State. This new and destructive disease affects sweet orange as well as other species, varieties, and hybrids when grafted on Rangpur (Citrus limonia). Then years after the first report on CSD, the causal agent and possible vector(s) have not been precisely identified. It is known, however, that all disease trees are infected with Citrus tristeza virus (CTV) and Citrus sudden death associate virus (CSDaV), which is a member of the Genus Marafivirus, Famíly Tymoviridae. Due to this, it is necessary to separate these pathogens, in order to complete Kochs postulated for the CSDaV. The main purpose of the present work was to try to isolate the CSDaV to verify its role on CSD disease. In addition, attempts were done to purify the virus and produce polyclonal antiserum for disease diagnosis. Purification was carried out as described for Potato leaf roll virus, but results were not suitable due to the constant presence of CTV. Efforts to remove CTV by immunoprecipitation with homologous antiserum did not succeed. The produced antiserum reacted indistinctly with extracts of plants infected with both viruses. SCDaV was mechanically transmitted to Nicotiana sp., N. clevelandii, Chenopodium amaranticolor, and C. quinoa, causing mainly local infection. Infection was confirmed by RT-PCR and virus identity was determined by nucleotide sequence of the amplified fragment. Efforts to transmit CSDaV, using as inoculum extract from field infected plants, by means of Cucucuta sp., by incisions on the trunk of the test-plants, with Toxoptera citricida apparently viruliferous only for the tymovirus, and by means of citrus seed inoculation gave negative results.

Orientador: João Pessoa Araújo Junior / Banca: José Paes de Oliveira Filho / Banca: Clarice Weis Arns / Resumo: O herpesvírus canino (HVC-1) causa doença infecto-contagiosa que acomete cães em todo o mundo. É considerado o responsável por causar problemas reprodutivos, respiratórios, oculares, alterações neurológicas podendo levar à morte neonatos e adultos imunossuprimidos. Assim, o presente trabalho teve como objetivos isolar o HVC-1 a partir de amostras biológicas, padronizar uma técnica diagnóstica para detecção do herpesvírus canino, verificar a presença de cães infectados no estado de São Paulo e comparar 3 modificações na técnica de diluição limitante em relação à quantificação molecular pela qPCR (quantitative Polymerase Chain Reaction). Para isso, foram coletadas 139 amostras sangue, fragmentos de órgãos de 12 neonatos que foram a óbito e 5 amostras de suabes de secreção genital, de animais provenientes de canis e domicílios. Após a obtenção do DNA das amostras colhidas, foram submetidas à qPCR e snPCR (seminested Polymerase Chain Reaction) para o teste de detecção do HVC-1. Foi encontrada positividade em nove foram amostras de sangue e em fragmentos de órgãos de um animal, as amostras de secreções genitais foram todas negativas. O isolamento viral foi realizado a partir de fragmento de rim. As células infectadas apresentaram efeitos citopáticos compatíveis com as do HVC-1. As amostras positivas foram sequenciadas apresentando identidade de 100% com o HVC-1 (Genbank ™: X75765). As técnicas moleculares para a detecção do agente mostraram-se sensíveis e específicas, tanto a snPCR como a qPCR. Nessas técnicas foi possível detectar até 15,35 cópias/ μL de DNA de amostras de sangue e 1,15 cópias/μg de tecido. O uso de técnicas moleculares possibilitou a detecção de infecções ativas por HVC-1 com ótimo limiar de detecção. No entanto, mais estudos são necessários para conhecer a epidemiologia da infecção do HVC-1 na população canina no Brasil / Abstract: The canine herpesvirus (CHV-1) causes contagious disease affecting dogs worldwide. It is considered the responsible for cause reproductive problems, respiratory, eyes, neurologic changes may lead to death newborns and immunocompromised adults. Therefore, our study aimed to isolate CHV-1 from biological samples, standardize a diagnostic techniques for the detection of canine herpesvirus, verify the presence of infected dogs in São Paulo state and compare three modifications in the technique of limiting dilution in relation to molecular quantification to qPCR (by quantitative PCR to determine the viral titer). Thus, we collected 139 blood samples, organ's fragments from 12 neonates death and 5 genital secretions samples with swabs of animals from kennels and households. After obtaining the DNA from samples were accomplished the qPCR (quantitative PCR) and snPCR (seminested PCR) assay for detection of CHV-1. It was found positivity in nine blood sample and organ's fragments of an animal, the genital secretions samples were all negative. The viral isolation was performed from kidney. The infected cells showed cytopathic effects compatible with the CHV-1. Positive samples were sequenced showed 100% identity with the CHV-1 (Genbank™: X75765). The molecular techniques for detection agent were sensitive and specific, both the snPCR as the qPCR. In these techniques was enabled to detect DNA 15.35 copies/mL of blood samples and 1.15 copies/μg of tissue. The use of molecular techniques allowed the detection active infections of of HCV-1 with optimal detection limit. More studies are needed to understand the infection epidemiology of CHV-1 in the canine population in Brazil / Mestre

