Orientador: Antonio Carlos Paes / Banca: Marcio Garcia Ribeiro / Banca: Osimar de Carvalho Sanches / Resumo: A cinomose é uma doença infecto-contagiosa viral, sistêmica e letal causada pelo Morbilivírus que acomete cães de todas as idades e apresenta sinais clínicos respiratórios, gastrintestinais, dermatológicos, oftálmicos e neurológicos, que podem ocorrer sequencialmente ou isoladamente. Este estudo teve como objetivo comparar lesões encefálicas utilizando a histopatologia e avaliar a quantidade de vírus pela imunoistoquímica em cães tratados com Ribavirina na dose de 30mg/Kg por via oral uma vez ao dia por 15 dias e animais não tratados com esse fármaco, e que devido a evolução desfavorável da doença foram eutanasiados. Os animais foram atendidos e tratados durante a rotina do Setor de Enfermidades Infecciosas dos Animais Domésticos- FMVZ- UNESP/ Botucatu, SP. A análise histopatológica constituiu na avaliação de córtex cerebral, cerebelo, tálamo, tronco encefálico, hipocampo e visualização de manguitos perivasculares em meninges. A partir dessas regiões foi possível identificar e classificar lesões como: desmielinização; presença de manguitos perivasculares, edema, necrose neuronal, alterações vasculares (congestão, hemorragia e trombose) e alterações inflamatórias (meningite, meningoencefalite e meningoencefalomielite). Foi observado que a desmielinização se manteve altamente presente nos dois grupos, e que quando comparados estatisticamente não existe diferença significante entre os grupos, assim como na formação de manguitos perivasculares. Houve diminuição considerável do edema em todas as estruturas avaliadas, e a ribavirina® se mostrou eficaz na redução da inflamação encefálica, assim como a necrose neuronal foi menor em quase todas as estruturas avaliadas no grupo tratado, com exceção do tálamo. A análise estatística comparativa da marcação viral entre os grupos se mostrou significante somente para o cerebelo, tálamo e tronco encefálico / Abstract: Distemper is a viral infectious disease, caused by systemic and lethal morbillivirus that affects dogs of all ages and clinical signs respiratory, gastrointestinal, dermatological, ophthalmic and neurological disorders, which may occur sequentially or separately. This study aimed to compare brain lesions using histopathology and assess the amount of virus by immunohistochemistry in dogs treated with Ribavirin at a dose of 30mg/Kg orally once daily for 15 days and animals not treated with this drug, and that due to the unfavorable development of the disease were euthanized. The animals were treated and handled during routine Division of Infectious Diseases of Domestic Animals-FMVZ-UNESP / Botucatu, SP. Histopathologic analysis was to evaluate the cerebral cortex, cerebellum, thalamus, brainstem, hippocampus and viewing perivascular cuffs in the meninges. From these regions it was possible to identify and classify lesions such as demyelination, the presence of perivascular cuffing, edema, neuronal necrosis, vascular changes (congestion, hemorrhage and thrombosis) and inflammatory changes (meningitis, meningoencephalitis and meningo-encephalomyelitis). It was observed that the demyelination remained strongly present in both groups, and compared statistically no significant difference between groups, as well as the formation of perivascular cuffs. There was a considerable decrease of the edema measured in all structures, and ribavirin ® was effective in reducing brain inflammation, and neuronal necrosis was lower in almost all structures evaluated in the group treated with the exception of the thalamus. The comparative statistical analysis of the viral marking between groups was significant only for the cerebellum, thalamus and brainstem / Mestre

Orientador: Hélio José Montassier / Banca: Antônio Carlos Paulillo / Banca: Ricardo Luís Moro de Sousa / Resumo: Nesse estudo, um isolado de campo do vírus da bronquite infecciosa (VBI) no Brasil (IBVPR-12), previamente classificado como um genótipo variante, foi caracterizado de forma comparativa com a estirpe M41 do VBI, sendo levantadas as características de patogenicidade em diferentes órgãos como a traqueia, o pulmão, os rins, as gônadas e as tonsilas cecais (patotipo) e a imunidade cruzada com relação à estirpe vacinal H120 do VBI (protectotipo), incluindo as respostas imunes humorais sistêmicas e locais induzidas. Para tanto, foram utilizados grupos experimentais de galinhas "specific pathogen free" (SPF) previamente vacinadas ou não com a estirpe H120 do VBI e depois desafiados com essa variante viral, ou com a estirpe M41. Para essas duas estirpes virais foram avaliadas a capacidade de replicação e as lesões produzidas em diferentes órgãos, a atividade inibidora do movimento ciliar no epitélio traqueal e as respostas imunes humorais desenvolvidas no soro sanguíneo e na secreção lacrimal dessas aves. Foram observadas diferenças marcantes na patogenicidade e no tropismo tecidual desses vírus, sendo que a estirpe M41 apresentou replicação mais intensa e lesões mais pronunciadas no trato respiratório, especialmente na traqueia, enquanto que a estirpe variante foi encontrada de forma mais distribuída em vários dos órgãos analisados, tendo-se replicado e provocado menos lesões na traqueia, mas alcançando maior replicação e tendo causado lesões mais severas nos rins e nos testículos. Nas regiões teciduais mais afetadas por lesões, a presença do VBI foi detectada por marcação específica com anticorpos policlonais contra a nucleoproteína do VBI pela técnica de imuno-histoquímica. As aves vacinadas com a estirpe H120 do VBI, revelaram proteção parcial contra a estirpe variante em órgãos como traqueia e... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: In this study a Brazilian field isolate of infectious bronchitis virus (IBV), previously classified as variant genotype, was characterized comparatively with the M41 strain of IBV, by evaluating the pathogenicity in different organs (trachea, lung, kidney, gonads and caecal tonsil) and the cross-immunity with H120 vaccine strain, including the systemic and local humoral immune responses. Experimental groups of specific pathogen free (SPF) chickens were vaccinated or not with H120 strain of IBV and challenged with this variant isolate. The viral replication and histopathology in different tissues and organs, the ability to inhibit ciliar movement of tracheal epithelial cells, and local and systemic humoral immune responses were evaluated in these chickens. The pathogenicity and tissue tropism of these IBV strains showed marked differences, and while the M41 strain damaged more the respiratory tract, specially the trachea, the variant isolated has a wide tissue distribution, showing less replication and lesions in the trachea, but affecting more severely the kidney and the testicles. In the most affected tissue regions, the presence of IBV was detected by immunohistochemistry technique, using an anti-nucleoprotein polyclonal antibodies. The H120 vaccine induced against this variant isolate a partial protection with regard to the infection of trachea and kidney and no cross-protection to the infection of testicles. In conclusion, a new pathotype and a new protectotype of a variant genotype of a Brazilian IBV isolate were characterized in this study with regard to Massachusetts genotype and serotype strains of IBV, indicating the importance for... (Complete abstract click electronic access below) / Mestre

Orientador: Tereza Cristina Cardoso / Banca: Roberto Gameiro de Carvalho / Banca: Camila da Silva Frade / Resumo: Pertencente a família Herpesviridae, subfamília Alphaherpesvirinae, o Herpesvírus bovino tipo 5 (BoHV-5), é o agente causal da meningoencefalite bovina não supurativa, que atinge majoritariamente bovinos jovens. No Brasil, os casos de encefalite por BoHV-5 são a segunda maior causa de óbitos no rebanho. A infecção viral geralmente resulta em alteração no processo de morte celular programada, pela via mitocondrial, estimulando ou inibindo genes relacionados à apoptose, favorecendo a replicação e disseminação viral. O presente estudo teve como objetivo a análise da expressão de genes relacionados à apoptose (Apaf-1, α, FasL e citocromo c) em células MDBK infectadas ou não com o BoHV-5. Foi possível observar um aumento (p<0,05) na expressão dos genes estudados para o grupo infectado comparado ao controle, em diferentes momentos pós-infecção experimental. No caso da família Herpesviridae, a modulação da apoptose parecer ser uma etapa chave para sua patogênese, apesar da associação das atividades anti e pró-apoptóticas com o BoHV-5 ser completamente desconhecida. Este estudo demonstra a capacidade do BoHV-5 em modular, seja ativando ou inibindo, a via extrínseca da apoptose / Abstract: Belonging to the family Herpesviridae, subfamily Alphaherpesvirinae, bovine Herpesvirus type 5 ( BoHV-5) is the causative agent of bovine non-suppurative meningoencephalitis, which predominantly affects young cattle. In Brazil, cases of encephalitis caused by BoHV-5 are the second leading cause of deaths in the herd. Viral infection usually results in a change in the programmed cell death via mitochondrial, stimulating or inhibiting apoptosis related genes, promoting viral replication and dissemination process. The present study aimed to analyze the expression of apoptosis related genes (Apaf-1, TNFα-R1, FasL and cytochrome c) in infected or uninfected MDBK cells with BoHV-5. We observed an increase ( p < 0.05 ) in expression of the genes studied for the infected group compared with controls, in different times post-infection experimental. In the case of the Herpesviridae family, modulation of apoptosis appears to be a key step in its pathogenesis, despite the association of anti and pro-apoptotic activities with BoHV-5 is completely unknown. This study demonstrates the ability of BoHV-5 in modulating either activating or inhibiting the extrinsic pathway of apoptosis / Mestre

