Dissertations / Theses on the topic 'Virology'

Ljungan virus är ett virus som isolerades i Sverige i mitten på nittiotalet. Under perioden 1989-1992 avled flera svenska elitorienterare plötsligt i hjärtmuskelinflammation. Man misstänkte att orienterarna kunde ha utsatts för en vektorburen infektion eftersom de exponeras för djur som finns i skog och mark under träning och tävling. Det är sedan tidigare känt att sorkar är den naturliga reservoaren för ett annat virus (Puumala virus) som kan orsaka njurskada hos människor. Sorkantalet i vissa delar av Sverige varierar kraftigt från år till år i ett cykliskt förlopp. Man fann ett samband mellan antalet sorkar och förekomsten av hjärtmuskelinflammation, typ 1 diabetes och Guillain-Barre's syndrom vilket ledde till att ett tidigare okänt virus, Ljungan virus, kunde isoleras från sorkar. Detta virus är ett litet RNA-virus som tillhör familjen Picornavirus. Till denna familj hör också flera kända virus såsom många av våra vanligt förekommande förkylningsvirus, men också virus som kan orsaka svåra sjukdomar, till exemel poliovirus och mul- och klövsjukevirus. Ljungan virus är ett nyupptäckt virus och därför är kunskapen om viruset begränsad. För att öka vår förståelse om viruset så har arvsmassan för tre svenska isolat (87-012, 174F och 145SL) av Ljungan virus kartlagts (artikel IV). Denna studie visade att Ljungan virusets arvmassa har flera unika egenskaper. Släktskapstudier visade att Ljungan virus är endast avlägset släkt med redan kända picornavirus och viruset bör därför utgöra en egen undergrupp i familjen (artikel III). Med kunskap om Ljungan virusets arvsmassa så var det möjligt att visa att Ljungan virus förekommer även på andra ställen än i Sverige (artikel VI). I mitten på 60-talet isolerades ett virus, M1146, från sork som fångats i Oregon, USA. Baserat på egenskaper hos proteinhöljet (kapsiden) så antog man då att M1146 var ett picornavirus. Studier av arvsmassan för detta virus visade att M1146 är närmast besläktat med de svenska Ljungan virus isolaten och har samma unika egenskaper i sin arvsmassa (artikel VI). Dessa studier har varit möjliga eftersom vi tidigare har utvecklat en metod för att producera stora mängder av hela arvsmassan (artikel I och II). Slutligen har det proteinhölje som innesluter och skyddar Ljungan virusets arvsmassa studerats (artikel V). Dessa studier visade att kapsiden är uppbyggd av tre proteiner och inte fyra som hos de flesta picornavirus. Dessa studier underlättades av att en enkel och effektiv metod för att odla Ljungan virus i provröret har utvecklats (artikel V). Sammantaget så har dessa studier försett oss med nya kunskaper om Ljungan virus som möjliggör fortsatta studier av dess biologi och eventuella förmåga att orsaka sjukdom hos människor och djur.

It is today well known that the outcome of a certain infection depends on factors of both the host and the pathogen. Studies of host genetic susceptibility to infectious diseases aim to increase the understanding of why some individuals are more susceptible than others, to a certain infection. Knowledge of genetic susceptibility to a viral disease may be used in development of new therapeutic means, and also to recognize individuals who are at increased risk of severe symptoms if infected with a pathogen. It seems however that a risk factor for one disease may play a protective role in another situation; like a double-edged sword. In this thesis I have studied genetic factors affecting susceptibility to norovirus (NoV) and factors affecting the risk of developing tick-borne encephalitis (TBE) after infection with TBE virus (TBEV). NoV is the cause of the “winter vomiting disease”, affecting millions of people every year, and causing up to 200,000 fatalities among children in developing countries, each year. It is today recognized that the secretor status of an individual, i.e. the ability to express ABO blood groups and related antigens, in secretions and on mucosa, affect the risk of being infected by NoV. By studying authentic NoV outbreaks in Denmark, Spain and Sweden and by comparing the secretor status of affected and unaffected individuals we were able to confirm that secretor status have indeed great impact on susceptibility to some NoV strains, but also that there are strains circulating, which infect individuals regardless of secretor status. TBEV is endemic in many parts of Europe and Asia but studies have shown that 70-95% of all infections are asymptomatic or sub-clinical. Some individuals do however develop TBE, a severe disease including meningitis or encephalitis with or without myelitis. Also, many patients suffer from long-time sequelae and TBEV infections may in worst case be fatal. The reason for difference in disease outcome is not known and we have chosen to study if genetic factors affecting the immune response may play a role in disease outcome. To do this we used a prospectively collected Lithuanian material with samples from patients with TBE, AME (aseptic meningoencephalitis) and matched healthy controls. So far we have found that a deletion in chemokine receptor 5 (CCR5), a gene encoding a receptor involved in cell migration, is a risk factor for developing disease. We have also data showing that toll-like receptor 3 (TLR3), a receptor recognizing double stranded RNA (dsRNA), which is a product of TBEV replication, may instead of being protective increase the risk of TBE.

The splicing pattern during an adenovirus infection is shifted at the late phase towards using weaker splice sites, splicing out larger introns. Splicing of weak 3´ splice sites usually requires recognition of the 3´AG dinucleotide before the first catalytic step of splicing. Such splicing events are said to be AG-dependent and requires an interaction of both subunits of the cellular splicing factor U2AF with the 3´ splice site. We show that splicing of transcripts that are AG-dependent in uninfected nuclear extracts (NE) becomes AG-independent in nuclear extracts prepared form adenovirus late-infected HeLa cells (Ad-NE). Further we demonstrate that the first step in splicing of a model transcript, IgM, becomes completely U2AF-independent in Ad-NE. This finding supports our working model that 3´ splice site recognition in Ad-NE is altered, and in fact might be U2AF-independent. We further show that the adenovirus late protein L4-33K acts as a virus encoded alternative splicing factor. L4-33K activates splicing of both cellular and viral transcripts containing weak 3´ splice sites. This supports the hypothesis that adenovirus alter splicing during the infection to favour usage of weak, suboptimal 3´ splice sites. However, we were unable to find an alternative U2AF-related factor that could stimulate L4-33K splicing enhancer activity. Furthermore, we demonstrate that the serine residues in the C-terminal part of L4-33K are important for the splicing enhancer activity but also for its nuclear localisation. The adenovirus major late promoter is highly activated after the onset of viral genome replication. Protein complexes binding to downstream elements of the promoter are required for full enhancement of this promoter. We show that an L4-33K-related protein, L4-22K, stimulates transcription from the major late promoter. This stimulation is mainly via the downstream elements and does not require the viral IVa2 protein, which is a transcription factor of the major late promoter.

Winter vomiting disease is caused by norovirus (NoV) and affects millions of people every year resulting in 200.000 deaths among children in developing countries. It was observed early that not all individuals exposed to the norovirus became ill. The reason for this is now recognized to be dependent upon the secretor status of an individual. The secretor status determines the ability of an individual to express histo-blood group antigens (HBGA) on mucosa and in saliva. A non-secretor is unable to express HBGAs due to a mutation in a gene called FUT2. In this thesis, I have investigated the antibody prevalence and titer in humans in Sweden and Nicaragua to the most common GII NoV and the correlation to secretor status, Lewis status and ABO. I found that secretors had significantly higher antibody prevalence and titer to GII NoV than non-secretors suggesting that non-secretors are less prone to be infected by the GII NoV. In Nicaragua, I also found several different NoV strains circulating at the same time. The NoVs have been circulating and evolving in the human population for some time and the same individuals seems to be infected over and over again with the same virus. This suggests that there is no long-term immunity present but possibly short-term immunity, which would make it very difficult to produce a vaccine against NoV. However, recent studies have shown the possibility of using virus like particles as a vaccine candidate and have demonstrated long-term immunity. The bovine NoV (boNoV) cause gastroenteritis in cattle and are closely related to the human NoV. The possibility of zoonotic transfer to humans is currently being investigated. I found that 26% of Swedish blood donors have antibodies to the boNoV suggesting that they have been exposed to the virus. The human NoV has been observed to be able to infect and cause disease in cattle, could the boNoV do the same in humans? To date, no boNoV strain has been found in humans. The proposed receptor structure for boNoV is the αGal epitope, which is present in many mammals like cow, pig, horse, sheep and rabbit but not in humans. This indicates that humans are not at risk for boNoV infection because we lack the proper receptor structure. However, recombinations between different NoV strains have been demonstrated and the possibility of more than one receptor being present has been suggested. I found that aa position 365-379 on the boNoV capsid seems to be important for binding to erythrocytes. In this thesis, I hope to add some new pieces to the Norovirus Puzzle.

Human papillomavirus type 16 (HPV-16) is the major cause of cervical cancer. We speculate that inhibition of HPV-16 late gene expression is a prerequisite for establishment of persistence and progression to cervical cancer. This is based on the findings that the late proteins are found only in the nuclei of terminally differentiated epithelium, and are never detected in human papillomavirus infected cervical cancer cells. It is therefore of great importance to understand how HPV-16 controls the onset of the immunogenic proteins L1 and L2 in an infected cancer cell. HPV-16 late gene expression is tightly regulated by differentiation-dependent transcription as well as by post-transcriptional mechanisms. The long-term goal of these studies was to understand how HPV late gene expression is regulated. The specific aim of this thesis was to identify cellular and viral factors that force the virus to switch on the late genes, and to determine the mechanism of action of these factors. This will help us to understand under which circumstances HPV establish persistent infections that could progress to cancer. We found three cellular factors; PTB, ASF/SF2 and SRp30c, and one viral factor; AdE4orf4, that in four distinctive ways were involved in the regulation of HPV-16 late gene expression. Interestingly, over-expression of PTB, AdE4orf4 or SRp30c produced different types of spliced late mRNAs. PTB induced the unspliced L2/L1 mRNA, while AdE4orf4 and SRp30c induced the spliced L1 and L1i mRNA, respectively. The three proteins had different mechanisms of action and different target sites within the HPV-16 genome, which revealed the many and complex pathways in HPV-16 gene regulation. These findings have contributed to a broader understanding of how the expression of HPV-16 late genes is controlled.

Dengue virus (DENV) is a mosquito-transmitted flavivirus that threatens approximately half of the world's population. In this dissertation, the use of bioorthogonal chemistry as a tool for researching emerging viral diseases, including DENV is explored. To this end, a bioorthogonally-modified amino acid was successfully installed within the proteome of DENV, which was used for the pull down of a known virus-protein interaction. This technology is intended to be broadly used for the determination of any virus-host interaction, through the installment of a non-perturbing modification that 1) does not hinder viral infectivity and 2) can be selectively discriminated by any complimentary probe. En route to using this technology, a new viral purification strategy was developed for DENV that reduces the overall purification time by 10 hours, and improves retention of virion infectivity. This method and a survey of other viral purification methods used with DENV is contained herein. Furthermore, a chemical scaffold that was repurposed for exploration of protein-protein crosslinking, namely for release of a reactive chemical warhead under acidic conditions, was used for the surface modification of DENV. This triazabutadiene probe was found to be activated by light. In this dissertation is reported the first time aryl diazonium ions for protein crosslinking have been generated on a protein or viral surface through UV-irradiation. The advantages and limitations of this chemistry are presented herein.

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From about 200 virus that cause respiratory infections, only 8 are responsible for severe illness in children, immunodeficient adults and elderly, inc1uding Adenovirus, Influenza A and B, Parainfluenza 1, 2 and 3, Human RespÍratory Syncytial Vírus (HRSV) and Human Metapneumovirus (hMPV). Three of these virus are responsible for a significant morbidity in elderly: Influenza, HRSV and hMPV. The nosocomial infection caused by these vÍruses can be fatal in hospitalized children and patients with other pathologies. With the advance of the of molecular biology techniques, the diagnosis and the characterization of these vírus became more effective. Beside vírus detection by PCR, isolation of vírus in specific cellular cultures to increase the amount of pathogens have been used in severallaboratories. Studies on prevalence of respÍratory vírus in elderly are rare. The objective of this research was to evaluate the occurrence of Influenza, HRSV and of the hMPV and the risk factors involved in the diseases caused by these vÍruses. Nasopharyngeal swabs were collected and used for inoculation in cells culture and dÍrect analysis by RT-PCR. The results from RT-PCR of Influenza Vírus and RSV were negative. We also tested the samples with GeneScan, that is a technique based on fluorescent primers specific to the studied vírus. Results for Flu virus and HRSV were the same ofRT-PCR, but 1 MPV sample was positive. 55,32% ofthe 47 studied elderly were vaccinated against Influenza; 14,9% had tabagism habits and 70,2% were women. These risk factors might had influenced in the absence of positive samples for the Influenza vírus andHRSV.