Orientador: Alexandre Secorun Borges / Banca: João Pessoa Araújo Junior / Banca: Paulo Eduardo Martins Ribolla / Banca: Eduardo Furtado Flores / Banca: Marcelo Mendes Brandão / Resumo: Herpesvirus bovino (BoHV-5) é um alfaherpesvirus que causa meningoencefalite não-supurativa preferencialmente em bovinos jovens. Esta enfermidade ocorre naturalmente em surtos ou casos isolados, apresentando baixa morbidade e alta letalidade. A epidemiologia, as características anatomopatológicas e o diagnóstico do BoHV-5 são bem conhecido. No entanto, as interações moleculares entre a células hospedeiras e o BoHV-5 são pobremente compreendidas. Técnicas moleculares como a PCR quantitativa e o sequenciamento de RNA (RNA-seq) são importantes ferramentas para o estudo de interações entre vírus e células hospedeiras. Neste estudo células MDBK foram infectadas pelo BoHV-5, cepa SV507-99 e o mRNA extraído em 0, 6, 12, 18, e 24 horas após a infecção (pi) foi utilizado para avaliar a expressão de genes do vírus e da célula hospedeira por qPCR e o mRNA de 1h, 6h e 24h pi foram utilizado no estudo por RNA-seq. Células MDBK não infectadas foram usadas como controle. A qPCR demonstrou que os três genes do BoHV-5 estudados (bICP0, UL9 e US4) apresentaram perfil de expressão semelhante nos diferentes momentos após infecção e que o gene bovino GAPDH teve sua expressão diminuída pela infecção viral. Ao menos 900 genes foram diferencialmente expressos para cada momento de infecção na análise por RNA-seq. Estes genes estavam principalmente relacionados a vias celulares de controle de ciclo celular, produção de interleucinas, quimiotaxia para células inflamatórias, reparo e dano ao DNA, resposta a vírus, apoptose, processo oxidativo, e ubiquitização de moléculas. Estes resultados representam novos conhecimentos sobre a interação entre o BoHV-5 e as células bovinas e podem representar um ponto de partida para novas pesquisas e melhor compreensão da patogenia deste vírus / Abstract: Bovine herpesvirus 5 (BoHV-5) is an Alphaherpesvirus that causes nonsuppurative meningoencephalitis mainly in young cattle. This disease occurs naturally in either outbreaks or isolated cases, and exhibits low morbidity and high lethality. Besides BoHV-5 epidemiology, pathological findings and diagnosis were well known, the molecular interactions between host cell and BoHV-5 are poorly understood. Molecular biology techniques such as quantitative PCR (qPCR) and RNA sequencing (RNA-seq) are important tools to study virus and host cell interactions. In this study we infected MDBK cells and use the extracted mRNA at 0, 6, 12, 18 and 24h post infection (pi) to analyse BoHV-5 (bICP0, UL9 e US4) and host cell gene (GAPDH) expression using qPCR and 1h, 6h and 24h pi in the RNA-seq study. Mock-infected cells were used to control purpouse. The qPCR releveled that the three BoHV-5 genes showed the same expression behavior during the infection and the GAPDH gene were down-regulated in the infected group. At least 900 genes were differentially expressed during BoHV-5 in each moment analysed by RNA-seq. These genes were up- or down-regulated and mainly associated with cell cycle, interleukin production, inflammatory cells chemotaxis, DNA damage and repair, response to virus, apoptosis, oxidation-reduction process, and ubiquitination pathways. The results demonstrate new insights about BoHV-5 and bovine cells interactions and could be a starting point to new researches and to better understand the pathology of this virus / Doutor

As hepatites B e C constituem ainda importante problema de saúde pública no mundo, com cifras de portadores crônicos estimados em cerca de 350 milhões e de 170 milhões, respectivamente, fazendo com que número elevado de pessoas se encontrem sob risco de cronificação e evolução para cirrose hepática e hepatocarcinoma. Entre os fatores de risco mais conhecidos para hepatite B estão às transfusões de sangue e derivados, o contato com sangue e/ou com secreções, por relações sexuais desprotegidas, compartilhamento de seringas e/ou agulhas para uso de drogas injetáveis, tatuagem e piercing com material contaminado. A hepatite C é transmitida primordialmente por via parenteral e a sua prevenção faz-se de modo semelhante à hepatite B. Diversos fatores de risco para ambas parecem ser fazer presentes na população surda, a qual é historicamente marginalizada em termos de acesso a informações e serviços de saúde e sobre a qual não existem referências de investigações sobre hepatites virais. Esse trabalho teve como objetivos estimar a prevalência de sorologia positiva para hepatite B e C e investigar possíveis fatores de risco entre a população surda de Ribeirão Preto. Foram estudados 88 surdos, aos quais foi apresentado DVD explicativo sinalizado em Libras sobre características e riscos das hepatites