ABSTRACT Adenovirus Death Protein (ADP) expression during late stages of a lytic infection releases mature virions to promote viral spread, thus leading to death of the host cell. We sought to investigate ADP expression patterns in persistently infected human lymphocytes cells. We hypothesized that low expression of ADP allows the virus to persist while high expression would promote lytic infection in lymphocytes. Accordingly, we found ADP expressed in low amount in BJAB and KE37 cells, while lytically infected Jurkat cells demonstrated higher ADP expression in both protein and transcript levels. ADP overexpression in persistently infected lymphocytes did not alter the viability of these cells, or their level of ADP expression. In contrast, Jurkat cells infected with an ADP-deleted virus had increased survival and maintained viral DNA for greater than 1-month, suggesting conversion to a persistent infection. Also manipulating ADP expression had minimal impact on the total virus yield from infected lymphocytes.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2006. / Härtill 4 uppsatser.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Biotecnologia e Biociências, Florianópolis, 2015. / Made available in DSpace on 2015-10-06T04:06:59Z (GMT). No. of bitstreams: 1 334675.pdf: 2103795 bytes, checksum: ccd250eef9810ed9db2bf44c58134fb1 (MD5) Previous issue date: 2015 / Com a criação do Programa de Controle Higiênico Sanitário de Moluscos Bivalves pelo governo brasileiro em 2012, o processo de purificação de moluscos pelo método da depuração passou a ser exigido quando há a detecção de patógenos bacterianos nos animais. Entretanto, não é fornecida a forma ou eficácia desse método. Mais estáveis e prevalentes em amostras ambientais que bactérias, os vírus entéricos são comumente associados a surtos de gastroenterites relacionados ao consumo de moluscos contaminados. Porém, sua detecção em geral é baseada em métodos de detecção de genoma, sem relação com a infecciosidade viral. Dessa forma, esse trabalho teve como objetivo avaliar a aquisição de patógenos virais por ostras Crassostrea gigas artificialmente contaminadas, a estabilidade desses patógenos frente a condições controladas de temperaturas e a eficácia do processo de depuração na inativação desses micro-organismos. Foram utilizados como modelos virais o adenovírus humano tipo 2 (AdVH-2) e o norovírus murino tipo 1 (NVM-1) e metodologias de detecção de vírus infecciosos. O comportamento entre os dois modelos virais foi diferente no processo de bioacumulação artificial pelas ostras, com o AdVH-2 atingindo o ápice de concentração viral no tecido digestivo após 8h, e o NVM-1 após 24h. Os dois modelos virais se mostraram estáveis por até 96h em ostras resfriadas a 4°C, porém foram inativados em amostras de ostras cozidas "ao bafo" na temperatura de 100°C por até 6 min. A depuração se mostrou eficiente para a inativação do AdVH-2 utilizando a metodologia de placa de lise de detecção de vírus infecciosos, e o uso da lâmpada U.V. acelerou o processo. Após 24h de depuração com luz U.V., 2,92 log de redução foram encontrados (correspondendo a mais de 99% de inativação viral), não sendo mais detectado nenhum virus infeccioso após esse período. E com 48h de depuração no tanque controle, 2,65 log de redução foram detectados. O uso de métodos de desinfecção (como a luz U.V.) em sistemas de depuração fechados é exigido pelas agências reguladoras internacionais, uma vez que a permanência de patógenos na água do tanque pode re-contaminar os animais, e o descarte irregular dessa água pode levar à contaminação ambiental. As análises da depuração do NVM não puderam ser concluídas, pois as amostras de ostras foram coletadas durante o período reprodutivo dos animais, e o estresse causado pela coleta e contaminação artificial levou os animais a desovarem e morrerem. Dessa forma, novos experimentos serão conduzidos no inverno para complementar os dados do atual trabalho.<br> / Abstract : The process of shellfish purification through depuration started to be required by the Brazilian government with the establishment of the Hygienic Control Program of Bivalve mollusks in 2012, but recommendation on how it should be performed or on its efficiency has not been suggested. More stable and prevalent in environmental samples than bacteria, enteric viruses are generally related to gastroenteritis outbreaks associated with the consumption of contaminated oysters. However, virus detection methods are usually based on genome detection rather than infectious virus. Therefore, this work aimed at evaluating the accumulation of viral pathogens on oysters Crassostrea gigas artificially contaminated; the stability of these pathogens maintained in controlled conditions of temperature and the depuration efficiency in inactivating these microorganisms. Human adenovirus type 2 (ADVH-2) and the murine norovirus type 1 (NVM-1) were used, as well as detection methodologies of infectious viruses. The behavior of the two viruses were different in the oyster?s artificial bioaccumulation process, with the AdVH-2 reaching its peak of viral concentration after 8h, and the NVM-1 in 24h. The two viruses were stable for 96 hours in oysters maintained under 4°C, but they were completely inactivated in steamed oyster samples cooked for up to 6 minutes in 100°C. The depuration process was efficient in inactivating the AdVH-2, evaluated by the plaque assay for infectious virus detection, and the U.V. light accelerated the process. After 24h of depuration with U.V. light, a reduction of 2,92 log was found (which represents an inactivation of more than 99% of the viruses), with no infectious virus being detected after this period. After 48h in the tank without U.V. light, a reduction of 2,65 log was found. The use of disinfection methodologies in closed depuration systems is required by international regulation agencies, once these pathogens can re-contaminate other animals, and the irregular discharge of the tank water can lead to environmental contamination. The NVM-1 depuration assays could not be concluded because the oysters samples were collected during the reproductive period of the animals, and the stress caused by the collection and artificial contamination lead the animals to spawn and die. Thereby, new experiments will be conducted in the winter to complete this work data.