Le virus HTLV-1 est le seul rétrovirus humain oncogène découvert à ce jour à l’origine d’une lymphoprolifération maligne à cellules T, la leucémie aiguë de l’adulte (ATL). L’oncoprotéine Tax du virus HTLV-1 joue un rôle majeur dans l’établissement de la leucémie mais aussi dans la réplication du virus. Tax régule la transcription du promoteur viral situé au sein du LTR 5’ du virus, gouvernant ainsi sa propre production. Pour ce faire, Tax recrute des dimères du facteur de transcription cellulaire CREB, sous forme phosphorylée, au niveau d’éléments de réponse à l’AMP cyclique (vCRE) situés dans la partie U3 du LTR. La phosphorylation de CREB est un processus crucial pour la transactivation du promoteur viral. CREB est aussi ciblé et régulé par OGlcNAcylation, modification post traductionnelle impliquée dans de nombreuses pathologies comme le cancer, dont la régulation dépend de deux enzymes, l’OGA et l’OGT, formant le complexe OGlcNAzyme. Du fait que Tax dérégule de nombreuses machineries de modifications post traductionnelles et que la OGlcNAcylation régule avec la phosphorylation la stabilité et l‘activité de facteurs de transcriptions, nous avons évalué pour la première fois le statut de la OGlcNAcylation de CREB dans le cadre de l’infection par HTLV-1 et son implication sur la régulation du promoteur viral. Nous avons pu montrer que dans le cadre de l’infection par HTLV-1, la protéine Tax dérégulait la machinerie de OGlcNAcylation en inhibant l’activité de l’OGA. Pour ce faire, Tax interagit avec le complexe OGlcNAzyme entrainant une augmentation de la OGlcNAcylation de CREB favorisant ainsi la transcription du LTR 5’. Ainsi nous avons pu établir pour la première fois une relation entre Tax et la OGlcNAcylation permettant de proposer un nouveau modèle de régulation du LTR viral
HTLV-1 virus is the only oncogenic human retrovirus discovered to date. It is responsible for a T cell malignant lymphoproliferation named Adult T cell Leukemia. HTLV-1 Tax oncoprotein plays a major role in the development of the leukemia and also in the viral replication. Tax regulates the transcription from the viral promoter located in the virus 5’LTR, favoring its own transcription. In order to do this, Tax recrutes dimers of the phosphorylated cellular transcription factor CREB to viral cyclic AMP response elements (vCRE) situated in the U3 part of the LTR. CREB phosphorylation is a crucial process to allow the transactivation of the viral promoter. CREB is also targeted and regulated by OGlcNAcylation which is a post-translational modification implicated in many pathologies such as cancer. Its regulation depends on two enzymes, OGA and OGT forming the OGlcNAzyme complex. As Tax deregulates many post-translational modification machineries and as OGlcNAcylation regulates the stability and activity of transcriptional factors together with phosphorylation, we have evaluated for the first time the OGlcNAcylation status of CREB in the context of HTLV-1 infection and its implication in the regulation of the viral promoter. We have shown that in the context of HTLV-1 infection, Tax protein was deregulating the OGlcNAcylation machinery leading to an increase in CREB OGlcNAcylation which favors the transcription from the 5’LTR. Thus we have established for the first time a relationship between Tax and OGlcNAcylation allowing us to propose a new model of regulation of the viral LTR

O Vírus da influenza, apesar de todas as epidemias e pandemias referirem-se a infecções em seres humanos, não está restrita a espécie humana e é capaz de causar debilidade ou mortalidade em várias outras espécies, incluindo cavalos, suínos, mamíferos marinhos e aves, entre outros. Estudos ecológicos das viroses de influenza conduziram a hipótese que todas as que acometem mamíferos derivam de reservatórios destes vírus em aves. Mesmo com programas de monitoramento contínuo de aves silvestres em alguns países do mundo que possuem casos originados pelos vírus aviário H5N1, pouco foi feito na Antártica e por isso, o presente trabalho foi realizado nas estações de verão antártico de 2006, 2007 e 2008 em duas localidades no território Antártico, a Península Keller, localizada na Ilha Rei George e na ilha Elefante 61°08S, 55°07W, a primeira onde está situada a Estação Antártica Comandante Ferraz-EACF e a segunda onde está localizada uma base de apoio a estudos avançados. Para este estudo foi realizada a coleta de 283 amostras de quatro diferentes espécies de pinguins: Pygoscelis adeliae; P. papua; P. antarctica; Aptenodytes patagonicus. Para o diagnóstico das amostras colhidas, foi aplicada a detecção direta dos produtos amplificados pelo método de RT-PCR em gel de agarose confirmados pelo método de Real-Time PCR (Applied Biosystems) e pelo RT-PCR-GeneScan no laboratório de Virologia Clínica e Molecular, do Departamento de Microbiologia, da Universidade de São Paulo. Os resultados obtidos em nosso estudo foram 8 amostras positivas em pinguins para o vírus Influenza A. As amostras positivas por RT-PCR foram encaminhadas para o laboratório de Influenza do Department of Infectious Diseases, St. Jude Children\'s Research Hospital, Memphis, TN, USA, para isolamento em ovos embrionados, não havendo crescimento de vírus da influenza A. Quatro destas amostras positivas puderam ser sequenciadas e comparadas com sequências de Influenza A depositadas no Genbank apresentando uma identidade de 96,8 % a 100 % entre elas e o controle tendo este último uma identidade de 100% com as do banco de dados, confirmando a presença do vírus nestas aves.
Epidemics and pandemics of influenza usually refer to infections in human beings. The influenza virus is not, however, restricted to humans and can cause infirmity and death in other species including horses, swine, marine mammals, birds, and others. Ecological studies of viral infections have led to the hypothesis that the influenza viruses that attack mammals have their origin in the accumulation of these viruses in birds (avian flu). In some countries with influenza cases caused by the avian H5N1 virus, there was monitoring of wild birds but little had been done in Antarctica. The present work was therefore carried out during the Antarctic summer seasons of 2006, 2007, and 2008 in two Antarctic locations: The Commander Ferraz Antarctic Station, on the Keller Peninsula of King George Island, and at the Base of Advanced Studies located on Elephant Island (61°08S, 55°07W). Two hundred eighty-three (283) samples from four different penguin species Pygoscelis adeliae, Pygoscelis papua, Pygoscelis antarctica; and Aptenodytes patagonicus were collected for this study. Diagnoses of the samples were performed not only by application of direct detection and amplification according to the RT-PCR method in agar-gel, but also by Real-Time PCR (Applied Biosystems), and by RT-PCR gene scan at the Laboratory of Clinical and Molecular Virology of the Department of Microbiology of the University of Sao Paulo. Eight of the penguin samples tested positive for the Influenza-A virus. The positive samples, as determined by RT-PCR, were sent to the Influenza Laboratory of the Department of Infectious Diseases of the St. Jude Research Hospital in Memphis, Tennessee, USA, to be isolated in egg embryos where no further growth of the Influenza-A virus took place. Four of these positive samples could be sequenced and compared with those of Influenza-A on deposit at the Gene Bank and ranged from 96.85 to 100% when compared with the control samples (100% positive), thus confirming the presence of the virus in the tested birds.

Les deux virus d’ARN VIH et VHC attirent beaucoup d’attention de la santé publique parce qu’ils partagent des mêmes facteurs de risque de transmission par le sang et par des contacts sexuels. En plus, les maladies associées aux infections par le VIH et par le VHC sont des causes principales de mortalité et de morbidité globalement. Néanmoins, grâce à l’introduction des thérapies d’antirétroviraux pour le traitement de l’infection par le VIH et les antiviraux à action directe (AADs) pour le traitement de l’infection par le VHC, les patients infectés par ces virus constatent une amélioration significative de leur qualité de vie. Cependant, le taux de réplication élevé et l'absence de mécanisme de correction d'erreur de ces virus génèrent une population virale diversifiée appelée des quasi-espèces. Sous la pression sélective de traitement, les quasi-espèces virales sélectionnent des variants de résistance au médicament correspondant et rendent la thérapie inefficace, en particulier dans les cas où une surveillance appropriée du traitement n'est pas assurée. Afin de réserver un large choix de traitement antirétroviral tout au long de la vie chez les patients infectés par le VIH et, parallèlement, de réduire le coût de traitement des infections par le VIH et le VHC, il est fondamental de mener des recherches ciblées sur la détection, la surveillance et la transmission des mutations de résistance aux antiviraux. Dans cette thèse, nous avons utilisé les technologies de séquençage haut-débit (UDS) ou de séquençage de nouvelle génération (NGS) pour chercher des variants de résistance minoritaires (MiRVs) qui sont classiquement considérés comme représentant moins de 15%-25% de la population virale et ne sont pas trouvés par la technique de séquençage Sanger. La présence des MiRVs à baseline est responsable possiblement de l'échec de traitement et leur présence à l'échec peut limiter les options des traitements ultérieurs. Dans cette thèse, nous avons évalué la prévalence et l’impact clinique des MiRVs sur le gène de l’intégrase chez les patients infectés par le VIH qui ont été en échec d’un régime thérapeutique contenant un inhibiteur de l’intégrase. Nous avons également évalué l’impact des MiRVs chez les patients infectés par le VHC de génotype 3 et de génotype 4 et ayant échoué aux AADs. De plus, nous avons utilisé la technique UDS pour identifier et caractériser les chaines de transmission du VHC parmi une population clé d'hommes ayant des rapports sexuels avec des hommes, co-infectés par le VIH ou ayant un risque élevé d'acquisition du VIH. Nous avons également observé plusieurs cas d’infections mixtes de génotype du VHC dans cette population, probablement à cause du risque élevé de multiples expositions au VHC. Les questions sur l’avantage de l'UDS dans la recherche en virologie et l'applicabilité de cette technique en clinique ont été posées et vérifiées au travers de multiples types de projets dans cette thèse. L’UDS n’a pas été établi de manière concluante comme étant plus intéressant et bénéfique que la technique de séquençage de Sanger pour prévenir l’échec au traitement chez les patients infectés par le VIH ou le VHC et pour identifier les chaînes de transmission à grande échelle si le coût et le temps de l’expérience pour l’analyse des données sont pris en compte. Cependant, le développement dynamique des nouvelles technologies de l’UDS et les efforts sans cesse afin d’optimiser des procédures d'analyse montrent un rôle prometteur de l’UDS. Et l'applicabilité de l'UDS en pratique clinique devra encore être élucidée dans différents types de projets de recherche
The two RNA viruses HIV and HCV are getting a lot of public health concerns because both of them have overlapping risk factors for transmission through direct blood and sexual contacts. Furthermore, HIV and HCV infections are the leading cause of mortality and morbidity globally due to related diseases. However, with the introduction of antiretroviral therapy (ART) for the treatment of HIV infection and direct-acting antivirals (DAAs) for the treatment of HCV infection, patients infected by these viruses are witnessing significant improvement in their quality of life. However, the high replication rate and the lack of error correction mechanism of these viruses result in a diverse viral population referred to as quasispecies. Under drug- selective pressure, the viral quasispecies select resistance variants against corresponding drug and render the therapy ineffective especially in cases an appropriate treatment monitoring is not ensured.To reserve a wide range of possibilities for a life-long ART in HIV-infected patients and in parallel to reduce cost for treatment of both HIV and HCV infection, research focusing on detection, surveillance and transmission of resistance mutations is fundamental to prevent treatment failure on antivirals. In this PhD, we employed the ultra-deep sequencing (UDS) or next-generation sequencing (NGS) technologies to look for minority resistant variants (MiRVs) which are conventionally considered to represent less than 15%-25% of viral population and undetectable by Sanger sequencing. The presence of MiRVs at baseline is possibly responsible for the treatment failure and their presence at failure may limit options for subsequent therapies. In this PhD, we evaluated the prevalence and clinical impact of MiRVs on integrase gene in HIV-infected patients failing an integrase inhibitor containing regimen. We also evaluated the impact of MiRVs in HCV genotype 3 and genotype 4-infected patients failing DAAs. Furthermore, we used the UDS technique to identify and characterize the HCV transmission networks among a key population of men having sex with men either co-infected with HIV or at high risk of HIV acquisition. We also discovered several cases of mixed HCV genotype infections in this population probably for their high risk of multiple HCV exposures. The advantages of UDS in virology research and the applicability of this technique in clinic have been questioned and verified throughout multiple types of projects in this PhD. UDS has not been conclusively established to be more interesting and beneficial than Sanger sequencing in prevention of treatment failure in patients infected by HIV or HCV and in identifying the viral transmission networks at large scale if taking into account the experiment cost and time for data analysis. However, the dynamic development of UDS technologies and the continuing attempts in optimizing analysis procedures display a promising role of UDS. And the applicability of UDS in clinical practice still needs to be elucidated in different kinds of research projects

Orientador: João Manuel Grisi Candeias
Resumo: Não disponível.
Abstract: From about 200 virus that cause respiratory infections, only 8 are responsible for severe illness in children, immunodeficient adults and elderly, inc1uding Adenovirus, Influenza A and B, Parainfluenza 1, 2 and 3, Human RespÍratory Syncytial Vírus (HRSV) and Human Metapneumovirus (hMPV). Three of these virus are responsible for a significant morbidity in elderly: Influenza, HRSV and hMPV. The nosocomial infection caused by these vÍruses can be fatal in hospitalized children and patients with other pathologies. With the advance of the of molecular biology techniques, the diagnosis and the characterization of these vírus became more effective. Beside vírus detection by PCR, isolation of vírus in specific cellular cultures to increase the amount of pathogens have been used in severallaboratories. Studies on prevalence of respÍratory vírus in elderly are rare. The objective of this research was to evaluate the occurrence of Influenza, HRSV and of the hMPV and the risk factors involved in the diseases caused by these vÍruses. Nasopharyngeal swabs were collected and used for inoculation in cells culture and dÍrect analysis by RT-PCR. The results from RT-PCR of Influenza Vírus and RSV were negative. We also tested the samples with GeneScan, that is a technique based on fluorescent primers specific to the studied vírus. Results for Flu virus and HRSV were the same ofRT-PCR, but 1 MPV sample was positive. 55,32% ofthe 47 studied elderly were vaccinated against Influenza; 14,9% had tabagism habits and 70,2% were women. These risk factors might had influenced in the absence of positive samples for the Influenza vírus andHRSV.
Mestre