Orientador: Roberto Calderon Gonçalves / Coorientador: Ivan Roque de Barros Filho / Banca: Simone Biagio Chiacchio / Banca: José Paes Oliveira Filho / Banca: Sílvia Cristina Osaki / Banca: Andreza Amaral da Silva / Resumo: Realizou-se levantamento sorológico dos seguintes vírus respiratórios Herpesvírus Bovino 1 (BoHV-1), Vírus da Diarreia Viral Bovina (BVDV), Vírus da Parainfluenza Bovina Tipo 3 (bPIV-3) e Vírus Respiratório Sincicial Bovino (BRSV) em bovinos da raça Holandesa variedade Preta e Branca (HPB), criados no estado do Paraná. Colheram-se 714 amostras de sangue da veia jugular de bovinos, não vacinados para os agentes estudados, com mais de seis meses de idade, de 26 propriedades leiteiras, distribuídas em 17 municípios do estado do Paraná. Paralelamente à colheita, aplicou-se um questionário para cada propriedade estudada, com a finalidade de avaliar os fatores de risco associados aos vírus examinados. As amostras sorológicas foram submetidas ao teste de diagnóstico de virusneutralização no Laboratório de Viroses de Bovinos do Instituto Biológico de São Paulo. Foram observadas prevalências de 22,3%; 35,8%; 80,4% e 93,5% de bovinos sororreagentes e 65,3%, 88,5%, 96,1% e 100% das propriedades reagentes para BoHV-1, BVDV, bPIV-3 e BRSV, respectivamente. A coinfecção que ocorreu com maior prevalência foi a bPIV-3 e BRSV (37,1%). Após análise de regressão logística, os fatores de risco associados ao BoHV-1 foram: bovinos com idade maior que 48 meses (OR=15,012; IC95% 7,500 - 30,051), área da propriedade 26 a 50 hectares (OR=11,328; IC95% 2,482 - 51,697), lotação maior de três bovinos por hectare (OR=1,026; IC95% 0,370 - 2,847), percentual de entrada maior que 10% de bovinos (OR=52,520; IC95% 13,269 - 207,876); para o BVDV foram: bovinos com idade maior que 48 meses (OR=4,407; IC95% 2,456 - 7,906), região Sudoeste (OR=52,388; IC95% 4,629 - 5892,873), sistema de produção semi-intensivo (OR=1,333; IC95% 0,261 - 6,815), propriedade com número de animais menor ou igual a 50 bovinos (OR=16,682; IC95% 3,218 - 86,481), percentual de entrada maior que 10% de bovinos (OR=17,56; IC95% 7,613 - 40,506) e ... / Abstract: A serological survey of the following respiratory viruses: Bovine Herpesvirus 1 (BoHV-1), Bovine Viral Diarrhea Virus (BVDV), Bovine Parainfluenza Virus Type 3 (bPIV-3) and Bovine Respiratory Syncytial Virus (BRSV) in Holstein cattle black and White (HPB), raised in Paraná state was performed. 714 blood samples were collected from the jugular vein of cattle not vaccinated for the studied agents, over six months of age, from 26 dairy herds, distributed in 17 municipalities of Paraná state. Alongside the collection, a questionnaire was used for each property studied in order to assess the risk factors associated with the viruses examined. The serum samples were subjected to the diagnosis test of virus neutralization at the Laboratory of Viral Diseases of Cattle of the Biological Institute of São Paulo. Frequencies of 22.3%; 35.8%; 80.4% and 93.5% of seropositive cattle were observed and 65.3%, 88.5%, 96.1% and 100% of the seropositive properties BoHV-1, BVDV, bPIV-3 and BRSV, respectively. The co-infection that occurred most frequently was the bPIV-3 and BRSV (37.1%). After logistic regression analysis, risk factors associated with BoHV-1 were cattle with more than 48 months of age (OR = 15.012; 95% CI 7.500 to 30.051), property area 26-50 hectares (OR = 11.328; 95% CI 2.482 - 51.697), stocking higher than three animals per hectare (OR = 1.026; 95% CI 0.370 to 2.847), cattle entry percentage higher than 10% (OR = 52.520 entry; 95% CI 13.269 to 207.876); for BVDV were cattle older than 48 months (OR = 4.407; 95% CI 2.456 to 7.906), Southwest region (OR = 52.388; 95% CI 4.629 to 5892.873), semi-intensive production system (OR = 1.333; 95% CI 0.261 to 6.815),property with fewer animals than or equal to 50 (OR = 16.682; 95% CI 3.218 to 86.481), a percentage of cattle entry higher than 10% (OR = 17.56; 95% CI 7.613 - 40.506) and neighboring pasture sharing (OR = 9.148; 95% CI 3.751 to 22.310); for bPIV-3 cattle older than 48 months ... / Doutor

INTRODUÇÃO: Indivíduos infectados pelo HIV apresentam maior risco de quadros graves de infecção por influenza sazonal e, portanto, devem receber doses anuais da vacina contra gripe. No entanto, a capacidade dos indivíduos responderem às vacinas com títulos apropriados de anticorpos depende de variáveis como tipo de antígeno vacinal, idade e grau de comprometimento imunológico no momento da imunização. OBJETIVOS: 1) Avaliar a imunogenicidade da vacina contra influenza sazonal em 37 pacientes infectados pelo HIV, em comparação com 29 indivíduos não infectados pelo HIV 2) Realizar a vigilância dos episódios de infecções respiratórias durante o período de acompanhamento após a vacinação. MÉTODOS: Ambos os grupos receberam a vacina contra o vírus da influenza sazonal recomendada para o hemisfério sul em 2008. A resposta de anticorpos contra os antígenos H1N1, H3N2 e B foi medida em amostras de sangue extraídas 1-2h antes da vacinação (T0), após 1 mês (T1) e após 6 meses (T6; apenas no Grupo HIV). A vigilância dos sintomas respiratórios foi realizada através de telefonemas semanais, durante 6 meses após a vacinação. Em indivíduos sintomáticos para infecções respiratórios foram coletadas amostras de lavado nasofaríngeo para pesquisa de vírus respiratórios por Imunofluorescência e PCR: influenza A e B, parainfluenza 1, 2 e 3, adenovírus, metapneumovírus, vírus sincicial