<div>Viruses are a group of contagious microbes that have compact structures, containing a nucleic acid core and a protein shell. The replication of viruses requires assistance from hosts which can be almost any cellular organism. Viral infections are often associated with diseases and have been a major threat to the human race. To cope with viral diseases, we need to understand viruses, including their structures, life cycle, pathogenesis and interactions with their hosts. The first structure of a human virus was determined by the Rossmann lab in 1985 using X-ray crystallography.</div><div>Thanks to the recent advances in both hardware and software, cryo-electron microscopy (cryo-EM) has emerged as a powerful tool to study virus structures. Cryo-EM allows structural determination for a wide range of specimens to high resolution comparable to what can be achieved by X-ray crystallography. Currently two techniques of cryo-EM are commonly used in structural virology: single particles analysis (SPA) and electron tomography (ET). </div><div>Single particle analysis has been used to determine the structures of viruses complexed with host factors in three studies that are to be discussed with more details in chapters 2-4. </div><div>The structure of B19 parvovirus complexed with Fabs of a neutralizing human antibody was determined to 3.2 Å resolution. This structure showed that amino acids from three neighboring VP2 proteins form a quaternary structure epitope. In addition, the structure of human rhinovirus-C (RV-C) complexed with its cellular receptor, CDHR3, was determined to 3.9 Å resolution. Despite the low occupancy of the receptors, a “powerful” localized 3D classification procedure helped to select viral particles that had more bound receptors. Furthermore, structures were determined to 10 Å resolution of bacteriophage ΦX174 bound to lipopolysaccharide (LPS) bilayers, before and after genome ejection. These structures showed a series of conformational changes that occurred when a phage penetrated the bacterial membranes. These studies are good examples of applying cryo-EM to investigate virus-host interactions.</div><div>However, single particle analysis requires samples to be isolated, homogenous and monodispersed. On the contrary, tomography allows in situ studies and is applicable to samples with more flexibility and more heterogeneity. In the case of ΦX174, the structural changes that are involved in the assembly of the H-tube during infection remains a huge mystery. To provide an environment that is more similar to the surface of a bacterial cell, LPS-containing liposomes were mixed with ΦX174 viruses. It was then observed that the ΦX174 particles bound to these liposomes in a very compact manner which was impossible interpret with single particle analysis. Using cryo-ET, 3D volumes of liposome-ΦX174 complexes were reconstructed and structural details were visualized by sub-tomogram classification and averaging.</div><div>The emergence of cryo-EM has not only made high-resolution structural studies possible but also broadened the scope of samples with which virologists could work. Moreover, studies on flexible and heterogeneous complexes between viruses and host factors are now possible using either single particle analysis or electron tomography. These techniques will help us to understand virus-host relationships and finally, to develop effective anti-viral therapies.</div>

O eritrovírus humano (parvovírus), gênero Erytrovírus, é o único representante da família Parvoviridae responsável por um amplo espectro de doenças. Estudos recentes têm demonstrado variações entre o eritrovírus e orientam a reclassificação destas variantes em três genótipos distintos: genótipos 1, 2 e 3. O papel do eritrovírus na etiopatogenia de doenças hematológicas em humanos permanece incerto. Este estudo teve como objetivo principal avaliar a relação etiopatogênica dos genótipos do eritrovírus e as citopenias de origem desconhecida. Materiais e Métodos: Participaram do estudo 285 indivíduos procedentes do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo. Destes, 120 apresentavam citopenias de origem desconhecida (grupo 1 Casos), 45 eram doadores de medula óssea (grupo 2 Controles Saudáveis) e 120 eram pacientes com doenças oncohematológicas crônicas (grupo 3 Controles com Neoplasias Hematológicas). A pesquisa do vírus foi realizada pelo método de semi-nested PCR (Reação em Cadeia da Polimerase) em amostras de medula óssea e de sangue periférico. As fitas complementares foram seqüenciadas diretamente do produto da PCR. Amostras de plasma de todos os indivíduos incluídos no estudo foram testadas para presença de anticorpos IgG e IgM específicos contra o eritrovírus por ensaio imunoenzimático. Resultados: Dos 40 indivíduos com resultado positivo na PCR em amostra da medula óssea, o genótipo 1 foi encontrado em 22 (55%), o genótipo 2 em 5 (12,5%), o genótipo 3 em 13 (32,5%). Quando comparadas as freqüências de positividade entre os casos e controles (Grupo 1 VS Grupos 2 e 3), não encontramos diferença significativa com relação ao genótipo 1 (p=0, 192) nem com relação aos genótipos 2 e 3 (p= 0.143). A soroprevalência encontrada na amostra foi de 71%. Conclusão: Concluímos que a infecção isolada pelo eritrovírus, independente do genótipo encontrado, não tem relação etiopatogênica com as citopenias de origem desconhecida, uma vez que o vírus foi encontrado com a mesma freqüência nos casos e nos controles estudados / The human erythrovirus (parvovirus), genus Erytrovirus, is the only representative of the family Parvoviridae responsible for a broad spectrum of diseases. Recent studies have shown variations within the erythrovirus and guide the classification of these variants in three distinct genotypes: genotypes 1, 2 and 3. The role of the erythrovirus in the etiopathogenesis of hematological diseases in humans remains uncertain. This studys main objective was to evaluate the etiopathogenic relationship between the genotypes of the erythrovirus and the cyptopenias of unknown origins. Methods and Materials: 285 individuals coming from the Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo participated in the study. Of these, 120 represented cytopenias of unknown origins (group one Cases), 45 were bone marrow donors (group two - Healthy Controls), and 120 were patients with chronic oncohematological diseases (group three Controls with Hematological Disorder). The research of the virus was done through the semi-nested PCR method (polymerase chain reaction) in bone marrow and peripheral blood samples. The complementary strands were sequenced directly from the product of the PCR. Plasma samples from all of the individuals included in the study were tested through immunosorbent assay for the presence of lgG and IgM antibodies specific to the eritrovírus. Results: Of the 40 individuals that had positive PCR bone marrow results, the genotype 1 was found in 22 (55%), the genotype 2 in 5 (12.5%), and genotype 3 in 13 (32.5%). When the frequency of positivity was compared between the cases and the controls (Group 1 vs. Groups 2 and 3), we did not find a significant difference in relation to genotype 1 (p=0.192), nor did we find a significant difference in relation to genotypes 2 and 3 (p=0.143). The overall seroprevalence found in the samples was 71%. Conclusion: We conclude that the infection isolated by the erytrovirus, independent of the genotype found, does not have a etiopathogenic relationship with the cytopenias of unknown origins, hence the virus was found with the same frequency in the cases and the controls studied