En esta tesis se aborda la identificación de factores del pulgón Myzus persicae que podrían participar en el proceso de transmisión del potyvirus del grabado del tabaco, Tobacco etch virus (TEV), y se explora la posibilidad de alterar la expresión de genes particulares en el insecto vector como una manera de dificultar o impedir la diseminación de este virus. Se conoce la intervención de un factor auxiliar de origen viral, la proteína HCPro, en la transmisión de potyvirus, actuando como un puente molecular reversible para retener las partículas de virus en el aparato bucal del pulgón. La proteína HCPro interacciona con la proteína de la cápside viral, CP, y previsiblemente con receptores específicos en el pulgón. Después de analizar un conjunto de productos capaces de interaccionar con la proteína HCPro de TEV (interactoma), se han considerado dos proteínas candidatas. La primera, MpRPS2, presenta homología con proteínas ribosomales, y la segunda MpRR1Cp2 es una proteína cuticular. La interacción entre HCPro y MpRPS2 se ha verificado en ensayos Far Western Blot y en el sistema de doble híbrido de levaduras. Los intentos para confirmar la localización de esta proteína en estiletes diseccionados de pulgón no fueron concluyentes, aunque con un antisuero específico sí se ha podido detectar la presencia de MpRPS2 en la cutícula de mudas de pulgón aisladas. Para validar la participación de estos hipotéticos receptores en el proceso de transmisión viral, se han explorado estrategias de interferencia con la expresión génica de MpRPS2 y MpRR1Cp2 en pulgones. Tras analizar sus niveles de expresión durante a lo largo del desarrollo, se consideraron dos sistemas para inducir respuestas de silenciamiento (RNAi) basados en la alimentación. El uso de dietas artificiales suplementadas con dobles cadenas de RNA específicas sintetizadas in vitro no produjo en general reducciones de la acumulación de RNA mensajeros, mientras que la alimentación de pulgones sobre plantas infectadas con un vector viral basado en el virus del cascabeleo del tabaco, Tobacco rattle virus (TRV), que contiene un fragmento de la secuencia del gen, tuvo un efecto más pronunciado. Las reducciones de expresión observadas fueron variables entre los dos genes, con el efecto más fuerte en el caso del gen MpRR1Cp2, en pulgones alimentados en plantas de tabaco infectadas con una variante de TRV que incorporaba un fragmento de este gen. Este sistema de RNAi nos ha permitido ensayar el efecto de la reducción de la expresión de los dos candidatos seleccionados en experimentos de transmisión de TEV. Estos resultados podrían ayudar a mejorar la comprensión de las interacciones moleculares necesarias durante la transmisión, a identificar factores en el vector que participan en el proceso, y eventualmente podrían usarse para el diseño de estrategias innovadoras de bloqueo de la diseminación viral basadas en la interferencia de la expresión de dichos factores.
This thesis addresses the identification of Myzus persicae aphid factors that could be involved in the transmission process of Tobacco etch virus (TEV), a potyvirus, and explores the possibility of altering the expression of specific genes in the insect vector as a way to prevent virus spread. An auxiliary factor of viral origin, the HCPro protein is known to participate in potyvirus transmission. HCPro acts as a reversible molecular bridge retaining virus particles in aphid mouthparts. HCPro interacts with the viral coat protein, CP, and predictably with specific receptors in aphid mouthparts. After analyzing a set of products able to interact with the TEV HCPro (interactome), we have considered two candidate proteins. The first one MpRPS2, shows homology with ribosomal proteins, and the second one, MpRR1Cp2 is a cuticular protein. The interaction between HCPro and MpRPS2 has been verified in Far Western Blot assays and in yeast two-hybrids. Attempts to confirm the localization of this protein in dissected aphid stylets were not conclusive, although using an specific antiserum it has been possible to detect the presence of the MpRPS2 in the cuticle of isolated aphid moults. To confirm the involvement of these hypothetical receptors in the viral transmission process, strategies of interference with expression of MpRR1Cp2 and MpRPS2 have been explored in aphids. After analyzing their expression levels along development, two systems based on feeding were considered to induce silencing responses (RNAi). The use of artificial diets supplemented with specific in vitro synthesized dsRNA did not produced reductions in mRNA accumulation, while aphids fed on plants infected with a viral vector based on Tobacco rattle virus (TRV), which contains a fragment of the targeted genes, showed more clear effects. The observed reductions in expression were rather variable between the two genes considered, with a stronger effect in the case of MpRR1Cp2 in aphids fed on tobacco plants infected with a TRV variant that incorporates a fragment of this gene. This RNAi system allowed us to test the effect of knocking down the expression of the two selected candidates in TEV transmission experiments. Our results might help to improve our understanding of the molecular interactions during transmission, to identify factors in the vector that participate in the process, and eventually could serve to design new strategies to interfere with viral dissemination based on specifically interfering with their expression.

This thesis is devoted to the modeling of malicious behaviors inside malevolent codes, commonly called malware. The thesis work follows two directions, one operational, one theoretical. The objective is to eventually combine these two approaches in order to elaborate detection methods covering most of existing malware, while offering formal security guarantees against appearing ones. The operational approach introduces an abstract behavioral language, independent from implementation. The language itself relies on the attribute-grammar formalism, capable of expressing the behavior semantics. Within the language, several behavior descriptions are specified in order to build a multi-layered detection method based on parsing. Its deployment has shown satisfying results in terms of coverage. On the basis of the same language, some techniques of behavioral mutation have been formalized using compilation techniques. These mutations have proved themselves interesting tools for the evaluation of antiviral products. The theoretical approach introduces a formal viral model, no longer based on functional paradigms, but on process algebras. This new model enables the description of self-replication as well as other more complex behaviors based on interactions. It supports the redemonstration of fundamental results such as the detection undecidability or the prevention by isolation. Moreover, the model supports the formalization of several existing techniques of behavioral detection, thus allowing the formal evaluation of their resilience
Cette thèse s'intéresse à la modélisation des comportements malicieux au sein des codes malveillants, communément appelés malwares. Les travaux de thèse s'articulent selon deux directions, l'une opérationnelle, l'autre théorique. L'objectif à terme est de combiner ces deux approches afin d'élaborer des méthodes de détection comportementales couvrant la majorité des malwares existants, tout en offrant des garanties formelles de sécurité contre ceux susceptibles d'apparaître. L'approche opérationnelle introduit un langage comportemental abstrait, décorrélé de l'implémentation. Le langage en lui-même repose sur le formalisme des grammaires attribuées permettant d'exprimer la sémantique des comportements. A l'intérieur du langage, plusieurs descriptions de comportements malicieux sont spécifiées afin de construire une méthode de détection multicouche basée sur le parsing. Sur la base de ce même langage, des techniques de mutation comportementale sont également formalisées à l'aide de techniques de compilation. Ces mutations se révèlent un outil intéressant pour l'évaluation de produits antivirus. L'approche théorique introduit un nouveau modèle viral formel, non plus basé sur les paradigmes fonctionnels, mais sur les algèbres de processus. Ce nouveau modèle permet la description de l'auto-réplication ainsi que d'autres comportements plus complexes, basés sur les interactions. Il supporte la redémonstration de résultats fondamentaux tels que l'indécidabilité de la détection et la prévention par isolation. En outre, le modèle supporte la formalisation de plusieurs techniques existantes de détection comportementale, permettant ainsi d'évaluer formellement leur résistance

La terapia antirretroviral ha cambiado la perspectiva sobre la infección por VIH-1 de enfermedad letal a crónica. Aun así, el reservorio latente del VIH-1 es una de las mayores barreras para lograr una cura. La estrategia “shock and kill” se basa en inducir la transcripción viral del provirus latente del VIH-1 seguido de la muerte selectiva de las células reactivadas. Aunque muchos agentes reversores de la latencia (LRAs) han sido identificados y testados, ninguno ha logrado erradicar eficazmente dicho reservorio. Dada la necesidad de nuevos agentes y estrategias capaces de eliminar el reservorio latente, hemos propuesto moduladores de la respuesta inmune innata o del ciclo celular para dicho fin. El estudio de moduladores de la inmunidad innata puede representar una alternativa dadas sus funciones intrínsecas, i. e., protección y eliminación de infecciones. Acitretina, regulador de la inmunidad innata aprobado contra la psoriasis, ha sido propuesto como inductor de la reactivación del VIH-1 y la muerte de las células infectadas. A pesar de ello, el efecto de acitretina en la reactivación fue muy modesto en la mayoría de modelos celulares que testamos, aunque se detectó la activación de la vía de RIG-I, y una ligera inducción de la reactivación viral en un modelo no clonal de células T. Además, acitretina tampoco promovió la eliminación de las células infectadas. Los compuestos anti-cáncer también han sido propuestos como estrategia contra el reservorio, debido a la habilidad de ciertos agentes para modificar la transcripción génica o promover la apoptosis. El cribado de una librería de compuestos anti-cancer reportó varias dianas cuya inhibición reactivó el VIH-1, incluyendo histona deacetilasas (HDACs), Janus quinasas (JAKs), IκB quinasas (IKKs) y proteínas “heat-shock” (HSPs). Entre los nuevos LRAs identificados, los inhibidores de Aurora kinasas (AURKi) representaron la mayor familia de compuestos, no descritos como LRAs, que mostraron capacidad de reactivación del VIH-1 de forma significativa y consistente. Los AURKi mejoraron la reactivación mediada por los HDACi, sugiriendo la habilidad para reactivar distintos provirus insertados. Curiosamente, AURKi restringió la replicación aguda del VIH-1, insinuando un papel dual para dichos compuestos en la infección por VIH-1. Midostaurin, un inhibidor de multi-quinasas aprobado contra la leucemia, también se identificó como LRA. Midostaurin reactivó el VIH-1, tanto por si solo como en combinación con otros LRAs, corroborando previos reportes que asociaron esa actividad con la activación de la vía de NF-κB. Además, también se observó una inhibición de la infección aguda del VIH-1 en células primarias dependiente de SAMHD1 no descrita. El hecho de que los AURKi y midostaurina mejoren la reactivación del VIH-1 en combinación con otros LRAs, corrobora la idea de que distintos compuestos pueden ser necesarios para reactivar todos los provirus integrados, presentando así distintas especificidades para la reactivación del provirus que dependan de su lugar de integración en el genoma. Estas observaciones plantean dudas sobre los modelos usados para estudiar la latencia del VIH-1, pues los modelos clonales podrían ser inadecuados por la falta de heterogeneidad de lugares de integración. En conjunto, nuestros resultados sugieren que la modulación de la inmunidad innata y ciclo celular podrían incluirse en el desarrollo de futuros LRAs para la estrategia “shock and kill”; aun así, investigaciones adicionales siguen siendo necesarias con tal de avanzar hacia la cura del VIH-1.
Current antiretroviral therapy has changed the perspective of HIV-1 infection from a lethal illness to a chronic disease. However, the HIV-1 latent reservoir is a major hurdle to achieve a cure for HIV-1. The “shock and kill” strategy is based on inducing viral transcription of latent HIV-1 provirus followed by the selective killing of reactivated cells. Although several latency-reversing agents (LRAs) have been identified and tested, none of them has been able to efficiently eradicate the HIV-1 latent reservoir. Based on the need of novel agents and strategies to efficiently clear the latent reservoir, we evaluated compounds developed as modulators of the innate immune response or designed to modulate the cell cycle progression as novel agents able to purge the viral reservoir. The study of innate immune modulators as agents able to clear the HIV-1 reservoir might represent an alternative due to its intrinsic functions, i. e., protection and clearance of infections. The innate immune regulator acitretin, an FDA-approved compound for psoriasis, has been proposed to induce HIV-1 reactivation and selective killing of the infected cells. However, the effect of acitretin on HIV-1 reactivation was negligible in the vast majority of models tested, albeit activation of RIG-I pathway was detected and a mild induction of viral reactivation was observed in a non-clonal T cell model. Moreover, acitretin treatment did not induce the selective killing of the infected cells. Anti-cancer compounds have also been proposed as candidate therapies targeting the latent reservoir, mainly due to the ability of certain agents to modify gene transcription or to promote cell apoptosis. The assessment of the HIV-1 reactivation potential of an anti-cancer compound library reported several molecular targets whose inhibition promoted HIV-1 latency reversal, including the histone deacetylases (HDAC), Janus kinases (JAK), IκB kinases (IKKs) and heat shock proteins (HSPs). Among the new identified LRAs, Aurora kinases inhibitors (AURKi) represented the largest family of compounds not previously described as LRA that significantly and consistently showed HIV-1 reactivation capacity. AURKi were able to enhance the HDACi-mediated reactivation, suggesting that AURKi are able to target a distinct set of integrated provirus than that reactivated by the well-described HDAC inhibitors. Interestingly, AURKi restricted acute HIV-1 infection, suggesting a dual role for these compounds on HIV-1 infection. Midostaurin, a multi-kinase inhibitor approved for leukemia treatment, was also identified as an LRA. Midostaurin induced HIV-1 latency reactivation, either alone or in combination with other LRAs, consistent with previous reports that associated this activity with the activation of the innate immune NF-κB pathway. Moreover, we also observed a non-yet-reported and SAMHD1-dependent inhibitory effect of HIV-1 replication in primary cells. The enhanced capacity to promote HIV-1 reactivation of AURKi and midostaurin in combination with other LRAs supports the idea that different agents are needed to reactivate all latent provirus, presenting different specificities towards HIV-1 provirus reactivation depending on its integration site in the host genome. Furthermore, these observations also raise concerns on the models used to study HIV-1 latency, as clonal models might not be suitable due to the lack of heterogeneity in proviral insertion site, characteristic of non-clonal models. Altogether, our results suggest that modulation of innate immunity and cell cycle may be taken into account for the design of future LRAs for the “shock and kill” strategy; however, further research is still necessary before it can lead to an HIV-1 cure.