respiratório, rinovírus e coronavírus. RESULTADOS: A idade mediana da população de estudo foi de 12 (10-18) anos. No momento T1, ambos os grupos mostraram aumento significativo nos TMGs para todos os antígenos. Contudo, o grupo controle apresentou valores mais elevados para os antígenos A/H1N1 e A/H3N2 (p = 0,002 e 0,001, respectivamente). Houve maior aumento na porcentagem de indivíduos não infectados pelo HIV com títulos protetores A/H1N1 (96,6%) em comparação aos infectados pelo HIV (67,6%). No T1 (p=0,004). A porcentagem de indivíduos do grupo controle com aumento de quatro vezes ou mais nos títulos de anticorpos para A/H1N1 e A/H3N2 foram mais elevadas que no grupo HIV (p = 0,03 e 0,01, respectivamente). Agentes virais foram detectados em 39/60 (65%) dos episódios de infecção respiratória no grupo HIV e em 17/32 (53,1%) no grupo controle. Os vírus diagnosticados no grupo HIV e grupo controle foram respectivamente: adenovirus (8,6%), metapneumovirus (1,2%), rinovirus (16,8%), coronavirus (14,0 %) e influenza B (0,1%).CONCLUSÕES: A vacina sazonal contra os vírus da influenza foram imunogenicas em ambos os grupos. Ocorreram diferença nas taxas de soroproteção entre os grupos somente para o antígeno H1, que foi mais elevadas no grupo controle. O grupo controle também mostrou valores mais altos nos TMGs para os antígenos H1 e H3 depois da imunização. Os rinovirus (27,7%) e coronavirus (22,5%) foram os agentes mais prevalentes identificados no grupo infectado pelo HIV. No grupo controle, os vírus mais freqüentes foram os rinovirus (24,2%) e adenovirus (21,2%) / INTRODUCTION: Individuals infected with HIV are at higher risk for severe cases of seasonal influenza infection and therefore should receive annual doses of influenza vaccine. However, the ability to respond to vaccines respond appropriate antibodies titres depends on variables such as vaccine antigen, age and degree of immune impairment at immunization. OBJECTIVES: 1)To evaluate the immunogenicity of a seasonal influenza vaccine in 37 HIV-infected patients (HIV Group), compared to 29 uninfected individuals (Control Group) 2) To carry out a clinical and virological surveillance of influenza in this population during a follow-up period of six months. METHODS: Both groups received the vaccine against seasonal influenza virus recommended for the southern hemisphere in 2008. The antibody response against the antigens H1N1, H3N2 and B were measured in blood samples drawn at vaccination (T0), after 30 days (T1) and after 6 months (T6; only for HIV Group). Antibody titres >1:40 were considered protective against influenza infection A surveillance of respiratory symptoms was performed weekly by telephone calls for a post-vaccination follow-up period of 6 months. Samples were collected (nasal wash) if respiratory symptoms. DFA and real time PCR was used to diagnose influenza A virus (FLU A) and B (FLU B), respiratory syncytial virus (RSV), parainfluenza virus types 1, 2 and 3 ( Paraflu 1, 2 or 3), adenovirus, coronavirus, rhinovirus, metapneumovirus and bocavirus. RESULTS: The median age of the study population was 12 (10-18) years. At T0, there were no significant differences in the antibody geometric mean titres (GMTs) against all vaccine antigens between groups. One month after vaccination (T1), both groups showed significant increases in the antibody GMTs for all antigens. However, healthy controls showed higher values for antigens A/H1N1 and A/H3N2 (p = 0.002 and 0.001, respectively). There was a higher increase in the percentage of HIVuninfected subjects with protective A/H1N1 antibodies (96.6%) comparing to HIVinfected vaccinees (67.6%) at T1 (p = 0.004). The percentage in subjects control group with a fourfold or greater increase of A/H1N1 and A/H3N2 antibody titres was higher than that found in HIV group (p = 0.03 and p = 0.01, respectively. Viral agents were identified in 39/60 (65%) episodes of respiratory infections in HIV-infected group and in 17/32 episodes (53.1%) from the control group (P=0.273). The virus diagnosed in HIV group and control group were, respectively: Adenovirus (8;6), Metapneumovirus(1;2) Rinovirus(16;8), Coronavirus(14 ;0); Influenza B(0;1). CONCLUSIONS: The seasonal influenza vaccine was immunogenic in both groups. There were differences in seroprotection rates between groups only for AgH1, which was higher in the control group. The control group also showed a greater increase in GMTs for H1 and H3 antigens after immunization. Viral agents were identified in respiratory symptoms during the follow-up: Rhinoviruses (27.7%) and coronavirus (22.5%) were the most prevalent agents identified in HIV-infected individuals. In the control group, the viruses most frequently found were rhinoviruses (24.2%) and adenovirus (21.2%)