Diss. (sammanfattning) Stockholm : Karol. inst., 2001. / Härtill 6 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 5 uppsatser.

Parvovirus B19 is a common human pathogen that typically infects erythroid progenitors and causes hematological problems such as anemia and aplastic crises. The clinical presentation depends mainly on the immunological status of the patient. PVB19 can cause serious clinical disorders in immunocompromised patients after transplantation. More than 1500 samples from 90 patients who passed the HSCT in 2015 were tested for the presence of PVB19 in this work. This work describes the incidence of the virus and two typical periods of onset of infection in patients after the transplantation. Although several sources report the negative effect of PVB19 infection on the survival of allogeneic graft patients, this work did not confirm this assertion. Also, the results of this work suggest that allogenic grafts are not the main source for transmission, but that it is likely to be reactivated after long-term persistent or latent PVB19 infections. PVB19 is divided into 3 genotypes. Genotype 1 is the most widespread, genotype 2 is very rare in Europe for the last 10 years, and genotype 3 occurs mainly in tropical localities. This work as the first describes the distribution of genotypes in the Czech Republic. More than 130 samples from 125 PVB19 positive patients, stored in the Motol University Hospital from 2004...

A fragmentação e a invasão de florestas tropicais e a crescente concentração de assentamentos humanos aumentaram exponencialmente as chances de exposição a vírus emergentes e emergentes. Dado o grande potencial de espalhamento de patógenos em população humanas, a identificação e caracterização de agentes patogênicos circulantes podem melhorar a atenção primária e as capacidades de diagnóstico para um agente emergente futuro. As abordagens moleculares e metagenômicas que utilizam as tecnologias de sequenciação da próxima geração levaram a descoberta e caracterização de muitos vírus emergentes na última década. Além disso, as abordagens in silico também podem ajudar a identificar vírus emergentes usando apenas dados de sequenciamento publicamente disponíveis. Além disso, estimar a ascendência filogenética e até mesmo analisar as mudanças no uso de codons são ferramentas adicionais que podem melhorar a nossa compreensão de vírus emergentes ou reemergentes. Este projeto visou aplicar essas ferramentas em ambos os vírus que poderiam estar circulando no Brasil: Parvovírus B19 e vírus da Febre Amarela. Também exploramos as aplicações de modelos ocultos de Markov e índice de adaptação de codons usando dados publicamente disponíveis. Esperamos que este trabalho forneça uma prova de conceito para futuros projetos metagenômicos e demonstre a utilidade das várias técnicas moleculares e bioinformáticas no estudo de vírus emergentes. / Fragmentation and encroachment of tropical rainforests and the growing concentration of human settlements have exponentially increased chances of exposure to re-emerging and emerging viruses. Given the large potential for pathogens to spillover and spread in a population, identifying and characterizing circulating human pathogens could improve the readiness and diagnostic capabilities for a future emergence. Molecular and metagenomic approaches using next-generation sequencing technologies have led to the discovery and characterization of many emerging viruses over the last decade. In complement, in silico approaches can also help identify emerging viruses using only publicly available sequencing data. Moreover, estimating the phylogenetic ancestry and even analyzing changes in codon usage are additional tools that can improve our understanding of an emerging or re-emerging virus. This project aimed to apply these tools to two viruses that could be circulating in Brazil: Parvovirus B19 and Yellow Fever virus. We also explored the applications of Hidden Markov models and codon adaptation index using publicly available data. We expect this work to provide a proof-of-concept for future metagenomic projects, and demonstrate the utility for several molecular and bioinformatics techniques in the study of emerging viruses.