La protéine cellulaire BST2/Tetherin est un médiateur de l’immunité innée qui exerce son activité antivirale contre la dissémination de nombreux virus enveloppés. Ce facteur de restriction retient physiquement les virus néoformés à la surface cellulaire de la cellule infectée, empêchant ainsi le relâchement des virions du VIH-1. La protéine Vpu du VIH-1 est l’un des acteurs viraux capable de contrer cette restriction : elle diminue le niveau d’expression de BST2/Tetherin présent au site de bourgeonnement viral et restaure ainsi une production virale efficace. A ce jour, les mécanismes exacts par lesquels Vpu contre cette barrière cellulaire ne sont pas bien caractérisés. Des études récentes ont montré que HRS et Rab7A, deux protéines contrôlant la voie endo/lysosomale tardive sont impliquées dans le mécanisme déployé par Vpu pour contrer la restriction imposée par BST2. De manière intéressante, ces protéines sont également requises pour l’achèvement des étapes finales de l’autophagie. L’autophagie (macroautophagie) est un mécanisme cellulaire hautement conservé, qui permet la dégradation de composants cytoplasmiques via la formation d’une vésicule à double membrane, appelé autophagosome. L’autophagie est contrôlée par une trentaine de protéines ATG (AuTophaGy-related genes) qui sont recrutés au site de formation du phagophore qui conduira, suite à son élongation, à la formation de l’autophagosome. Il a récemment été décrit que certaine protéines ATG sont également impliquées dans d’autres fonctions cellulaires indépendante de l’autophagie, comme la résistance contre certains pathogènes. C’est le cas notamment de la phagocytose médiée par LC3 (LC3-associated phagocytosis, LAP), un mécanisme indépendant du complexe de pré-initiation de l’autophagie au cours duquel certaines protéines ATG modifient la membrane du phagosome et accélèrent la dégradation des éléments phagocytés. L’objectif de ma thèse était donc de définir si l’autophagie ou certaines protéines de l’autophagie sont impliquées dans le mécanisme moléculaire déployée par Vpu pour contrer la restriction imposée par BST2/Tetherin sur la production virale. Au cours de ma thèse, nous avons démontré l’implication de la protéine LC3C dans le mécanisme par lequel Vpu contrecarre la restriction imposée par BST2 sur la libération des particules virales du VIH-1 dans le milieu extracellulaire. Plus précisément, nos résultats montrent que les protéines ATG5 et Bécline 1, et non tous les composants de l’autophagie, agissent avec la protéine LC3C pour faciliter l’action de la protéine virale Vpu sur la restriction imposée par BST2. L’expression de la protéine LC3C favorise l’élimination par Vpu des molécules de BST2 présentes au site du bourgeonnement, permettant ainsi une libération plus efficace de particules virales VIH-1. Nos expériences d’immunofluorescence montrent que les protéines BST2 et Vpu sont présentes dans des compartiments marqués par LC3, protéine décorant les phagophores, autophagosomes et phagosomes impliqués dans la LAP. Enfin, nos données biochimiques révèlent une interaction spécifique et directe entre la protéine Vpu et la protéine LC3C via un motif LIR non-canonique. L’intégrité de ce motif présent dans le domaine cytoplasmique de Vpu est nécessaire pour la levée par Vpu de la restriction imposée par BST2. En conclusion, mes travaux de thèse montrent que la protéine Vpu du VIH-1 via son interaction avec la protéine d’autophagie LC3C utilise un mécanisme de LAP pour contrer la restriction induite par BST2 sur la production de virus VIH-1
BST2/Tetherin is a key mediator of the innate immune system that restricts the dissemination of enveloped viruses. This restriction factor impedes the release of de novo formed HIV particles by physically retaining them at the surface of infected cells. The HIV-1 protein Vpu promotes the release of virus by counteracting this restriction. Vpu removes BST2 present at the budding site and downregulates BST2. The mechanisms by which Vpu counteracts BST2 are still not well understood. Recently, we showed that HRS and Rab7A, two regulators of the endocytic and autophagic pathway participates to the mechanism by which Vpu counteracts BST2-mediated restriction on HIV-1 release. Interestingly, these two proteins are also required in the autophagy pathway. Autophagy (macroautophagy) is a highly conserved degradative mechanism that leads to degradation of cytosolic components through the formation of double-membrane vacuoles called autophagosomes that sequester cytosolic material. This process is tightly regulated by the ATG proteins that are hierarchically recruited at the phagophore assembly site to form the autophagosome. Some ATG proteins are additionally involved in non autophagic cell functions involved in maintenance of cell homeostasis and resistance of pathogens. Notably, they participate in microbe clearance through LC3-associated phagocytosis, a process independent of autophagic preinitiation complex in which some ATG proteins directly modify the phagosomal membrane to enhance degradation of phagocytosed elements. The aim of my thesis was to explore if the autophagy pathway or some ATG proteins could be involved in the molecular mechanism by which Vpu counteracts BST2/Tetherin on HIV-1 release. Here, we reveal that the protein LC3C is required in the Vpu-induced antagonism of BST2 restriction. Our results show that only ATG5 and Beclin-1, and not all the components of the autophagy pathway, act with LC3C to favor the counteraction of Vpu on BST2 restriction, and thus enhance HIV-1 release. We report that BST2 and Vpu are present in LC3-positive compartments. We found that Vpu selectively interacts with the ATG8 ortholog, LC3C, through a non-canonical LIR motif by immunoprecipitation and GST pulldown assays. This motif is required for Vpu to antagonize BST2 restriction. LC3C expression favors the removal of BST2 from HIV-1 budding site, and thus HIV-1 release in BST2 expressing cells. Altogether, our data support the view that Vpu uses a non-canonical autophagy pathway reminiscent of LC3-associated phagocytosis to counteract BST2 restriction

Several viruses from different virus families are known to cause ocular infections, e.g. members of the Adenoviridae, Picornaviridae and the Herpesviridae families. These infections are spread by contact and in the case of adenoviruses (Ads) and picornaviruses they are also highly contagious. The ocular infections caused by Ads and picornaviruses are called epidemic keratoconjunctivitis (EKC) and acute hemorrhagic conjunctivitis (AHC), respectively. Historically, EKC is caused mainly by three types of Ads from species D: Ad8, Ad19 and Ad37. The infection is characterized by keratitis and conjunctivitis but also involves pain, edema, lacrimation and blurred vision. AHC is caused mainly by two types of picornaviruses: coxsackievirus A24v (CVA24v) and enterovirus 70 (EV70), and is characterized by pain, redness, excessive tearing, swelling and subconjunctival hemorrhages. In addition, blurred vision, keratitis, malaise, myalgia, fever, headache and upper respiratory tract symptoms can also be experienced. Both infections are problematic in many parts of the world, affecting millions of people every year. Despite the great need, the only treatment available today is supportive treatment; no antiviral drugs are available to combat these common viral infections. Ad37 has previously been reported to use sialic acid (SA) as its cellular receptor. Since there is no antiviral treatment available against EKC we wanted to evaluate the inhibitory effect of SA-based antiviral compounds on Ad37 binding to and infection of ocular cells. We found that multivalent compounds consisting of SA linked to a globular carrier molecule, in this case human serum albumin, efficiently blocked Ad37 binding and infection at low concentrations. Further attempts were then made to improve the effect by chemically modifying SA monosaccharides. However, no enhanced inhibitory effect was accomplished and the conclusion was that the best inhibitors are based on unmodified SA. We next hypothesized that development of efficient SA-based binding inhibitors may require detailed knowledge about the structure of the SA-containing receptor. Using a battery of biological and biochemical experiments, including glycan array, binding and infection assays, X-ray crystallography and surface plasmon resonance (SPR); we identified a specific glycan involved in the binding and infection of Ad37. This glycan turned out to be a branched, di-SA-containing motif corresponding to the glycan motif of the ganglioside GD1a. However, the ganglioside itself did not function as a cellular receptor, as shown by a number of binding and infection assays. Instead, the receptor consisted of one or more glycoproteins that contain the GD1a glycan motif. This glycan docked with both its SAs into the trimeric Ad37 knob resulting in a very strong interaction as compared to most other protein-glycan interactions. Hopefully, this finding will aid development of more potent inhibitors of Ad37 binding and infection. The receptor for CVA24v, one of the main causative agents of AHC, has been unknown until now. We showed that this ocular virus, like Ad37, is also able to use SA as a receptor on corneal cells but not on conjunctival cells. This suggested that CVA24v may use two different receptors. As for Ad37, the receptor used by CVA24v on corneal cells also appears to be one or more sialic acid-containing glycoproteins. We believe that these findings may be a starting point for design and development of candidate drugs for topical treatment of AHC.

The innate type I interferon antiviral response is the first line of defence invoked to limit the spread of viral infections. Hence a number of viruses including Kaposi's sarcoma-associated herpesvirus (KSHV) have evolved defence strategies against this antiviral response. KSHV is the aetiologic agent of KS and almost one quarter of the KSHV genes specify either demonstrated or potential immunomodulatory activity including the four viral interferon regulatory factors (vIRFs). vIRFs 1, 2 and 3 have previously been shown to inhibit type I IFN signalling, whereas the inhibitory role of vIRF4 is yet to be reported. A previous stable isotope labelling of amino acids in cell culture (SILAC) study coupled to LC-MS/MS identified USP7 and ribosomal proteins as binding partners of both vIRF2 and vIRF4. The aim of the present study was to investigate the role of these binding partners in type I IFN signalling and to determine the regulatory mechanisms behind the effects of these partner proteins on the functions of the vIRF2 and vIRF4 proteins. Polysome profiling and microarray studies were carried out on vIRF4 expressing cells and suggested a novel regulatory role for vIRF4 in translation. USP7 was also characterised as a positive regulator of IFN signalling and the mechanism behind this effect was explored.

Includes bibliographical references.
This study confirms the widespread existence of highly divergent HIV lineages in Cameroon. While the genetic complexity of the Cameroonian HIV-1 epidemic has potentially serious implications for the design of biomedical interventions, detailed analyses of divergent Cameroonian HIV-1 group M lineages could be crucial for dissecting the earliest evolutionary steps in the emergence of HIV-1 group M. In addition, the central nature of HIV-1 consensus M sequences resulted in their broad recognition, but failed to identify highly immunodominant peptides between homogeneous and diverse HIV epidemics. Further refinement of these immunogens may contribute to the development of a globally relevant vaccine. Finally, the use of PTE peptides did not increase the breadth of T cell recognition in Abstract Page xvi this divergent population when compared to consensus M peptides. This underlies the need to include more mosaic peptides representing the variety of viruses that circulate in the region.

Non-crystallographic symmetries are ubiquitous in physics, chemistry and biology. Prominent examples are quasicrystals, alloys with long-range order but no translational periodicity in their atomic organisation, and viruses, micro-organisms consisting of a protein shell, the capsid, that in most cases displays icosahedral symmetry. Group theory plays a crucial role in understanding their structures and their physical and geometrical properties. In this thesis new group theoretical methods are introduced, to characterise virus organisation and model structural transitions of icosahedral quasicrystals. It is shown that these problems can be described via the embedding of non-crystallographic groups into the point group of higher dimensional lattices. Indeed, the analysis of orbits of such embeddings, akin to the construction of quasicrystals via the cut-and-project method, provides a rigorous mathematical construction of finite nested point sets with non-crystallographic symmetry at each distinct radial level. In the case of icosahedral symmetry, it is shown that the point arrays thus obtained can be used to provide constraints on the geometry of viral capsids, encoding information on the organisation of the capsid proteins and the genomic material collectively. Moreover, structural transitions of icosahedral quasicrystals can be analysed in a group theoretical framework through continuous rotations in the higher dimensional space connecting distinct copies of the embedded icosahedral group, sharing a common maximal subgroup. These rotations induce in projection continuous transformations between aperiodic point sets that preserve the symmetry described by the common subgroup. Theoretical methods as well as applications are discussed, with emphasis on the computational and geometric aspect of group theory.