There are several reasons to celebrate the latest advances in the treatment of the infection with HIV. According to the Joint United Nations Programme on HIV/AIDS, the number of new infections dropped by 15%; there is also a decrease by 22% in the number of deaths related to HIV/AIDS. Nevertheless, there are new emerging challenges, i.e. the transmission of drug-resistant HIV-1 strains. Therefore, there is a demand for the continued research for new and more potent antiretroviral agents. The entry of HIV into the cell implies a complex and well-orchestrated series of steps in which both viral and cellular molecules are implied, ending with the production of new viral particles. The HIV gp120 glycoprotein binds to the cellular CD4 receptor and to a chemokine receptor, inducing structural rearrangements that continue with the cellular and viral membrane fusion mediated by the HIV glycoprotein gp41. Hence, the entry of HIV is an essential step of the viral replication that offers an open path for the design of new antiviral compounds that could be added to the repertory of drugs used in the treatment of HIV infection. In coincidence with the recent and highly relevant information of the fusion mechanism occurring during the viral entry, the design of new fusion inhibitors has become one of the most promising and debated areas in the study of entry inhibitors. ADS-J1 was originally selected to bind to gp41 and to inhibit the fusion of membranes. In several assays, including the generation of HIV strains resistant to ADS-J1, our laboratory has proved that ADS-J1 interact with gp120 instead of gp41. A more recent publication suggested that ADS-J1 binds to the pocket region of gp41 preventing the infection by the virus. Here, we confirmed that ADS-J1 interacts with gp120 instead of gp41. Recombination of gp120 into a wild type HIV-1 backbone restored the resistant phenotype. Moreover, time of addition assays clearly demonstrated that ADS-J1 does not interact with gp41. VIRIP was identified as a natural peptide present in human hemofiltrate that inhibits the HIV gp41-mediated membrane fusion. It was suggested that VIRIP interact with the fusion peptide in gp41, therefore blocking the fusion of membranes. With the objective to determine the precise mode of action of VIRIP, we generated a HIV-1 virus resistant to VIR-353, an analogue of VIRIP. Additionally, we determined the most relevant combination of mutations for the resistant phenotype. Recent studies have shown the effectivity of VIR-576, a peptide closely related to VIRIP and VIR-353 in a clinical trial phase I/II. The resistance to VIRIP/VIR-353 took a long time to emerge, suggesting a high genetic barrier to resistance. The mutations responsible for the resistant phenotype affected in large scale the replicative capacity of the virus, nevertheless, several compensatory mutations restored the viral fitness, while the resistance to VIR-353 was unaltered. The antiviral combination of VIR-353 and T20 showed an additive effect in inhibiting viral replication, indicating that VIR-353 appeared no to affect the binding of T20 to gp41 in its antiviral activity, the combination of the two fusion inhibitors showed an additive effect in inhibiting viral replication. In general, our results evidence the plasticity of the HIV envelope glycoproteins. This plasticity is highly remarked when the virus replicates under drug selective pressure, which imposes an additional genetic barrier for the virus to overcome. / ADS‐J1 ha estat seleccionat per unir‐se a gp41 i inhibir la fusió de les membranes. A través de diversos assajos, incloent la generació de soques resistents a ADS‐J1, el nostre laboratori va demostrar que ADS‐J1 interactua amb gp120 i no amb gp41. Una publicació posterior va suggerir que ADS‐J1 s’uneix a la ‘pocket‐region’ de gp41, prevenint l’infecció pel virus. En el present treball, nosaltres confirmem que ADSJ1 interactua amb gp120 i no amb gp41 i que la recombinació de gp120 en un VIH silvestre restitueix el fenotip resistent. Assajos de temps de addició van demostrar clarament que ADS‐J1 no interactua amb gp41. VIRIP va ser identificat com un pèptid natural present en el hemofiltrat humà capaç d’inhibir la fusió de membranes operada per gp41 del VIH. Es va suggerir que VIRIP interactua amb el pèptid de fusió de gp41, bloquejant la fusió de les membranes. Nosaltres hem generat un virus resistent a VIR‐353, un anàleg de VIRIP. Addicionalment, hem determinat la combinació de mutacions que generen el fenotip resistent. Estudis recents van mostrar l'efectivitat de VIR‐576, un pèptid amb alta similitud a VIRIP i VIR‐353 en un assaig clínic fase I/II. La resistència a VIRIP/VIR‐353 va requerir un període de temps llarg per emergir, la qual cosa suggereix una elevada barrera genètica a la resistència. Les mutacions responsables del fenotip resistent van afectar en greument la capacitat replicativa del virus, no obstant això, diverses mutacions compensatòries van restaurar‐ne la capacitat replicativa, mantenint intacta la resistència a VIR‐353. L’activitat antiviral de T20 no sembla afectada per VIR‐353, la combinació dels dos inhibidors de fusió van mostrar un efecte additiu en la inhibició de la replicació. En general, els nostres resultats evidencien la plasticitat de les glicoproteïnes de l'embolcall del VIH. Aquesta plasticitat es realça quan el virus replica sota la pressió selectiva imposada per fàrmacs que inhibeixen la replicació viral, la qual cosa afegeix una barrera genètica addicional a ser superada pel virus.