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The expression of the adenovirus (Ad) genome is co-ordinately regulated and the various early regions of the genome are known to exert feedback controls on each other. The region E1B is known to encode functions which are required for oncogenic transformation by adenovirus yet its potential regulatory role is poorly understood. In this study the regulatory role(s) of the region E1B was investigated at the level of RNA accumulation. The Northern blot technique using various cloned early regions as probes and RNAs from E1B mutants Ad 12, Ad 2 and Ad 5 were used. The levels of region E1A specific RNAs were found to be aberrant. Thus for Ad 12 cyt 68 and Ad 2 dl 250 the levels were higher than wild type ones at late times whereas for Ad 5 dl 313 hr-6 the levels were consistently lower than Ad 5. This implies that the region E1B normally encodes functions which are involved either directly or indirectly in the efficient accumulation of region E1A specific RNA. Other regions also seemed to be affected in these mutants. In cyt 68, region E1B RNA was higher than wild ones at late time, indicating an auto-regulatory mode of control for this region. The expression of region E3, E4 and LS RNA were all perturbed by the E1B mutation as some of the mutants either accumulated higher or lower than wild type levels of RNA. / Thesis / Master of Science (MS)

The adenovirus genome is a linear double stranded DNA molecule and the current widely accepted model of viral DNA replication proposes linear viral DNA intermediates at all stages of replication. Although the experimental evidence for this mechanism of replication is very strong, circular forms of adenovirus serotype 5 (Ad5) DNA molecules have also been detected in both permissive and non-permissive cell lines. In some experiments the circular structures were detected before the onset of viral DNA replication and thus suggested a possible role for circular forms of Ad5 DNA in the life cycle of the virus after infection. This study was undertaken to better understand the role of circular forms of the adenovirus genome in virus replication. The approach was to subclone the viral junction internally into the linear genome thus creating mutant viruses with embedded terminal sequences and to study the effect of such inserts on DNA structure. A total of five mutant viruses were constructed containing a variety of inserts and viral DNA from infected cells and banded viruses was analyzed by Southern Blot hybridization. The data clearly showed that embedded viral junctions have biological activity in that they generated novel, rear ranged viral DNA molecules, and that embedded single ITR sequences were also biologically active, but to a lesser extent. It was also observed that the copy numbers of the rearranged molecules were variable. It appeared that the embedded viral junction was active in recombination and replication of the viral genome, creating the rearrangements through these two processes. However, the results suggested that circular forms are not obligatory intermediates in DNA replication. The analysis of banded viral DNA in this study suggested that the encapsidation signal from the left end of the linear genome was required in cis for packaging of the viral genome, confirming previous results which identified an encapsidation signal for viral DNA packaging. The banding experiments also showed that truncated viral DNA molecules containing 75% of the viral genome were packaged. / Thesis / Master of Science (MS)

The 55K product of early region 1B (E1B) of human adenoviruses is required for viral replication and participates in cell transformation. Both biochemical and genetic approaches have been used to show that this 496-residue (496R) protein of adenovirus (Ad5) is phosphorylated at both serine and threonine residues at sites near the carboxy terminus within sequences characteristic of a casein kinase II. Mutations which converted serines 490 and 491 to alanine residues decreased virus replication and reduced the efficiency of transformation of primary baby rat kidney cells, suggesting that phosphorylation at these sites is of importance in the regulation of 55K biological activity. / Thesis / Master of Science (MS)

Previous studies have shown that a functional Ad 5 E1A protein from the 13 S mRNA is required during lytic infection for activation of early viral gene expression (Jones and Shenk, 1979b; Berk et al, 1979; Montell et al, 1982). The mechanism by which E1A exerts this regulatory function is still unclear and has been the subject of intense investigation (Persson et al, 1981a; Katze et al, 1981, 1983; Nevins, 1981; Shaw and Ziff, 1982). Most of these recent investigations have relied on the use of metabolic inhibitors such as cycloheximide to eliminate protein synthesis as a means of determining the role of viral and host proteins in the regulatory process. The results from these studies have been inconsistent. The purpose of this research project has been to re-examine the regulation of Ad 5 early gene expression without the use of drug inhibitors. In this study tsH1 cells, a mutant CHO cell line which at temperatures above 37°C are inhibited in protein synthesis, were used. At critical times during the course of wild type or host-range 1 infection of tsH1 cells, protein synthesis was inhibited and Early Region 4 expression was examined. In every case, efficient E4 expression was dependent on functional E1A protein and this requirement could not be replaced by simply inhibiting protein synthesis. The results are discussed in relation to models proposed to explain the regulation of Ad 5 Early Gene Expression. / Thesis / Master of Science (MS)

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