O Vírus Respiratório Sincicial humano (hRSV human Respiratory Syncytial Virus) está entre os principais causadores de doenças do trato respiratório. O hRSV pertence à família Paramyxoviridae. Os sintomas da infecção podem variar de simples estado gripal a doença respiratória grave, e eventualmente levar a óbito. Atualmente não há vacina licenciada ou droga eficaz contra esse vírus. Anteriormente foram obtidos, em nosso laboratório, vetores com genes otimizados. Com essas ferramentas a proteína M foi produzida e purificada tendo sido possível obter anticorpos policlonais específicos e eficientes na sua detecção. Com esses anticorpos confirmamos a interação da proteína M com as proteínas celulares tropomiosina e nucleofosmina. Também foi demonstrado por imunofluorescência e por western blotting que a proteína M quando expressa fora do contexto de infecção apresenta localização nuclear e citoplasmática. Foram feitos testes de imunização com a proteína M purificada ou com um vetor eucariótico que a expressa (DNA). A imunização com proteína M resultou apenas em resposta humoral, enquanto com a vacina de DNA obtivemos apenas resposta celular. Nenhum desses imunógenos, entretanto, foi capaz de conferir proteção contra hRSV. / The human Respiratory Syncytial Virus (hRSV) is among the main causes of respiratory tract diseases, hRSV belongs to the Paramyxoviridae family. The symptoms of the infection can range from simple flulike state to severe respiratory disease eventually leading to death. Currently there is no licensed vaccine or effective drug against this virus. Vectors with genes optimized for of the hRSV Matrix protein (M) expression in bacteria and in eukaryotic cells were previously obtained in our lab. With these tools the M protein was produced and purified. Specific and efficient polyclonal antibodies could then be obtained and used for its detection. Using these antibodies we confirmed M interaction with cellular proteins tropomyosin and nucleophosmin. It was also demonstrated by immunofluorescence and western blotting that protein M when expressed out of viral infection context, presents cytoplasmic and nuclear localization. Immunization tests were made with the purified M protein or with a eukaryotic vector that expresses it (DNA). Immunization with M protein resulted only in humoral response, while with the DNA vaccine only cellular response was obtained. None of these antigens, however, was able to confer protection against hRSV.

O Brasil é o país líder no uso do fungo entomopatogênico Metarhizium anisopliae para o controle pragas agrícolas. Pouco se conhece sobre a diversidade e o impacto dos micovírus em M. anisopliae sensu stricto. Este trabalho mostra a riqueza dos micovírus associados a isolados de M. anisopliae da coleção de entomopatógenos da Universidade de São Paulo, usinas sucroalcooleiras e produtos microbianos à base do entomopatógeno. RNAfd foram encontrados em 55% dos 36 isolados de Metarhizium e apresentaram 16 diferentes padrões eletroforéticos consistindo de 3 a 18 bandas de RNAfd em gel de poliacrilamida. RNAfd não foram detectados em nenhum dos produtos comerciais utilizados no presente estudo. Os diferentes padrões de vírus encontrados nos isolados aqui estudados, aparentemente, não têm relação com os locais onde foram coletados. O inibidor de síntese protéica ciclohexamida não foi eficiente na eliminação dos vírus de RNAfd em nenhum dos isolados de fungos testados. Colônias dos isolados de M. anisopliae ESALQ 866, M. anisopliae ESALQ 1256 e M. anisopliae CTC F8 foram curadas por meio do cultivo monoconidial ou isolamento de ponta de hifas. Alguns segmentos do isolado M. anisopliae PL26 foram perdidos após o subcultivo de ponta das hifas. Colônias de M. anisopliae ESALQ PL26 obtidas por meio do cultivo monoconidial ou subcultivo de ponta de hifas apresentaram grande variabilidade morfológica, no entanto, essa variação não foi correlacionada com a presença de micovírus. Colônias isogênicas de M. anisopliae ESALQ 1256 mostraram diferenças no crescimento, na produção de conídios e na virulência, no entanto, essas diferenças não foram associadas à presença de RNAfd. Não foram observadas diferenças na tolerância aos raios ultravioleta e ao calor entre as colônias de M. anisopliae ESALQ 1256, com e sem vírus de RNAfd. Colônias oriundas de setores formados no isolado M. anisopliae PL26 produziram menor quantidade de conídios e apresentaram menor quantidade de vírus RNAfd em comparação com as colônias oriundas de outras regiões da colônias originais com características normais. A repicagem sucessiva dos isolados de M. anisopliae ESALQ PL26, ESALQ 1256, CTC F8 e CTC F15, infectados por diferentes vírus de RNAfd, nos meios de cultura BDA, SDAY e meio de arroz, bem como a passagem dos fungos em larvas de Tenebrio molitor, em geral, não afetaram a replicação dos micovírus. / Brazil is the leading country in the use of the entomopathogenic fungus Metarhizium anisopliae against agricultural pests. Little is known about the diversity and the impact of mycovirus in M. anisopliae sensu stricto. This