En el primer estudi de la present tesi, es van estudiar brots de malaltia respiratòria compatible amb virus de la influença de tipus A (IAV) així com granges que no mostraven simptomatologia clínica. Per a l'estudi dels brots, es van recollir mostres de hisops nasals d'animals amb signes respiratoris i febre (≥40 ° C), mentre que a les granges sense simptomatologia clínica, es van recollir hisops nasals de garrins de maternitat, transició i porcs d'engreix (20 per grup). Es va estudiar un total de 211 brots i 19 granges aparentment subclíniques. La presència i llinatge es van determinar per RT-qPCR, i es va fer l'aïllament de mostres seleccionades usant cèl·lules MDCK. Els aïllats van ser seqüenciats (genoma complet) mitjançant la tecnologia Illumina Miseq. Es va confirmar la presència de IAV en 145 casos de brots (68.7%), i en 15 granges aparentment subclíniques (78.9%). Els llinatges majorment detectats van ser H1avN2hu (33.6%), H1avN1av (24.3%) i H1huN2hu (18.7%). Es va obtenir un total de 60 aïllats, i els seus genomes van ser completament seqüenciats. Els genotips majoritàriament detectats van ser el tipus D i l'A, que es corresponen als llinatges H1avN2hu i H1avN1av, respectivament. Es van detectar un total de 14 genotips diferents, dels quals, 7 d'ells no havien estat prèviament reportados.En el segon estudi de la present tesi, es va estudiar la dinàmica de transmissió de IAV en les transicions d'una granja endèmica abans i després de l'aplicació de diferents esquemes de vacunació a les truges. Es van realitzar un total de tres estudis longitudinals: abans de la vacunació, després de la vacunació amb una vacuna comercial polivalent inactivada H1N1-H1N2-H3N2 i després de la vacunació amb una vacuna comercial monovalent pandèmica H1N1. Es van recollir mostres setmanals de hisops nasals dels garrins des de les 3-9 setmanes de vida, i mostres de sang a les 3, 6 i 9 setmanes de vida. En el primer longitudinal abans de la vacunació, es va avaluar la circulació vírica basal en 50 garrins de 4 lots consecutius. En el segon longitudinal, es va realitzar vacunació en llençol de truges usant la vacuna comercial polivalent (grup control) i la meitat d'aquestes van ser revacunades 3 setmanes abans de el part (grup tractament). Es va seleccionar un grup aleatori de 10 truges de cada grup i es va fer el seguiment setmanal de 5 garrins per truja. L'estudi va ser repetit en 4 lots consecutius. En el tercer estudi longitudinal, el procediment va ser el mateix que en l'anterior, però fent servir la vacuna inactivada pandèmica H1N1. Hisops nasals van ser examinats per RT-qPCR i els sèrums van ser analitzats utilitzant un ELISA comercial (CIVTEST ©-Suis Influenza). En el segon longitudinal després de l'aplicació de la primera vacuna, l'inici de la infecció es va retardar en dues setmanes, però no es van observar diferències significatives entre els dos grups; i en el tercer, l'inici de la infecció es va moure cap a l'esquerra en tots els grups, sense diferències significatives entre ells. En els tres estudis, es van detectar animals que excretaron virus en dos o fins a en tres mostrejos consecutius, així com alguns casos de re-infeccions. El llinatge present a la granja durant els dos primers estudis longitudinals es correspon a un H1avN1av. No obstant això, durant el tercer estudi, es va detectar circulant en tots els grups d'animals 1 H3huN2hu que portava un nou llinatge H3 humà derivat d'un virus de la grip estacional humana.
En el primer estudio de la presente tesis, se estudiaron brotes de enfermedad respiratoria compatible con virus de la influenza de tipo A (IAV) así como granjas que no mostraban sintomatología clínica. Para el estudio de los brotes, se recogieron muestras de hisopos nasales de animales con signos respiratorios y fiebre (≥40°C), mientras que en las granjas sin sintomatología clínica, se recogieron hisopos nasales de lechones de maternidad, transición y cerdos de engorde (20 por grupo). Se estudió un total de 211 brotes y 19 granjas aparentemente subclínicas. La presencia y linaje se determinaron por RT-qPCR, y se hizo el aislamiento de muestras seleccionadas usando células MDCK. Los aislados fueron secuenciados (genoma completo) mediante la tecnología Illumina Miseq. Se confirmó la presencia de IAV en 145 casos de brotes (68.7%), y en 15 granjas aparentemente subclínicas (78.9%). Los linajes mayormente detectados fueron H1avN2hu (33.6%), H1avN1av (24.3%) y H1huN2hu (18.7%). Se obtuvo un total de 60 aislados, y sus genomas fueron completamente secuenciados. Los genotipos mayoritariamente detectados fueron el tipo D y el A, que se corresponden a los linajes H1avN2hu y H1avN1av, respectivamente. Se detectaron un total de 14 genotipos diferentes, de los cuales, 7 de ellos no habían sido previamente reportados.En el segundo estudio de la presente tesis, se estudió la dinámica de transmisión de IAV en las transiciones de una granja endémica antes y después de la aplicación de diferentes esquemas de vacunación en las cerdas. Se realizaron un total de tres estudios longitudinales: antes de la vacunación, después de la vacunación con una vacuna comercial polivalente inactivada H1N1-H1N2-H3N2 y después de la vacunación con una vacuna comercial monovalente pandémica H1N1. Se recogieron muestras semanales de hisopos nasales de los lechones desde las 3-9 semanas de vida, y muestras de sangre a las 3, 6 y 9 semanas de vida. En el primer longitudinal antes de la vacunación, se evaluó la circulación vírica basal en 50 lechones de 4 lotes consecutivos. En el segundo longitudinal, se realizó vacunación en sábana de cerdas usando la vacuna comercial polivalente (grupo control) y la mitad de estas fueron revacunadas 3 semanas antes del parto (grupo tratamiento). Se seleccionó un grupo aleatorio de 10 cerdas de cada grupo y se hizo el seguimiento semanal de 5 lechones por cerda. El estudio fue repetido en 4 lotes consecutivos. En el tercer estudio longitudinal, el procedimiento fue el mismo que en el anterior, pero usando la vacuna inactivada pandémica H1N1. Hisopos nasales fueron examinados por RT-qPCR y los sueros fueron analizados usando un ELISA comercial (Civtest-Suis Influenza). En el segundo longitudinal después de la aplicación de la primera vacuna, el inicio de la infección se retrasó en dos semanas, pero no se observaron diferencias significativas entre ambos grupos; y en el tercero, el inicio de la infección se movió hacia la izquierda en todos los grupos, sin diferencias significativas entre ellos. En los tres estudios, se detectaron animales que excretaron virus en dos o hasta en tres muestreos consecutivos, así como algunos casos de re-infecciones. El linaje presente en la granja durante los dos primeros estudios longitudinales se corresponde a un H1avN1av. Sin embargo, durante el tercer estudio, se detectó circulando en todos los grupos de animales un H3huN2hu que llevaba un nuevo linaje de H3 humano derivado de un virus de la gripe estacional humana.
In the first study of the present thesis, we investigated outbreaks of respiratory disease (n=211) compatible with influenza A virus (IAV) as well as farms without overt respiratory disease (n=19) for the presence of IAV. In the outbreak investigations, nasal swabs were taken from animals with respiratory signs and fever (≥40°C) while in the farms with no evident respiratory disease, nasal swabs were randomly taken from suckling piglets, weaners and fatteners (20 animals per phase). Presence of IAV and lineage determination were assessed by RT-qPCR and isolation was attempted in selected samples using MDCK cells. Isolates were sequenced (full genome) by using Illumina Miseq technology. IAV participation was confirmed in 145 (68.7%) of the outbreaks, and in 15 (78.9%) of the farms without overt disease. The most commonly detected lineages were H1avN2hu (33.6%), H1avN1av (24.3%) and H1huN2hu (18.7%). Sixty IAV isolates were obtained and the genomes were fully sequenced. Genotypes D and A, H1avN2hu and H1avN1av, respectively, were predominant but up to 14 genotypes were identified, of which seven had not been previously reported. Four isolates containing a new H3hu lineage derived from a human seasonal virus were detected, and isolates containing genes from the pandemic virus represented a 31.7 % of the total. In the second study of the present thesis, the transmission dynamics of IAV in the nurseries from an endemic farm were assessed before and after the application of different vaccination schemes for sows. Three follow-up periods were examined: before vaccination, after vaccination with a commercial inactivated polyvalent H1N1-H1N2-H3N2 and after vaccination with a monovalent pandemic H1N1. Nasal swabs of piglets were taken weekly from 3-9 weeks of age and blood samples were taken at three, six and nine weeks of age. In the first follow-up before vaccination, the basal IAV circulation was assessed by sampling 50 piglets in 4 batches. In the second longitudinal study, sows were blanket vaccinated with the polyvalent vaccine (control group) and half of them received an extra dose 3 weeks pre-farrowing (treatment group). A random cohort of 10 sows in each group was selected and 5 piglets per sow were weekly followed. The trial was replicated in 4 consecutive batches. In the third follow-up period, the procedure was the same as in the second, but using a pandemic H1N1 inactivated vaccine. Nasal swabs were examined by RT-qPCR and serum samples were analysed using a commercial ELISA (Civtest-Suis Influenza). Incidences and beta values per week and pen were calculated after the RT-qPCR results. Before applying any vaccination scheme, the patterns of incidence were diverse in the examined pens but often viral circulation was detected as early as 4 weeks of age. At three weeks of age, most of the analysed animals were positive with high S/P ratios. In the second follow-up period after the application of the first vaccination scheme, the onset of infection was delayed by two weeks but there were no other significant differences between both groups, and in the third, the onset of infection shifted to the left for all groups, without significant differences among them. In all of the three studies, animals that shed virus in two and even three consecutive sampling times were detected, as well as some cases of re-infection. Interestingly, an H1avN1av virus was initially detected in the farm, but during the third study, a H3huN2hu was found circulating in the batches, carrying a new H3 human-like derived from human seasonal virus.
Universitat Autònoma de Barcelona. Programa de Doctorat en Medicina i Sanitat Animals

Bacteriophages, or simply "phages," are the most abundant biological entities on the planet and are thought to be the largest untapped reservoir of available genetic information. They are also important contributors to both soil health and nutrient recycling and have significantly influenced our current understanding of molecular biology. Bacteria in the genus Streptomyces are also known to be important contributors to soil health, as well as producing a number of useful antibiotics. The genetic diversity of large (> 30) groups of other actinobacteriophages, i.e. phages infecting a few close relatives of the Streptomycetes, has been explored, but this is the first formal effort for Streptomyces-infecting phages. Described here are a group of 45 phages, isolated from soil using a single Streptomycete host, Streptomyces griseus ATCC 10137. All 45 phages are tailed phages with double-stranded DNA. Siphoviruses predominate, six of the phages are podoviruses, and no myoviruses were observed. Notably present are seven phages with prolate icosahedral capsids. Genome lengths and genome termini vary considerably, and the distributions of each are in line with findings among other groups of studied actinobacteriophages. Interestingly, the average G+C among the 45 phages is around 11% lower than that of the isolation host, a larger disparity than reported for other groups of actinobacteriophages. Eighteen of the phages carry between 17 and 45 tRNAs and 12 of those carry a single tmRNA. Forty-three phages were grouped into seven clusters and two subclusters based on dot plot analysis, average nucleotide identities, and gene content similarities. Two phages were not clustered with other phages in this dataset. A total of 5250 predicted genes were sorted into 1300 gene "phamilies," with about 8% of the total phamilies having only a single member. Analysis of gene content among the 45 phages indicates first that most clusters presented here appear to be relatively isolated from one another, with phages in any one cluster generally sharing < 10% of their genes with phages in other clusters described here. Secondly, most of the phages here are more than twice as likely to share genes with phages isolated on bacteria outside of the genus Streptomyces than they are other phages isolated using a Streptomycete as host. These observations suggest that (1) the phage clusters here have a distinct extended host range, (2) those host ranges share overlap, and (3) Streptomyces griseus is likely not the preferred natural host for all phages described.

El rotavirus (RV) és el principal causant de diarrea greu en nens menors de cinc anys i, encara que es necessiten més estudis estandarditzats, hi ha evidència de que els probiòtics poden ajudar a lluitar contra el RV i contra altres patologies infeccioses i intestinals. Sobre aquesta base, l'objectiu de la present tesi va ser el d’avaluar l'efecte protector de productes relacionats amb els probiòtics i els prebiòtics contra les infeccions per rotavirus, utilitzant models experimentals en rata lactant. Per aconseguir aquest objectiu, es va utilitzar un model d’infecció simple per RV ja existent, amb algunes noves variables. D'altra banda, es va desenvolupar un nou model de doble infecció per rotavirus en rates. Diverses variables immunològiques van demostrar que és adequat per a la realització d'estudis intervencionals, i sembla que les accions moduladores sobre la primera infecció tenen una influència sobre la resposta immunitària desenvolupada enfront a una segona infecció per RV. Això s'ha demostrat amb l'administració d’un calostre boví hiperimmune contra el RV (HBC) durant la primera infecció. Pel que fa a la suplementació amb la soca probiòtica Bifidobacterium breve M-16V durant la lactància, s’ha observat que millora el desenvolupament de la immunitat de la mucosa en rates per mitjà de la modulació de l'expressió de TLR, la millora del procés de reclutament dels limfòcits T verges als GLM i la retenció dels limfòcits activats al compartiment intraepitelial, així com la potenciació de la síntesi d’IgA intestinal. D'altra banda, modula la infecció i la reinfecció per RV en el model d’infecció simple i doble, respectivament, alleugerint les manifestacions clíniques durant la primera infecció, però permetent a l'hoste elaborar la pròpia resposta immunitària, que l'ajudarà a controlar una segona infecció. En quant a la suplementació prebiòtica durant la lactància en rates, la barreja prebiòtica scGOS/lcFOS 9:1 (Immunofortis), tot i presentar un efecte directe sobre la consistència de les femtes, ha mostrat efectes beneficiosos contra la infecció per RV i ha modulat la reinfecció en el model de doble infecció, mostrant una important acció immunomoduladora. Aquests efectes han estat comparables als del prebiòtic Bimuno® GOS. Com a simbiòtic, quan s’ha avaluat en combinació amb el probiòtic B. breve M-16V, s’ha mostrat molt eficaç en la modulació de la diarrea induïda per RV, així com en la modulació de la resposta immunitària i de la reinfecció per RV en el model preclínic de doble infecció. Per contra, la combinació d’Immunofortis® amb oligosacàrids acídics derivats de pectina (pAOS) no ha potenciat el seu efecte preventiu sobre el model de gastroenteritis per RV en rata. D'altra banda, la suplementació amb un postbiòtic (una llet fermentada per B. breve i Streptococcus thermophilus) durant la lactància en rates, ha estat capaç de prevenir gairebé totes les manifestacions derivades de la diarrea induïda per RV, mentre que també ha modulat la resposta immunitària anti-RV. L’addició d’Immunofortis® a aquest postbiòtic no ha mostrat efectes clars de sinèrgia. Com a conclusió general, tots els productes estudiats, essent el probiòtic i el postbiòtic els més eficaços, han mostrat efectes beneficiosos sobre la gastroenteritis induïda per RV en el model de rata lactant, modulant les variables clíniques i resposta immunitària en primeres etapes de vida. Així i tot, calen més estudis per comprendre millor el seu mecanisme d'acció i per a ser considerats per a la seva inclusió en preparats per a lactants o suplements, com estratègies per a la protecció contra la diarrea induïda per RV en nens.
Rotavirus (RV) is the leading cause of severe diarrhoea among infants and young children and there is evidence that probiotics can help to fight against RV infection. The aim of the present thesis was to evaluate the protective effect of probiotics, prebiotics and other related products against RV infections using the suckling rat as experimental model. To achieve this objective, an updated model of simple RV infection and a new RV double-infection model in rat were used. Regarding the probiotic effect, the supplementation with Bifidobacterium breve M-16V improved the development of mucosal immunity in early-life rats. Moreover, it attenuated RV infection and reinfection by ameliorating diarrhoea during first infection but allowing the host to elaborate its own immune response, which seems to help control the second infection. Regarding the prebiotic effect, scGOS/lcFOS 9:1 (Immunofortis) ameliorated RV infection in the single-infection model and modulated reinfection in the double RV infection model, showing a high immunomodulatory action. Its effect was comparable to that of the prebiotic Bimuno GOS. A synbiotic combining the scGOS/lcFOS prebiotic with the probiotic Bifidobacterium breve M-16V, was highly effective in modulating RV-induced diarrhoea as well as modulating immune response and RV reinfection in the double-infection preclinical model. Conversely, the combination of Immunofortis with pectin-derived acidic oligosaccharides (pAOS) did not potentiate its preventive effect. The postbiotic supplementation with a Bifidobacterium breve and Streptococcus thermophilus-fermented formula, and its combination with scGOS/lcFOS were able to prevent almost all features derived from the RV-induced diarrhoea and they also modulated the anti-RV immune response. Overall, all tested products showed beneficial effects on the RV-induced gastroenteritis in the neonatal rat model, modulating clinical biomarkers and immune system responses early in life, with the probiotic and the postbiotic being the most effective. Further studies are needed in order to better understand their mechanism of action and for them to be considered for inclusion in infant formulas or supplements as strategies for protecting against human RV-induced diarrhoea in children.