La infecció pel VHC és la principal causa d'hepatopatia crònica arreu del món. Els nens constitueixen una petita proporció de la població infectada i en l'actualitat la principal via d'adquisició de la infecció pel VHC en la infància és la transmissió vertical. És probable que els pacients infectats durant la infancia, tal com passa en els adults, es mantinguin asimptomàtics durant un llarg període de temps, tot i que s'han descrit casos de cirrosi en un temps inferior als 10 anys. Donada l'absència de manifestacions clíniques en les primeres fases de la infecció, s'han estudiat diversos marcadors analítics per a definir la progressió de la malaltia. En l'actualitat, es disposa d'un tractament antiviral eficaç (interferó pegil·lat més ribavirina) per a la infecció crònica pel VHC. Aquest tractament ha estat autoritzat recentment per a la població pediàtrica. L'absència de comorbilitats i el curt temps d'evolució de la infecció converteixen al nen infectat pel VHC en el candidat ideal per a obtenir altes taxes de curació de la malaltia. En els darrers anys, la infecció pel VIH ha esdevingut una patologia de curs crònic mentre que l'hepatopatia és actualment una de les principals causes de morbilitat i mortalitat en els pacients co-infectats. En el pacient pediàtric co-infectat les consideracions prèvies sobre l'inici de tractament específic esdevenen especialment rellevants, ja que les probabilitats d'èxit del mateix probablement es relacionin amb la seva precocitat. Els objectius principals del nostre estudi són definir les característiques de la transmissió vertical del VHC en el nostre medi, descriure la història natural de la infecció crònica pel VHC en l'edat pediàtrica i establir el paper de la co-infecció pel VIH en l'evolució natural de la infecció crònica pel VHC en el nen.Per això hem dissenyat dos estudis prospectius observacionals que inclouen els fills de mares infectades pel VHC nascuts al nostre centre entre gener del 1999 i desembre del 2006 (estudi 1) i els nens infectats pel VHC controlats en el nostre centre (estudi 2). Tots elspacients han estat seguit de forma prospectiva. Les principals conclusions dels estudis són les següents: Estudi 1: La taxa prevalença de la infecció pel VHC en les gestants és del 0.49%. La taxa de transmissió vertical del VHC és del 2.8%. Existeix una relació entre la co-infecció materna pel VIH i la transmissió vertical del VHC en el grup de pacients estudiats. Estudi 2: La majoria dels pacients romanen asimptomàtics i sense troballes clíniques al llarg del seguiment. La meitat dels pacients estan infectats pels genotips 1a o 1b; en els fills de mare UDVP, hi predominen les infeccions pels genotips 3 i 4. La biòpsia hepàtica mostra signes d'hepatitis crònica en la majoria dels casos. D'entre els pacients no tractats, un 12% evolucionen al clearence espontani de la infecció (tots ells són pacients infectats per transmissió vertical) mentre que el 82% dels pacients evolucionen a un patró d'hepatitis crònica. La taxa de prevalença de cirrosi és de l'1.8%. Només 4 de 27 pacients tractats amb interferó en monoteràpia durant 12 mesos assoleixen el clearence de la virèmia. El 86% dels pacients pateixen efectes secundaris del tractament, tot i que solen ser lleus. Els pacients co-infectats mostren unes xifres màximes d'ALT més altes que la resta i evolucionen en la majoria de casos seguint un patró d'hepatitis crònica. / HCV infection is the main cause of hepatopathy worldwide. Children represent a small proportion of the infected people and vertical transmision is the main way of acquisition in childhood. Patients infected during childhood will probably remain asymptomatic for a long time, as happens in adults, but some cases of cirrhosis have been described. Regarding the lack of clinical manifestations during the first years of the infection, several serum markers have been studied to define the progression of the illness. Nowadays an efective treatment is available (pegilated interferon plus ribavirin) for the chronic HCV infection and it has been recently authorised for childhood. The absence of comorbilities and the short course of the infection will probably lead to a higher rate of response in this group of patients. Early treatment will probably be effective also in HIV co-infected patients. Main objectives of our study are to define HCV vertical transmission in our media, to describe natural history of the chronic HCV infection in childhood and to stablish the role of HIV co-infection in the evolution of HCV infection. Two prospective studies have been conducted. They include children from HCV infected mothers born in our center between january 1999 and december 2006 (Study 1) and HCV infected children visited in our center (Study 2). Main conclusions of our study are: Study 1: Prevalence rate of HCV infection among pregnant women is 0.49%. HCV vertical transmision is 2.8%. There is a relation between vertical transmision and maternal HIV co-infection. Study 2: Most of the HCV infected patients remain asymtomatic during the follow up. Half of the patients belong to 1a or 1b HCV genotipes; in children born from drug users genotipes 3 and 4 are frequent. 12% of the patients that have'nt received treatment experiment spontaneous clearence of the infection (all are vertically infected) while 82% end in a chronic hepatitis. Prevalence rate of cirrhosis is 1.8%. Only 4 of 27 patients treated with interferon reach the clearence of the viremia and 86% suffer secundary effects, usually mild. Co-infected patients have higher ALT determinations and usually end in a chronic hepatitis.