study shows the richness of mycovirus associated with M. anisopliae isolates from the collections of entomopathogens of the University of São Paulo, from sugar-alcohol factories and microbial based products. dsRNA were found in 55% of the 36 Metarhizium isolates and showed 16 different electrophoretic patterns consisting of 3 to 18 dsRNA bands in polyacrylamide gels. dsRNA was not detected in any of the commercial products used in this study. The different viral patterns found in the isolates studied here apparently have no relation to the locations where they were collected. The inhibitor of protein synthesis cycloheximide culture was efficient in eradicating dsRNA virus from fungi. Colonies of isolates of M. anisopliae ESALQ 866, M. anisopliae ESALQ 1256 and M. anisopliae F8 were cured by monoconidial or by hyphal tip isolation of. Some segments of the M. anisopliae PL26 isolate were lost following hyphal tip subculture. Colonies both from monoconidial culture and hyphal tip subculture of M. anisopliae ESALQ PL26 showed great morphological variability; however, this variation was not correlated with the presence of mycoviruses. Isogenic colonies of M. anisopliae ESALQ 1256 showed differences in growth, conidia production and virulence; however, these differences were not associated to the presence of the dsRNA. No difference was observed regarding tolerance to ultraviolet rays and heat among the colonies of M. anisopliae ESALQ 1256, with and without the dsRNA virus. Sectors formed in the isolate M. anisopliae PL26 produced a smaller number of conidia and fewer dsRNA virus in comparison to the original colonies with normal characteristics. The repeated subculture of isolates of M. anisopliae ESALQ PL26, ESALQ 1256, CTC F8 and CTC F15, infected by different virus of dsRNA, in the PDA, SDAY culture media and in rice, as well as the fungi passage in larvae of Tenebrio molitor, in general, did not affect the replication of the mycovirus.

No presente trabalho, investigamos a filodinâmica de três modelos virais diferentes, utilizando técnicas baseadas em verossimilhança e inferência bayesiana. Dois desses são flavivírus com genoma de RNA fita simples e senso positivo. O terceiro é um bunyavírus com genoma tri-segmentado de RNA fita simples com senso negativo. Estes diferentes modelos permitiram estudar diferentes mecanismos promotores de diversidade viral, reagrupamento de segmentos genômicos (shift) e mutação (drift), que atuam em diferentes granularidades. Descrevemos pela primeira vez o espalhamento geográfico das linhagens de vírus Zika (ZIKV) em um nível continental, assim como ocorrência de recombinação e associação entre padrões de glicosilação e vetores. Para o flavivírus da encefalite transmitida por carrapatos (TBEV), investigamos seu espalhamento e encontramos evidências que corroboram a hipótese de circulação viral restrita a focos na Europa central. As análises sobre o vírus da Febre da Grande Fenda Africana (RVFV) apontaram a ocorrência de reagrupamento de segmentos genômicos e também ajudaram a elucidar sua dispersão do leste do continente africano para o oeste, encontrando-se diversas introduções no Senegal e Mauritânia. Aparentemente, este vírus teve a entrada facilitada nesses países por uma região que funciona como um centro de dispersão (hub) por ser encontro de rotas migratórias de animais. Ademais, investigamos a ocorrência de rearranjos de segmentos genômicos de RVFV e também estudamos as diferenças nas dinâmicas evolutivas de cada segmento. / In this study, we investigated the phylodynamics of three different viral models, using techniques based on maximum likelihood and Bayesian inference methods. Two of these viruses are flaviviruses, whose genomes are formed by a single-stranded positive-sense RNA molecule. The third is a Bunyavirus with tri-segmented single-stranded RNA genome with negative sense. These different models allowed us to investigate two different mechanisms to promote viral diversity, (i) recombination of genomic segments (\"shift\") and (ii) mutation (\"drift\"), therefore exploring different levels of granularity of evolutionary process. We described for the first time the geographic spread of Zika virus (ZIKV) strains in a continental level, as well as, the occurrence of recombination and association between glycosylation patterns and vectors. For the other Flavivirus, tick-borne encephalitis virus (TBEV), we investigated its spreading and found evidences to support the hypothesis that viral circulation is very constrained by the foci in central Europe. The analyses about the Rift Valley Fever Virus (RVFV) revealed the occurrence of reassortment of genomic segments and their dispersal from eastern Africa to the west, with several introductions to Senegal and Mauritania. Apparently, the entry of RVFV in these countries was facilitated by the region of Kedougou, where several migratory routes of animals converge. This place maybe works as a hub to spread RVFV for West Africa. Moreover, we also investigated the differences in evolutionary dynamics of each genomic segment of RVFV.