Els anellovirus són un grup de virus de cadena senzilla d’ADN amb una elevada diversitat genètica i que infecten a vertebrats. Els Torque teno sus virus (TTSuV) son ubics i és l’espècie d’anellovirus específica que infecta els porcs. S’han descrit 3 espècies víriques diferents, anomenades TTSuV1a i TTSuV1b dins del gènere Iotatorquevirus i TTSuVk2a, dins el gènere Kappatorquevirus. Aquests virus son genèticament molt diferents (>56% de diversitat de seqüencia nucleotídica) però comparteixen la mateixa organització del genoma i estratègia d’expressió. L ‘infecció de TTSuV en porcs es caracteritza per una virèmia persistent. Els TTSuVs no es consideren patogènics, però es creu que juguen un paper en la co-infecció amb altres agents infecciosos que causen malalties porcines importants. L'impacte real dels TTSuVs sobre la salut dels porcs, si n’hi ha , segueix sent objecte de debat. L'objectiu principal d'aquesta Tesi fou caracteritzar la variabilitat de TTSuV i explorar el desenvolupament de possibles prototipus vacunals enfront els TTSuVs. El primer estudi descriu la caracterització genètica d'una nova espècie de TTSuV, anomenada Torque teno sus virus k2b (TTSuVk2b). Segons l'anàlisi filogenètica, aquest nou virus pertany al gènere Kappatorquevirus, el mateix al qual pertany TTSuVk2a. Es van desenvolupar tècniques quantitatives de PCR basades en la tecnologia de SybrGreen pels estudis epidemiològics i per l’estudi de la distribució geogràfica. En concret, es va desenvolupar una tècnica que quantificava la càrrega total de TTSuV (TTSuV broad-spectrum qPCR) i tres més que quantificaven la càrrega viral específica de cada espècie de TTSuV. D'altra banda, es va estudiar la prevalença i la càrrega d'ADN vírica en animals afectats amb per circovirosi porcina (CP). L'estudi epidemiològic va revelar que TTSuVk2b es distribueix a tot el món, encara que és menys abundant que TTSuV1 i TTSuVK2a. TTSuVk2b es va associar amb la CP, una malaltia porcina d'importància econòmica molt significativa. El segon estudi contenia dos objectius: d'una banda, l'expressió de les proteïnes TTSuVs; per l'altra banda, l’avaluació de l'impacte d’una vacuna experimental multivalent enfront les 4 espècies de TTSuVs conegudes, utilitzant com a model l’infecció natural de TTSuV en porcs. Les proteïnes recombinants ORF1, ORF1-A, ORF2 i ORF3 dels quatre TTSuVs coneguts es van expressar amb èxit mitjançant un sistema d'expressió de baculovirus. Per altra part, la vacuna experimental multivalent (formada per les proteïnes dels gens ORF1 i ORF3) va ser administrada per via intramuscular i intradèrmica utilitzant dos esquemes de vacunació diferents (dues o tres dosis). La seroconversió i els títols vírics en sang es van mesurar a partir de 3 fins a 15 setmanes d'edat, utilitzant respectivament l'ELISA indirecte basat en proteïnes del virus expressades en baculovirus i tècniques de PCR quantitativa per detectar i quantificar els nivells de virèmia de cada espècie TTSuV, respectivament. Aquest estudi va mostrar que la vacuna multivalent induïa anticossos anti-TTSuV, però aquest prototipus vacunal no era capaç de controlar la virèmia durant l’infecció natural de TTSuV. Finalment, en el tercer estudi es va avaluar l’immunització enfront TTSuVk2a durant l’infecció natural en porcs. L’immunització va consistir en una combinació d'ADN i proteïnes víriques per augmentar les possibilitats d'activació de respostes immunitàries tant cel·lulars com humorals. Es van utilitzar tècniques de PCR quantitativa per detectar i quantificar els nivells de virèmia de cada espècie de TTSuV, mentre que l’inducció d'anticossos específics va ser monitoritzada mitjançant ELISA indirecte. El grup vacunat va mostrar una seroconversió i una reducció significativa de les càrregues víriques de TTSuVk2a en comparació amb el grup control. Aquest estudi va demostrar per primera vegada que la virèmia per TTSuV pot ser controlada a través de l’ús d’una combinació que inclou ADN i proteïnes recombinant. En general, aquesta Tesi Doctoral contribueix a augmentar el coneixement dels TTSuVs mitjançant la descripció d’una nova espècie vírica, la qual pot estar associada al desenvolupament de malalties del porc, com s’ha indicat per TTSuVk2a. A més, l’infecció per TTSuV pot ser controlada mitjançant l'administració d'una vacuna d'ADN i proteïnes recombinats del virus, mentre que la vacuna basada en múltiples proteïnes (multivalent) no va ser eficient.
Anelloviruses are a highly diverse group of circular single-stranded DNA viruses infecting vertebrates. Torque teno sus viruses (TTSuV) are ubiquitous pig-infecting anelloviruses. Three different viral species have been described, namely TTSuV1a and 1b within the genus Iotatorquevirus and TTSuVk2a, within the genus Kappatorquevirus. These viruses are genetically very distinct (>56% sequence diversity) but share similar genome organization and expression strategy. TTSuV infection in pigs is distributed worldwide, and is characterized by a persistent viremia. TTSuV themselves are considered non-pathogenic; however, it is believed that these viruses play a role in co-infection with other economically important viral porcine infections. Apparently, TTSuV infection can influence the development or may contribute to the pathogenesis of various porcine diseases during co-infection. The real impact of TTSuV on the pig health, if any, is still under debate. The present Thesis aimed to characterize a novel TTSuV species and to explore possible ways of vaccination against TTSuVs. The first study describes the discovery, genetic characterization and epidemiology of a novel TTSuV species, named Torque teno sus virus k2b (TTSuVk2b). According to phylogenetic analysis, this new virus belongs to the Kappatorquevirus genus, belonging to the same genus as TTSuVk2a. Quantitative PCR techniques based on SybrGreen technology were developed; one for quantification of total TTSuV load (TTSuV broad-spectrum qPCR) and others for quantification of each TTSuV species separately. These techniques were used for epidemiological studies and assess the geographical distribution. Moreover, prevalence and viral DNA load were determined in porcine circovirus type 2-systemic disease (PCV2-SD)-affected animals and healthy counterparts, since previous studies have associated another kappatorquevirus species, to the disease. The epidemiological study revealed that TTSuVk2b is worldwide distributed, although less abundant and displaying lower viral DNA titres in serum than TTSuV1 and TTSuVk2a. TTSuVk2b was associated with PCV2-systemic disease (PCV2-SD), which revealed that the two kappatorquevirus species are both genetically and biologically related. The second study contained two objectives. On one hand, TTSuV proteins were expressed in a baculovirus-based platform; on the other hand, the impact of a multivalent experimental vaccine was evaluated in a natural TTSuV infection model of pigs. The ORF1, ORF1-A, ORF2 and ORF3 recombinant proteins of all four known TTSuVs were successfully expressed in a baculovirus expression system. In additional, the multivalent experimental vaccine containing ORF1 and ORF3 proteins was administered by intramuscular and intradermal routes using two different vaccination schedules (twice or three times). Seroconversion and viral titres in blood were measured from 3 weeks until 15 weeks of age, using the indirect ELISA based on baculoviruses proteins and species-specific qPCRs, respectively. This study showed that vaccination induced anti-TTSuV antibodies; however the multivalent vaccine was not able to control viremia during TTSuV natural infection. Finally, in the third study, the immunization against TTSuVk2a during natural infection was evaluated using a different approach. The immunizations consisted of a combination of DNA and protein to increase the possibilities of activating both cellular and humoral immune responses. Quantitative PCR techniques were used to detect and quantify the viremia levels of each TTSuV species, while the induction of specific antibodies was monitored by indirect ELISA. The vaccinated group showed a seroconversion and a significant reduction of the TTSuVk2a viral loads compared to the control group. This study demonstrated for the first time that TTSuV viremia can be controlled by a combined DNA and protein immunization. Overall, the present Thesis contributes to increase the knowledge on TTSuV by means of describing a novel species, which may be involved in disease progression in co-infection with other pig pathogens. Moreover, TTSuV infection can be controlled by the administration of a combined DNA/protein immunization while a multivalent protein based vaccine was not efficient.

It has been suggested that the rising amounts of chemicals in the environment may affect host resistance and increase susceptibility to infections. Studies have also shown that infections can change the toxicity of pollutants. The aim of this thesis was to study interactions between environmental pollutant exposure in terms of polybrominated diphenyl ethers (PBDE) and a common human coxsackievirus B3 (CVB3) infection adapted to Balb/c mice. The studies focused on virus levels, cytokines, metabolising cytochrome P450 (CYP) enzymes and tissue distribution of PBDE. A novel finding was an organ-specific effect of CVB3 infection on the metabolising capacity of PBDE. The PBDE metabolising enzyme CYP2B10 was down-regulated by the CVB3 infection in the liver, up-regulated in the lungs, but not affected in the pancreas. Accordingly, CVB3 infection increased the concentration of PBDE in the livers of infected mice. However, serum levels of PBDE were not affected by the infection, indicating that serum does not reflect the actual organ exposure of PBDE in infected individuals. The change in metabolising capacity was likely mediated by infection-induced cytokines and associated effects on the nuclear factor-κB (NF-κB) pathway. PBDE drastically decreased serum levels of several cytokines and chemokines, an event that may create a slot for viruses to replicate. Accordingly, some results show that infected mice exposed to a high dose of PBDE had higher virus levels than mice exposed to a low dose. In conclusion, infected individuals showed organ-specific changes in metabolism and tissue levels of PBDE, which potentially could change the toxicity of PBDE. PBDE also seems to affect the fate of the infection. NF-κB activated pathways may mediate one possible mechanism underlying these effects. Thus, further investigations of this pathway are warranted. In addition, future studies should address how PBDE exposure affects viral replication.

Nucleic Acid Technology (NAT), introduced in the late 90s, is a molecular amplification method that can be used for the diagnosis and management of patients with infectious diseases. NAT test results are obtained quicker and are quantified, providing great.er information than the positive/negative results available from traditional techniques. However, NATs arc technically demanding, susceptible to contamination and hence results from associated diagnostic tests may be inaccurate. External Quality Assessment (EQA) services are programmes developed to assess and advance the quality performance of laboratories that use NAT kits to diagnose, manage and control human diseases. Quality Control for Molecular Diagnostics (QCMD) , an organisation that provides EQA, uses proficiency panels designed with samples containing no , weak, medium and strong microbial loads. The panels are distributed to participating laboratories who analyse them knowing the pathogen but blind to the microbial load.