Viruses are submicroscopic biological entities that need to infect a host cell in order to replicate. In their simplest form viruses are constituted by an infective genetic material and a protein shell (the capsid) that protects the viral genome. In this thesis we try to elucidate the general physical principles playing a major role in the morphology, stability, and assembly of viral capsids. Therefore, in the first part of the thesis, we develop a general theory that characterizes spherical and bacilliform (or prolate) capsids based on icosahedral symmetry under the same geometrical framework. In addition, we demonstrate that the structures derived in this geometrical study are obtained spontaneously from the free energy minimization of a very generic interaction among the viral capsomers of the capsid. In the second part of the thesis, we analyze the role of the discrete nature of capsids and the organization of capsomers in the actual mechanical properties of shells. We show that the icosahedral class P influences the stability and mechanical response of the quasi-spherical capsids. We also determine that under expansion, spherical shells tend to produce polyhedral structures (buckling), which are more resistant, and it is in consonance with the maturation process observed in some viruses. We also unveil the existence of built-in stress in the empty procapsids of the elongated bacteriophage φ29. This phenomenon is intimately related to the discrete nature of the structure, and reinforces the mechanical properties of the shell inverting the classical anisotropic response expected from continuum elasticity theory. In the last part of the thesis, we show that viral assembly and disassembly are activated processes controlled by nucleation barrier, which can be explained adapting classical nucleation theory (CNT). We focus on the case of spherical shells and confirm that the underlying assumptions of CNT are surprisingly good in characterizing the assembly of discrete shells, using the physical model introduced in the previous parts. Finally, we also unveil an interesting closure mechanism during the assembly of capsids.

Els tres primers capítols es dediquen al desenvolupament i aplicació del mètode Viraden. En el primer es prova d'optimitzar el mètode basant-nos en la modificació del medi overlay, per addició d'ions multivalents i/o de tripsina-EDTA o per addició del compost iode-deoxiuridina (IDU) al cultiu cel·lular en creixement. L'addició d'IDU millora la recuperació d'enterovirus.El mètode Viraden pot aplicar-se per a la detecció de clapes víriques damunt els filtres d'èsters de cel·lulosa emprant diferents tècniques moleculars, la hibridació amb sondes d'àcid nucleic i la immunodetecció. Ambdues tècniques permeten la detecció de les zones de lisi sobre el suport però hi ha una manca en la capacitat d'identificació que permeti una classificació ràpida dels diferents enterovirus.L'adaptació del mètode Viraden a filtres de 90 mm de diàmetre permet una major aplicació del mètode per a diferents tipus de mostres ambientals.Es desenvolupen diferents sistemes de clarificació i descontaminació basats en agents químics i en membranes filtrants. La descontaminació amb fenol combinada amb clarificació per filtració a través de filtres de baixa adsorció proteica (PVDF) de 0,65 µm de diàmetre de porus és un bon sistema per a la descontaminació de mostres d'efluent secundari quan s'aplica posteriorment l'enumeració de virus pel mètode Viraden. La filtració a través de filtres esterilitzants (0,22 µm) de baixa adsorció proteica (PVDF o PES) és un mètode vàlid per a l'obtenció de mostres descontaminades. Per a volums grans de mostres d'efluent secundari, riu i mar es recomana la clarificació per filtració a través de filtres de 0,65 µm PVDF. Es valora la possibilitat d'aplicar el mètode Viraden per a la realització de test de desinfectants i antisèptics amb els virus adsorbits sobre un suport. Es compara l'acció de diferents desinfectants i antisèptics (clor, iode i glutaraldehid) sobre els virus adsorbits i en suspensió. Es detecta una diferència de 2 logaritmes entre els virus adsorbits i els virus en suspensió. Els virus adsorbits amb HR alta (>80%) persisteixen més que els virus en suspensió i aquests més que els virus adsorbits i mantinguts amb HR baixa (<50%). Es descriu un nou mètode d'enumeració de virus cultivables consistent en la realització d'una doble capa. Sobre un cultiu confluent de cèl·lules es disposa una barreja composta per la suspensió viral a analitzar, una suspensió cel·lular (igual o diferent a la de la monocapa) i el medi overlay que conté agar. El mètode es compara amb altres mètodes d'enumeració de virus i en tots els casos apareix com el mètode que permet una major recuperació de virus, tant en suspensions de cultius purs de diferents enterovirus com en mostres ambientals. Quan s'utilitza la línia cel·lular CaCo-2 per a la preparació de la suspensió cel·lular s'obtenen majors recomptes de virus a partir de mostres d'aigua residual bruta i el tipus de virus diferents que s'identifiquen a partir d'aquest tipus de mostra és major. Els virus cultivables sobre BGM en aigua residual bruta presenten increments en les densitats de virus durant els mesos de primavera. En aigua de mar els enterovirus correlacionen amb colifags somàtics (0,98). En el 76% de les mostres d'aigua de platja que compleixen amb els paràmetres microbiològics de qualitat bacteriològics inclosos a la Directiva 76/160/CEE es detecten enterovirus infecciosos en 10 litres. A partir dels aïllaments d'enterovirus de mostres ambientals es procedeix a la seva identificació mitjançant tècniques de RT-PCR i RFLP i els resultats mostren que es detecten serotips d'enterovirus dels grups poliovirus, echovirus i coxsackievirus B.