A proteína de matriz do Virus Respiratório Sincicial humano foi o foco deste trabalho. Verificamos que o gene de matriz possui sítios internos de poliadenilação, sinais de instabilidade de RNA, baixo índice de adaptação de codons (CAI) e conteúdo GC, que podem impedir a expressão gênica vitro. Quando clonado sob controle do promotor de CMVie, o gene selvagem não apresenta expressão detectável, enquanto um gene sintético com a sequência do gene de matriz otimizada apresenta altos níveis de expressão em células transfectadas. Esse alto nível de expressão permitiu a confirmação da presença da proteína M no núcleo no início de sua expressão, por análise em microscopio confocal de varredura a laser, além de sua associação a membranas em regiões conhecidas como lipid rafts. Também foi observado que a proteína M é capaz de associar à proteína tropomiosina. Ainda foram analisados os possíveis domínios funcionais através de expressão de variantes da proteína M com deleções de trechos da proteína. Finalmente foi analisada a capacidade de indução de resposta imune. / The Human Respiratory Syncytial Vírus was the focus of this work. We found that matrix gene has internal polyadenilation sites, RNA instability motifs, low codon adaptation index (CAI) and GC content, that may impair its expression in vitro. When cloned under control of the ieCMV promoter, the wild-type M gene expression was not detectable, whereas a synthetic optimized matrix gene was highly expressed in transfected cells. This high level of expression made possible to follow M nuclear localization in the beginning of its expression by confocal laser scanning microscopy, and its association with membranes in regions known as lipid rafts. It has also been found that the matrix protein associates with tropomyosin. It was further analyzed the possible functional through expression of deviations of the M protein that lack portions of the protein. Finally it was analyzed its capacity to induce an immune response.

M. Tech. (Department of Biotechnology, Faculty of Applied and Computer Science), Vaal University of Technology. / Influenza-A virus is a single stranded negative sense RNA virus that is a member of a Orthomyxoviridae group. The virus is diverse and consists of 16 haemagglutinin (H) and 9 neuraminidase (N) glycoproteins subtypes that form a serotype. Avian influenza virus (AIV) has been detected in more than 100 bird species from 26 different families, although Anseriformes and Charadriiformes are considered the natural hosts of the virus. A 12-month study was conducted at the African Pride Irene Country Club lodge in Pretoria where the prevalence of AIV was monitored in a community of wild birds. The African Pride Irene Country Club lodge houses a population of wild bird species such as Egyptian geese (Alopochen aegytptiaca), Yellow-billed duck (Anas undulata), Red knobbed coot (Fulica cristata), African sacred ibis (Threskiornis aethiopicus) and Hadeda ibis (Bostrycha hagedash). A total of 3674 faecal samples were collected over a period of 12 months and screened for AIV group using the Matrix gene (M-gene) real time reverse-transcriptase PCR (rRT-PCR). Positive samples were submitted for virus isolation in embryonated chicken eggs. In addition, the RNAs were screened using H5 and H7 subtype specific rRT-PCR and a conventional universal RCR assay that targets the HA gene was also used. Polymerase Chain Reaction (PCR) products were requenced using Sanger sequencing and the viral isolates were subjected to Next Generation sequencing (NGS). Twenty percent of the samples tested positive for the AIV group and four virus subtypes were identified. One virus isolate was identified through NGS as H3N6; two through conventional PCR and Sanger sequencing as H9Nx and H6Nx. Of the twenty percent samples that tested positive for AIV 98% were identified as H7Nx by subtype specific through rRT-PCR. The highest frequency of AIV positive samples was detected between the months of January and February 2017 (20%), with smaller peaks detected in february and March 2016 (0.3%). Lower peaks were also detected between the months July and November 2016 (0.1%), respectively. A high prevalence of AIV was detected in the late summer months with a frequency of 65% positive, although a low prevalence was also detected in the autumn (0.6%) winter (0.6%) and spring 0.08%). Thus, the study provides a valuable insight into the seasonal prevalence of AIV in a heterogeneous wild duck population in Gauteng Province.