Le diagnostic virologique est un sujet d’actualité particulièrement du fait des récentes épidémiesou pandémies telles que la pandémie d’influenza A(H1N1) en 2009 ou la diffusion du virus Zika dansles Amériques et la région du Pacifique entre 2014 et 2017, associée à des cas de microcéphalie et dessyndromes de Guillain Barré. Encore plus récemment, en août 2018, le ministre de la santé de laRépublique Démocratique du Congo annonçait la 10e épidémie de virus Ebola dans le pays et endécembre 2019, le coronavirus SARS-CoV-2 est à l’origine d’une pandémie au départ de la Chine. Avecle nombre croissant de migrants et de voyageurs favorisant la dissémination des maladies virales, leslaboratoires diagnostiques doivent être parés à la fois pour l’identification des virus communs maisaussi de ceux importés.Les techniques les plus anciennes de diagnostic virologique tendent à devenir obsolètes suite audéveloppement rapide des techniques moléculaires depuis les années 90. Cependant, nous utilisonstoujours un mélange de techniques moléculaires et non moléculaires au sein de notre laboratoire.Les objectifs de ce travail sont de passer en revue les différentes techniques communémentutilisées pour la détection directe des virus avec leurs avantages et leurs inconvénients et de fournirune réflexion sur la place de chaque technique, en 2020, dans un laboratoire diagnostique.Nous aborderons tout d’abord les cultures cellulaires et nous insisterons sur leur polyvalence quipermet parfois de mettre en évidence des micro-organismes que l’on ne suspectait pas. Nousillustrerons ce point par un article relatant la mise en évidence de Chlamydia trachomatis du serovar Lresponsables de la lymphogranulomatose vénérienne dans des prélèvements envoyés pour suspiciond’infection herpétique.Le travail se focalisera ensuite plus particulièrement sur le diagnostic des infections viralesrespiratoires. Nous verrons les principes des tests de détection antigéniques et discuterons de leurslimites en se basant sur un article qui traite du diagnostic des virus influenza A et B par 3 différentstests immunochromatographiques. Cet article montre que la sensibilité des tests varie en fonction dela charge virale dans le prélèvement ainsi que du sous-type de virus.Nous poursuivrons avec les tests d’amplification d’acides nucléiques (tests moléculaires) enexpliquant la technique de PCR (Polymerase Chain Reaction) et une technique d’amplificationisothermique (Nicking Enzyme Amplification Reaction - NEAR). Nous illustrerons par un article portantsur l’évaluation du test Alere i influenza A&B (technique NEAR) en comparaison du test Sofia influenzaA+B (immunochromatographie). Cet article montre un gain de sensibilité de l’Alere i par rapport auSofia pour le diagnostic de l’influenza A mais pas pour l’influenza B. Il constitue également un travailpréliminaire sur l’appréciation de l’utilité d’une technique PCR rapide dans la prise en charge despatients. La conclusion est qu’il pourrait y avoir un apport de ce type de technique pour la diminutiondes hospitalisations, de la prescription des examens complémentaires et des antibiotiques. Celapermettrait également une prescription plus adéquate de l’oseltamivir pour le traitement de la grippe.Le point important est que l’impact du résultat est d’autant plus grand qu’il est délivré précocementdans la prise en charge des patients, idéalement lorsqu’ils sont encore aux urgences.Suite au travail sur l’Alere i, nous avons entrepris d’évaluer un test PCR multiplex (FilmArrayRespiratory Panel) pour le diagnostic des virus afin de voir si la détection d’un plus grand nombre depathogènes pourrait avoir un impact plus grand sur la prise en charge des patients. Cette évaluation adonné lieu à deux articles. Le premier détaille les avantages et inconvénients des différents outils dediagnostic pour la détection des virus respiratoires et sert d’état des lieux sur les tests utilisésactuellement dans les laboratoires de virologie. Le deuxième article porte plus particulièrement surl’apport du FilmArray dans la prise en charge des patients. La conclusion est que ce n’est pas le résultatdu test qui a un impact sur cette prise en charge mais plutôt d’autres facteurs notamment l’âge ou desmarqueurs inflammatoires biologiques.Nous terminerons ce travail par un aperçu des techniques de séquençage qui seront sans aucundoute de plus en plus utilisées pour le diagnostic en virologie.
Doctorat en Sciences médicales (Médecine)
info:eu-repo/semantics/nonPublished

Adenoviruses are common pathogens all over the world. The majority of the population has at some point been infected with an adenovirus. Although severe disease can occur in otherwise healthy individuals an adenovirus infection is most commonly self limited in these cases. For immunocompromised individuals however, adenoviruses can be life-threatening pathogens capable of causing disseminated disease and multiple organ failure. Still there is no approved drug specific for treatment of adenovirus infections. We have addressed this using a unique whole cell viral replication reporter gene assay to screen small organic molecules for anti-adenoviral effect. This RCAd11pGFP-vector based assay allowed screening without any preconceived idea of the mechanism for adenovirus inhibition. As a result of the screening campaign 2-[[2-(benzoylamino)benzoyl]amino]-benzoic acid turned out to be a potent inhibitor of adenoviral replication. To establish a structure-activity relationship a number of analogs were synthesized and evaluated for their anti-adenoviral effect. The carboxylic acid moiety of the molecule was important for efficient inhibition of adenovirus replication. There are 54 adenovirus types characterized today and these are divided into seven species, A-G. The receptors used by species B and other adenoviruses are not fully characterized. CD46 is a complement regulatory molecule suggested to be used by all species B types and some species D types but this is not established. We have designed a new bioassay for assessment of the interaction between adenoviruses and CD46 and investigated the CD46-binding capacity of adenovirus types indicated to interact with CD46. We concluded that Ad11p, Ad34, Ad35, and Ad50 clearly bind CD46 specifically, whereas Ad3p, Ad7p, Ad14, and Ad37 do not. CD46 is expressed on all human nucleated cells and serves as a receptor for a number of different bacteria and viruses. Downregulation of CD46 on the cell surface occurs upon binding by some of these pathogens. We show that early in infection Ad11p virions downregulate CD46 upon binding to a much higher extent than the complement regulatory molecules CD55 and CD59. These findings may lead to a better understanding of the pathogenesis of adenoviruses in general and species B adenoviruses in particular and hopefully we have discovered a molecule that can be the basis for development of new anti-adenoviral drugs.

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.

El uso de la terapia antiretroviral (ATR), ha sido durante décadas el tratamiento de elección utilizado en la inhibición de la propagación del VIH. La terapia ATR se centra en interrumpir las diferentes etapas del ciclo de replicación del virus en la célula susceptible de infección; sin embargo, el incumplimiento del régimen ATR por parte de los pacientes, ha facilitado la aparición de cepas multiresistentes que hacen que el virus cada vez sea más difícil de erradicar, convirtiéndose en un problema de salud pública. No obstante, nuevas estrategias basadas en el uso de las herramientas actualmente existentes tales como inhibidores de la fusión, son vanguardistas en el desarrollo de nuevas estrategias de inhibición en la propagación del virus. Se ha descrito que el GB virus C, tiene la capacidad de disminuir la progresión de VIH a SIDA, en individuos co-infectados, aumentando su tasa de supervivencia. Los mecanismos exactos mediante los cuales el GB-C es capaz de intervenir con la progresión del VIH a SIDA no son completamente conocidos, pero hallazgos realizados por diferentes grupos de investigación postulan que las proteínas de envoltura del GB-C tienen un importante papel en la inhibición de los primeros pasos de la fusión y entrada del VIH a la célula huésped. Es así, que péptidos derivados de las proteínas de envoltura E1 y E2 del GB virus C, se han utilizado como una nueva estrategia en la prevención y control del HIV-1. Debido a la susceptibilidad de estas moléculas a la degradación enzimática, a su limitada biodisponibilidad y deficiente capacidad de atravesar barreras biológicas; teniendo en cuenta que la principal puerta de entrada del virus del VIH-1 al organismo son las mucosas vaginales y rectales, en la presente tesis se llevó a cabo el desarrollo de NPs de PLGA recubiertas Glycol-Chitosan (GC-PLGA-NPs) para la incorporación de péptidos inhibidores de la fusión del VIH-1 derivados del GB virus C y su liberación en un modelo de mucosa vaginal. Para ello, se sintetizaron péptidos derivados de las proteínas de envoltura del GB virus C, E1P47 Y P6-2, los cuales fueron previamente descritos y caracterizados para su actividad anti- fusión del VIH. La síntesis de los péptidos se llevó acabo usando el método de síntesis en fase sólida SPPS y la estrategia Fmoc/tBut. Una vez obtenidos los peptidos deseados, estos fueron derivatizados con 5-6 carboxyfluoresceína, para su posterior utilización en los estudios in vitro, ex vivo e in vivo. Una vez obtenidos los péptidos y sus derivatizados, se desarrollaron y optimizaron mediante un diseño factorial central compuesto las GC-PLGA-NPs. Las Nps fueron caracterizadas tanto para su morfometría, morfología y eficiencia de encapsulación. Los principales hallazgos demostraron que las GC-PLGA-NPs, preparadas utilizando el método de la doble emulsión (w/o/w), fueron capaces de atravesar la principal barrera fisiológica que recubre el epitelio vaginal y alcanzar las capas subyacentes del epitelio, sin provocar daños del epitelio. La mucoadhesividad y bioseguridad, además del tamaño y carga positiva que otorgada a estos sistemas el GC, fue la principal característica de las GC-PLGA-NPs, Además, de acuerdo con los resultados obtenidos de los estudios de Rayos X y espectroscopía infrarroja por transformada de Fourier, los péptidos no sufrieron cambios conformacionales o interacciones con los componentes de la formulación que afectaran su efectividad, lo que cumplió con el principal objetivo de la encapsulación del estas moléculas en Nanoparticulas de PLGA, el cual fue proteger y mantener la eficacia de los inhibidores. Además, las GC-PLGA-NPs desarrolladas en esta Tesis demostraron ser seguras y no toxicas para el epitelio vaginal. Debido a estas razones, esta Tesis doctoral abre un nuevo campo de investigación, como lo es el desarrollo de microbicidas anti-anti-HIV basados en sistemas de liberación controlada.
The main drawbacks with current antiretroviral therapies are the occurrence of multi-drug resistance viruses and the reduced capacity of these drugs to access tissues. Hence, new therapeutic alternatives to fight the spread of HIV are based on peptides that inhibit the early steps of HIV-1 fusion in target cells, which avoids side effects such as therapy resistance. Unfortunately, the molecules used have weaknesses such as poor bioavailability; a short half-life; fast removal; and little capacity to cross physiological barriers such as vaginal fluid, which protects the vaginal epithelium from any foreign agents reaching it, especially biomolecules such as peptides. For these reasons, the main objective of this research was to develop, optimized and characterize polymeric Nanoparticles (NPs) covered with glycol chitosan (GC) to introduce and release peptides derived from GB virus C that inhibit HIV-1 into the vaginal mucosa. In vitro release and ex vivo studies were carried out using pig vaginal mucosa and the peptides were determined by an HPLC MS/MS-validated method. Moreover, the peptides were labeled with 5(6)-carboxyfluorescein and entrapped within the NPs to carry out in vivo studies and to evaluate the penetration and toxicity of the NPs in the pig vaginal mucosa. The results suggest that the fusion inhibitor peptides developed and loaded into the NPs coated with GC might be a new way to fight HIV-1, as the formulation could reach the human epithelial mucosa and release the peptide without any side effects.

Metapneumovírus Aviário (aMPV), família Pneumoviridae, gênero Metapneumovírus, é o agente etiológico responsável pela rinotraqueíte dos perus e também está associada à síndrome da cabeça Inchada em galinhas, duas importantes doenças respiratórias que acometem aves comerciais e levam a grandes perdas econômicas. O objetivo deste estudo foi detectar a presença de aMPV em amostras de aves silvestres e realizar a caracterização molecular dos isolados encontrados, com o intuito maior de contribuir para o entendimento da epidemiologia desse vírus. Para isso foram coletados no total, 448 suabes orofaringeanos (OP) e cloacais (C) oriundos de 234 aves silvestres em quatro locais do estado de São Paulo. Três tipos de amostras foram processadas e testadas: 1) 266 suabes agrupados de um ou até cinco animais que estavam no mesmo recinto e respeitando o mesmo tipo de suabe (OP ou C); 2)188 suabes foram agrupados na forma de pools de até dois animais, contendo os suabes OP e C de cada animal, formando então 48 pools; 3) amostras de tecido traqueal e pulmonar de três Egretta thula também foram coletados e testados. A purificação de RNA viral foi realizada utilizando o QIAmp RNA Mini Kit Kit (Qiagen). Para o teste de RT-PCR foi utilizado o kit OneStep RT-PCR (Qiagen) com primers baseados no gene N, previamente descritos, com fragmento esperado de 115 bp. As amostras foram também testadas por RT-PCR em tempo real (RRT-PCR) com primers específicos previamente descritos, para os subtipos A e B, com base no gene G com fragmentos de 116 e 135 bp, respectivamente. Os fragmentos das amostras positivas foram purificados e sequenciados pelo sequenciamento tipo Sanger para caracterização por análises filogenéticas. Das 126 amostras testadas pelo teste de RT-PCR baseado no gene N, quatorze foram positivas: oito amostras de Anseriformes (Aix sponsa, Aix galericulata, Dendrocygna viduata), três de Columbiformes (Columba livia), um de Falconiformes (Falco sparverius), um de Psittaciformes (Psittacara leucophthalma) e um de Pelecaniformes (Egretta thula). Das quatorze amostras positivas, treze eram provenientes de suabes, e a décima quarta era oriunda de tecido traqueal de Egretta thula. Pelo teste de RRT-PCR baseado no gene G nenhuma das 184 amostras testadas foi positiva. As análises filogenéticas realizadas com fragmentos de 44 e 54 bp do gene N de duas amostras positivas, que se agruparam com isolados pertencentes ao aMPV de subtipo A. Estes vírus apresentaram alta identidade com as estirpes derivadas de vacina e com estirpes vacinais, o que mostra uma possível ocorrência de escapes vacinais de aves de produção para as aves silvestres.
Avian metapneumovirus (aMPV), family Pneumoviridae, genus Metapneumovirus, it is the etiologic agent responsible for turkey rhinotracheitis and is also associated with swollen head syndrome in chickens, two important respiratory diseases in poultry which leads to large economic losses. The aim of this study was detect the presence of aMPV in wild birds samples, to perform the phylogenetic analysis of the isolates found with the major objective of contributing to the understanding of the epidemiology of this virus in poultry farms. In total, 448 oropharyngeal (OP) and cloacal (C) swabs from 234 wild birds collected in four different locations within the state of São Paulo. The samples were processed and tested in three different ways: 1) 266 swabs were in the form of pools of one up to five animals that were in the same enclosure and respecting the same type of swab (OP or C); 2) 188 remaining swabs were grouped into pools of up to two animals, containing the oropharyngeal and cloacal swabs of each animal; 3) tracheal and pulmonary tissue samples were also collected and tested. Purification was performed using the QIAmp RNA Mini Kit Kit (Qiagen). Viral detection was performed by conventional RT-PCR technique using the OneStep RT-PCR kit (Qiagen) with primers based on the N gene, previously described with expected fragment of 115 bp. The samples were also tested by a real time RT-PCR (RRT-PCR) with specific primers previously described, for subtypes A and B, based on the G gene with fragments of 116 and 135 bp, respectively. Of the 126 samples tested by the RT-PCR N gene based, fourteen were positive: eight samples of Anseriformes (Aix sponsa, Aix galericulata, Dendrocygna viduata), three Columbiformes (Columba livia), one Falconiformes (Falco sparverius), one Psittaciformes (Psittacara leucophtalma) and one Pelecaniformes (Egretta thula). Of the swab samples, five were derived from oropharyngeal swabs and four from cloacal swabs, the other four samples were detected in the samples processed in pools of up to two animals, which contained the oropharyngeal and cloacal swabs of each bird. The positive Egretta thula sample was from a tracheal tissue sample. Based on the RRT-PCR G gene based, none of the 184 samples tested were detected. Phylogenetic analyzes were performed on two positive samples that proved to belong to aMPV subtype A, showing high similarity with the strains derived from the vaccine and with vaccine strains.