CATALÀ:El medi aquàtic presenta concentracions molt variables de virus que constitueixen part de la comunitat microbiana. En zones properes a nuclis urbans el medi aquàtic rep aportacions amb elevades càrregues víriques excretades per l'home i els animals. Aquesta aigua a la vegada pot entrar en contacte altra vegada amb la població humana ja sigui per consum, per aigües de bany, etc. La qualitat de l'aigua i per tant la salut de la població es veu afectada per la presència de microorganismes patògens presents en l'aigua residual que entra en contacte amb l'aigua superficial.En aquesta tesi s'ha estudiat el possible rol dels poliomavirus i els adenovirus humans com a indicadors de la contaminació vírica d'origen humà. La tesi ha estat dividida en tres blocs.En el primer bloc s'ha estudiat el possible rol de SV40 com a virus humà i com a contaminant ambiental. SV40 es un virus de simi que va ser descobert com a contaminant de la vacuna de la poliomielitis distribuïda entre 1953 i 1963. Poc després es va veure que el virus era capaç de generar tumors en alguns animals, per això existeix la controvèrsia de si aquest virus pot generar aquest tipus de patologia en humans. En primer lloc es va estudiar l'excreció d'aquest virus per una colònia de micos naturalment infectats pel virus. Es va veure que els individus es seroconverteixen durant la joventut quan es mantenen en grups grans. L'anàlisi de les mostres ambientals de les gàbies va demostrar que SV40 es excretat per aquests micos en concentracions variables. En segon lloc es van analitzar mostres d'aigua residual del nord de la India, on la població humana coexisteix amb l'hoste natural del virus, per comprovar si realment SV40 es pot transmetre entre la població humana. Una de 30 mostres va ser positiva per SV40 però aquesta mostra contenia també contaminació fecal originada pels micos per la qual cosa no es pot concloure que la població humana excreti aquest virus.El segon bloc de la tesi esta enfocat al desenvolupament i aplicació de mètodes de concentració de poliomavirus i adenovirus humans a partir de mostres d'aigua superficial i de beguda. Es van comparar diversos mètodes per la concentració de virus a partir de diferents volums de usant mostres dopades amb quantitats conegudes de virus. El mètode que va presentar major eficiència es va aplicar en mostres de diferents punts de tres plantes de tractament d'aigües de beguda. La detecció i quantificació dels virus es va realitzar per PCR quantitativa. Les concentracions de virus es van usar per calcular l'eficiència d'eliminació de virus de les plantes. La quantificació d'adenovirus i poliomavirus humans usant PCR quantitativa va demostrar ser una eina útil per avaluar l'eficiència d'eliminació de virus en plantes potabilitzadores i com a índex de la qualitat virològica de l'aigua.El tercer bloc està centrat en l'anàlisi de la cinètica d'infecció del poliomavirus humà JC en diversos tipus de cultiu cel·lular. La transmissió d'aquest virus encara no ha estat definitivament establerta i també falta informació sobre sistemes eficients per la seva multiplicació in vitro. També es va explorar la possible interacció entre aquest virus i altres virus (adenovirus humans, poliomavirus BK i SV40). Es va veure que JC no interacciona amb adenovirus 2 en cèl·lules de càncer de colon, en canvi es va veure un efecte facilitador per part del poliomavirus BK en la infecció de JC en cèl·lules SVG (línia fetal glial humana), donant una possible explicació del perquè JC encara que és més freqüentment excretat que BK, infecta la població humana després que BK. / Water quality and public health is affected by the presence of pathogens present in wastewater which can be in contact with ground water used for recreational purposes or for consumption. The possible role of human polyomaviruses and adenoviruses as indicators of human viral faecal contamination has been the main objective of this thesis. The work has been divided in three thematic sections.The first section has been centred on the study of the possible role of SV40 as a human virus and as an environmental contaminant. SV40 is a simian virus discovered as a contaminant of the polio vaccine administered to millions of people between 1953 and 1963 and some studies have associated this virus with human cancer. The analysis of environmental samples in cages of a naturally infected macaque colony determined that it is excreted in variable concentrations. Waste water from northern India (where human population coexists with the natural host of the virus) was analyzed. SV40 is not excreted by the human population with the same pattern as human polyomaviruses do and it is not detected in the urban sewage analyzed.The second section is focused on developing and applying virus concentration methods from surface and drinking water. Several concentration methods were compared. The method that presented better performances was used to sample several steps of three drinking-water treatment plants for the presence of JC polyomavirus and human adenoviruses and the virus removal efficiencies of the plants. The quantification of human adenoviruses and JC Polyomavirus using quantitative PCR has showed to be a useful tool to evaluate the removal efficiency of viruses in drinking-water treatment plants.The third section is focused on the analysis of the multiplication kinetics of JC polyomavirus in different cell cultures. There is a lack of efficient in vitro multiplication systems of the virus. The possible interaction of JC and other viruses was studied. A significant BK helper function was observed when co-infecting SVG (human foetal glial cells) cells with JC that could be related to the fact that JC, being more frequently excreted, does not infect the human population until later that BK.