No description available.

Cornichon (CNI) proteins are a conserved family of proteins among eukaryotes, from Erv14 in the yeast Saccharomyces cerevisiae to CNI homologs (CNIHs) in mammals and plants. Erv14 functions as a cargo receptor of coat protein complex II (COPII) for protein trafficking from the endoplasmic reticulum (ER) to the Golgi apparatus, en route to their final destinations. By interacting with specific cargo proteins, CNI proteins regulate key steps of embryo polarity in Drosophila, budding in yeast, and synaptic transmission in the mammalian brain. However, we have very limited understanding of plant CNIHs. Positive-strand RNA viruses assemble their viral replication complexes (VRCs) at specific host organelle membranes. With a better understanding of host factors involved in targeting viral replication proteins to the preferred organelles, we expect to block trafficking of viral replication proteins and thus, viral infection, by manipulating the required host proteins. Brome mosaic virus (BMV) is a model of positive-strand RNA viruses and its replication can be recapitulated in yeast. Importantly, BMV replication protein 1a is the only required viral protein to form VRCs at the perinuclear ER membrane in yeast. I demonstrate that Erv14 and COPII coat proteins are required for targeting BMV 1a to the perinuclear ER in yeast, suggesting a novel function of COPII vesicles in protein trafficking to the perinuclear ER membrane and in the BMV VRC formation. As for cellular functions, I show that plant CNIHs complement the defective distribution of BMV 1a in yeast mutant lacking Erv14. Taking advantage of Arabidopsis thaliana knockout mutants and knockdown of gene expression in Nicotiana benthamina, I also discover that CNIHs unexpectedly play crucial roles in pollen development, infection of a bacterial pathogen, and maintenance of ER tubules. I further confirm that CNI proteins are also required for maintaining ER tubules in yeast, suggesting a novel and conserved role in shaping ER morphology. Therefore, these findings indicate the functional diversity and redundancy of CNI proteins in key cellular processes and suggest a novel strategy to control plant pathogenic viruses and bacteria by manipulating plant CNIHs. / Ph. D.

Le virus de la paralysie chronique de l'abeille (Chronic bee paralysis virus, CBPV) est l'agent étiologique d'une maladie infectieuse et contagieuse des abeilles adultes (Apis mellifera L.), appelée la paralysie chronique. Le CBPV est un virus à ARN simple brin positif qui contient 2 fragments d'ARN majoritaires. L'ARN 1 (3674 nt) et l'ARN 2 (2305 nt) codent respectivement 3 et 4 cadres ouverts de lecture (ORF). La séquence d'acides aminés de l'ORF 3 de l'ARN 1 partage des similitudes avec l'ARN polymérase ARN dépendante (RdRp) des virus des familles Nodaviridae et Tombusviridae. Par analogie avec ces familles virales, il a été suggéré que l'ARN 1 coderait les protéines non-structurales tandis que l'ARN 2 coderait les protéines structurales. Cependant, la réalité de ces protéines virales doit être démontrée expérimentalement afin d'étudier leurs fonctions, de mieux décrire ce virus et sa position taxonomique ainsi que d'améliorer les outils de diagnostic. Dans ce but, différentes approches expérimentales ont été utilisées. Une comparaison des protéomes d'hémolymphe d'abeilles non-infectées et infectées par le CBPV a été effectuée. Les protéines différentiellement exprimées ont été identifiées par empreinte peptidique massique (peptide mass fingerprint, PMF). Cette étude a permis d'identifier des protéines de l'abeille dont certaines contribueraient à une réponse immunitaire antivirale, mais aucune protéine virale n'a été identifiée par cette approche. Les ARN extraits du CBPV ont été utilisés dans des expériences de traduction in vitro. Malgré plusieurs essais réalisés en faisant varier les conditions expérimentales, cette approche s'est révélée infructueuse. / Chronic bee paralysis virus (CBPV) is the etiological agent that causes an infectious and contagious disease in adult bees (Apis mellifera L.), called chronic paralysis. CBPV is a positive single-stranded fragmented RNA virus which contains 2 major viral RNA fragments. RNA 1 (3674 nt) and RNA 2 (2305 nt) encode 3 and 4 putative open reading frames (ORFs), respectively. The amino acid sequence of ORF 3 on RNA 1 shares similarities with the RNA-dependent RNA polymerase (RdRp) of virus families Nodaviridae and Tombusviridae. By analogy with these viral families, it has been suggested that RNA 1 encodes non-structural proteins and RNA 2 encodes structural proteins. However, the reality of viral proteins needs to be experimentally demonstrated in order to study theirs functions, to describe CBPV biology and its taxonomic position and to improve diagnostic tools. With this aim, different experimental strategies have been used.A comparison of hemolymph proteomes between uninfected bees and bees infected with CBPV was performed. Differentially expressed proteins have been identified using peptide mass fingerprint method (PMF). This study allowed only identifying proteins of bees which could contribute to an antiviral immune response but viral proteins were not identified using this approach. Extracted CBPV RNAs were used for in vitro translation experiments. Despite several assays in varying experimental conditions, this approach has been unsuccessful. Another approach was to generate antibodies directed against different proteins or parts of viral proteins.