B e C. Aos que concordaram em participar foi aplicado questionário padronizado e coletada uma amostra de sangue para realização de testes imunoenzimáticos para detecção dos marcadores HBsAg, anti-HBs, anti-HBc e anti-HCV, realizados no Laboratório de Sorologia do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto. A análise sorológica revelou presença de infecção atual ou pregressa por hepatite B em sete participantes, correspondendo a 8% de prevalência total de marcadores (IC95%: 2,3 13,7). Ao se analisar as possíveis variáveis de risco, encontrou-se associação entre a infecção e as variáveis: ser nascido em outra Unidade Federada que não São Paulo e antecedente de encarceramento. Os participantes mostraram grande desconhecimento sobre aspectos básicos relacionados à transmissão das hepatites virais, indicando necessidade de políticas de saúde pública voltadas para esta população, e que levem em consideração suas particularidades linguística e cultural. / Hepatitis B and C are still an important public health problem in the world, with chronic carriers estimated at around 350 million and 170 million, respectively, causing a large number of people to be at risk of chronic forms and progression to liver cirrhosis and hepatocellular carcinoma. Among the most well-known risk factors for hepatitis B are blood and derivatives transfusions, contact with blood and/or secretions by unprotected sex, sharing needles and/or syringes in injectable drug use, tattooing and piercing with contaminated material. Hepatitis C is transmitted primarily by parenteral way and its prevention is similar to hepatitis B. Several risk factors for both seem to be present in the deaf population, which is historically marginalized in terms of access to health information and services and on which there are no references to research on viral hepatitis. This work aimed to estimate the prevalence of positive serology for hepatitis B and C and to investigate possible risk factors among the deaf population of Ribeirão Preto. An explanatory DVD about the features and risks of hepatitis B and C was presented to them in Brazilian Sign Language. Eighty eight deaf agreed to participate, signed a free and informed consent form and were included in the investigation. A standardized questionnaire was applied and a sample of blood was collected. Immunoenzymatic tests for detection of HBsAg, anti-HBS, anti-HBC and anti-HCV markers were carried out at the Serum Laboratory of the University Hospital of the Ribeirão Preto Medical School, University of São Paulo. The serological analysis revealed the presence of current or previous infection by hepatitis B in seven participants, representing 8% of total prevalence of markers (CI95%: 2,3 13,7). When analyzing the possible risk factors, it was found association between infection and the variables being born in another State other than São Paulo and past history of imprisonment. No positive samples for hepatitis C were found. The participants showed great ignorance about basic aspects related to the transmission of viral hepatitis, indicating need for public health policies directed to this population that takes into account their linguistic and cultural singularities.

Human papillomavirus (HPV) infection currently stands as the most common sexually transmitted disease in the world. With an estimated lifetime probability of disease acquisition of 84.6% for females and 91.3% for males with at least one sexual partner, HPV is an aggressive and ubiquitous virus that affects people from all walks of life. The virus generally resolves on its own within 1 year, but successful disease progression leads to complications ranging from genital warts to anogenital cancers. Globally, there are 530,000 new cases of, and 274,000 deaths caused by, cervical cancer in females each year, 99.7% of which are caused by HPV and 70% of which are caused by HPV types 16 and 18. With high rates of infection and 85% of cervical cancer cases concentrated in developing countries, the virus presents an immense threat to public health and global equity. Prophylactic vaccines demonstrate high efficacy against common HPV-related diseases, including cervical cancer, but the high cost and multiple-dose administration of the vaccine limit its full disease-fighting potential. This proposal seeks to determine if a single-dose prophylactic vaccine targeting HPV-16 and -18, when administered in preadolescence, demonstrates long-term efficacy against cervical cancer. Vaccine-induced antibody responses will be measured using geometric mean titers obtained from blood samples, and efficacy of the vaccine will be evaluated by persistent high-risk HPV (HR-HPV) infection, measured through Pap smears and HPV DNA tests. The study will extend 22 years post-vaccination. The single-dose vaccine is expected to provide protection against HR-HPV infection at rates comparable to those of multiple-dose vaccines currently in practice, with the implications of increasing accessibility of the vaccine and, thus, decreasing the global burden of cervical cancer.