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Raymond R. R. Rowland / Mechanisms for mutations in RNA viruses include random point mutations, insertions, deletions, recombination and re-assortment. Most viruses have more than one of these mechanisms operating during their life cycle. Impact of sequence divergence is seen in the areas of evolution, epidemiology and ecology of these viruses. Immediate negative consequences of genetic diversity include failure of vaccination, resistance to anti-virals, emergence and re-emergence of novel virus isolates with increased virulence or altered tropism. To identify specific sequence features that influence recombination, a new in-vitro system was developed using an infectious cDNA clone of PRRS virus that expressed fluorescent proteins. The in-vitro experimental system involved the co-transfection of a pair of closely related PRRSV infectious clones: a fully functional non-fluorescent PRRS virus infectious clone that possessed a single mutation in a green fluorescent protein (GFP) and a second infectious clone that contained a defective fluorescent virus. The readout for successful recombination was appearance of a fully functional fluorescent virus. The model system creates the opportunity to study several aspects of recombination, including the requirement for sequence homology between viruses undergoing recombination.

O estudo dos protozoários é importante por diversos motivos, entre eles sua diversidade, sua importância evolucionária e impacto na saúde pública. Muitos aspectos tornam o estudo desses seres vivo ainda mais fascinantes, como por exemplo, fenômenos como o trans-splincing e a presença de um vírus em algumas espécies de leishmanias. A proteína U5-15k é considerada de grande importância fazendo parte do spliciossoma. Neste trabalho tivemos o objetivo de iniciar a caracterização desta proteína empregando ferramentas de bioinformática para analisar sua sequencia de aminoácidos, sua estrutura secundária e modelar sua estrutura por homologia, também foi possível otimizar sua expressão, purificação, caracterizar sua auto clivagem e fazer estudos biofísicos de Espalhamento Dinâmico de Luz e Espectroscopia de Dicroísmo Circular. O Leishmania RNA vírus 1-4 (LRV1-4) é um vírus da família Totiviridae de capsídeo icosaédrico que codifica duas proteínas (proteína capsidial e RNA polimerase). Sendo pouco estudado tivemos o objetivo de iniciar a caracterização molecular deste vírus com objetivos futuros de empreender estudos estruturais e funcionais. Não foi possível obter expressão recombinante em nenhuma das duas proteínas virais, por isso se optou por se estabelecer um novo protocolo de lise celular e purificação com o qual se obteve imagens inéditas de Microscopia Eletrônica de Varredura, na qual o vírus é purificado ainda envolto em material genético. / The study of protozoa is important for several reasons, including their diversity, their evolutionary importance and public health impact. Many aspects make the protozoas study even more exciting, for example, phenomena such as trans-splincing and the presence of virus in some species of Leishmania. The U5-15k protein is considered of great importance as part of the spliciossome machinery. In this work we intended to characterize this protein , this using bioinformatics tools to analyze its amino acid sequence, its secondary structure and its structure by homology modeling, it was possible to optimize expression, purification, characterization and make biophysical studies of their self cleavage of Dynamic Light Scattering and Spectroscopy of Circular Dichroism. The Leishmania RNA virus 1-4 (LRV1-4) is a virus belonging to the Totiviridae family with an icosahedral capsid structure that encodes two proteins (a capsid major protein and an RNA polymerase). It was not possible to obtain the recombinant expression of any of the two viral proteins, so it was decided to establish a new protocol for cell lysis and purification with which it was obtained unprecedented images of Transmission electron microscopy, in which the virus is further purified wrap on genetic material.