Microbiology and Immunology
Ph.D.
The Adeno-Associated Virus(AAV) is a small, single stranded DNA virus that has been developed as a gene transfer vector. Early clinical trials using recombinant AAV vectors (rAAV) have identified the following concerns that need to be addressed in order to increase efficiency of these vectors. It has since been determined that AAV vector efficiency decreases due to the following mechanisms: ineffective endocytosis, endosomal degradation, inefficient trafficking, and the need to convert from a single-stranded AAV genome to a transcriptional active double-stranded form. The purpose of this study is to elucidate mechanisms that help stabilize the AAV genome in order to make it a more efficient vector for gene therapy. Previously, there have been studies using fluorescent labeling to track the movement of AAV into the nucleus. An integral part of AAV genomic stability may be the obstacles it encounters in the cytoplasm prior to entering the nucleus. Previous studies on improving AAV transduction have focused primarily on the nucleus. The study will hopefully shed light on the hurdles AAV encounters as it moves through the cytoplasm. Thus, this project has designed and utilized a new system for specifically tracking the status of AAV genomes in the cytoplasm as well as in the nucleus. This project utilizes a novel dual luciferase reporter system to track the movement of AAV particles from the cytoplasm into the nucleus to elucidate mechanisms that could contribute to the stabilization of the AAV genome. The novel dual reporter system is comprised of a single-stranded vector containing two different types of secreted luciferases: Cypridina Luciferase and Gaussia Luciferase. Cypridina Luciferase is placed under the control of a nuclear promoter and therefore it is expressed only in the nucleus. The second, Gaussia Luciferase, is under the control of a cytoplasmic promoter that will only be expressed in the cytoplasm upon the presence of T7 RNA polymerase. Using this dual reporter luciferase system along with RT qPCR quantification in a Hek293 cell line expressing the T7 RNA polymerase, demonstrated that genomes are present in the cytoplasm at 18 and 24 hours post infection. The second part of this study is using the dual reporter system to understand the trafficking patterns of rAAV and looking at ways to enhance transduction. One method could be administering rAAV vectors in conjunction with a drug; this approach may help overcome some of these cellular barriers encountered during infection as well as help stabilize the genome. Cidofovir (CDV) is a monophosphate nucleotide analogue that competitively inhibits the incorporation of deoxycytidine triphosphate into viral DNA via viral DNA polymerase. In vitro, CDV actively inhibits a number of DNA viruses including herpes viruses, adenovirus, polyomavirus, papillomavirus, and poxviruses. Cidofovir has already been approved by the Food and Drug Administration (FDA) for the treatment of cytomegalovirus (CMV) retinitis in patients with acquired immunodeficiency syndrome (AIDS). The effects of CDV on small DNA viruses that lack their own viral DNA polymerase, like AAV, has not been documented. Results have demonstrated that CDV is able to increase single-stranded rAAV transgene expression in the nucleus by 2 to 5 fold, depending on the cell type and concentration, in vitro, using both rAAV2 and rAAV8 luciferase reporter vectors. These results have been able to replicated using other reporter vectors: rAAV2-LacZ reporter, rAAV2-GFP, rAAV2-hAAT, and a rAAV8-GFP in vitro. Results have shown a dose-dependent increase in rAAV genomes with CDV pretreatment in HelaS3 cells via Southern Blot. Also southern blot analysis of cells pretreated with CDV then infected with rAAV revealed no difference in the amount of vector present between 0 and 2 hours post infection, suggesting CDV does not enhance viral entry but that CDV may enhance at steps downstream of viral entry. Using RNA sequencing and Ingenuity software analysis of HelaS3 cells pretreated with CDV, an increase in several genes of interest including those involved in the mechanism of viral exit were observed. These genes include Actin and vacuolar proteins, these molecules are involved in and associated with endosomal sorting complexes as well as required for transport inside the cell. This finding along with southern blot data supports the theory that CDV may enhance rAAV trafficking since we observed that CDV pretreatment enhances viral accumulation of rAAV vectors in both the cytoplasm and nucleus 24 hours post-infection. Also utilizing the dual luciferase reporter system an increase in transgene expression present with CDV pretreatment compared to PBS in both the cytoplasm and nucleus was observed suggesting that CDV may enhance with rAAV trafficking. These results taken together demonstrate that this dual reporter system is a powerful tool for understanding and improving rAAV trafficking. Also drugs like CDV can greatly contribute to the understanding of rAAV trafficking, and eventually lead to the development of novel strategies to increase overall efficiency of AAV transduction.
Temple University--Theses

Bibliography: pages 139-150.
The diversity of Subgroup I geminiviruses causing streak disease in maize, sugarcane, and indigenous wild grasses was investigated. The virus. isolates studied originated from maize (several southern African isolates), two sugarcane cultivars (from Natal province, South Africa, and from Mauritius), wheat, and three grasses (Panicum, Setaria, and Eleusine spp. from South Africa). The following methods were used: analysis of restriction fragment length polymorphisms (RFLPs) between viral genomes in individual infected plants; DNA cross-hybridization between virus isolates; restriction endonuclease mapping of whole virus genomes; and nucleic acid sequencing. The complete genome of the Natal sugarcane streak virus isolate was sequenced. Partial sequences were obtained for other isolates, either by sequencing the ends of cloned viral genomes, or by sequencing a 250 base pair fragment of a highly conserved open reading frame that had been amplified using the polymerase chain reaction technique. The viruses being studied were compared both among themselves and with other Subgroup I geminiviruses of known DNA sequence, on the basis of sequence (nucleotide and amino acid) and restriction map data. Distance matrix methods were used to infer phylogenetic relationships between Subgroup I geminiviruses from restriction map and sequence data. Phylogenies deduced from sequence data were considered to be more accurate than those deduced from map data. Regardless of the method of analysis used, however, the relationships between the Subgroup I geminiviruses studied here remained constant. Thus, three strains of MSV (maize, Setaria, and Eleusine strains) were distinguished. Streak viruses distinct from MSV were also identified: panicum streak virus (PanSV), and two distantly related strains (Natal and Mauritius) of sugarcane streak virus (SSV). Restriction mapping of different geographical isolates of the maize strain of MSV demonstrated that variation existed within a single strain of virus. RFLP analysis indicated that minor variation existed between virus genomes within single diseased plants. Methods used to. type Subgroup I geminiviruses were evaluated, and discrepancies in the serological typing of geminiviruses from Subgroups I and III were pointed out. A unified scheme was proposed for distinguishing between distinct Subgroup I geminiviruses and strains of geminiviruses. The origins of maize and sugarcane streak viruses were speculated upon.

The prevalence of HIV-1 is highest in Sub-Saharan Africa. Protective immune responses directed against HIV are complex and involve both cellular and humoral immunity. Based on the recent finding that the best correlate of protection against the first protective prophylactic RV144 vaccine were HIV-specific antibody responses, including those mediating natural killer (NK) cell antibody-dependent cellular cytotoxicity (ADCC), there has been considerable interest in measuring alternative roles for HIV-specific binding antibodies. The aim of this MSc dissertation was to optimise and compare two high-throughput flow cytometry based approaches - the GranToxilux and PanToxilux assays - to measure HIV-specific ADCC responses. To do this, NK cells from a panel of healthy HIV-negative individuals were screened for their ability to directly kill the tumour cell line K562, as a measure of direct NK cell cytotoxicity. The individual with the highest granzyme B and caspase activity against K562 cells was chosen as the universal NK cell donor for this study.

Includes bibliographical references.
Immune system pressure on HIV-1 replication drives the antigenic changes seen over time. The monitoring of changes in viral sequences can provide important information on the nature of the immune response and the correlates of protection. Viral diversification may also occur due to other selective pressures such as cell availability and differences in viral fitness. Information on the genetic characteristics of HIV-1 variants present in the mother and her infected infant are useful data for establishing whether any common features exist between source infection and transmitted genotypes. This helps in the understanding of the mechanism of transmission and the selective pressures occurring during and following transmission. The overall aim of this study was to explore alternative methods other than DNA sequencing for the monitoring of genetic diversity in the third variable region (V3) of the HIV-1 env gene of integrated HIV-I variants in peripheral blood mononuclear cells (PBMC's) derived from infected mother-child pairs. Two methods for displaying DNA differences were compared: I-leteroduplex Mobility Assay (I-IMA) and Base Excision Sequence Scanning (BESS). These methods were validated using sequence data. Extracted PBMC DNA from infected mother-child pairs were used to amplify the V3 region by nested PCR. DNA fragments were cloned into plasmid vectors and analyzed by HMA and BESS to establish subtype and intrasample genetic diversity. In addition, a PCR-ELISA quantitation system was developed to measure copy numbers of integrated HIV-1 genomes in order to confirm whether a sufficient number of template molecules were present to be representative of the total viral quasispecies. In conclusion, this study compared two methods (HMA and BESS) as cost-effective alternatives to DNA sequencing for HIV-1 diversity studies. in addition, a novel application of the BESS assay was demonstrated. Diversity studies are reliant on estimation of adequate input of amplifiable copies. The PCR-ELISA quantitation system developed provided an efficient and specific method for determining DNA copy number.

Introduction: Cytomegalovirus (CMV) drug resistance mutations, because of the widespread use of ganciclovir, have been widely reported in international literature, particularly in the post-transplant setting. However, a genotypic assay to detect CMV drug resistance is not available in South Africa and the prevalence of these mutations is therefore unknown. We aimed to document the prevalence and types of CMV UL97 mutations following exposure to ganciclovir in adult and paediatric oncology patients, transplant recipients and HIV-infected patients in the local tertiary level hospitals: Red Cross War Memorial Children's Hospital, Groote Schuur Hospital and Tygerberg Hospital. Methods: The study had two components, the first component being a retrospective cross-sectional study using stored extracted DNA from patients with serially elevated CMV viral load levels. Thirty-three samples were tested for this component. The second component was a prospective case series on patients who were referred by clinicians for genotypic testing in whom CMV drug resistance was suspected. Eight samples were tested for this component. The CMV UL97 gene was amplified by conventional nested polymerase chain reaction (PCR) and Sanger sequencing performed. Results: CMV UL97 mutations were identified in five of thirty-three (15%) retrospectively screened samples while the prospective testing of eight patient samples identified drug resistance mutations in three patients (38%). Overall 8/41 (20%) patients had CMV UL97 mutations. A trend of higher risk for development of drug resistance mutations among haematological oncology patients 7/23 (30%) compared to solid organ transplant recipients 1/10 (10%) was observed, however, this difference was not statistically significant (P=0.306). Conclusion: This study, the first of its nature in South Africa, identified the presence of CMV UL97 mutations conferring resistance to ganciclovir in the haematological oncology, primary immunodeficiency and solid organ transplant patients in the Western Cape. The assay successfully detected CMV UL97 drug resistance mutations in whole blood and cerebrospinal fluid clinical samples. Ongoing viral replication in the background of intensive immunosuppression and prolonged antiviral therapy selects for the emergence of CMV UL97 drug resistance mutations.

Introduction: HIV viral load testing is the preferred monitoring approach for HIV infected patients on combination antiretroviral therapy (cART) as it is more sensitive than CD4 count and clinical monitoring. In resource limited settings, timely plasma separation and transportation to testing laboratories is a major barrier to the access of HIV viral load testing. The 2015 World Health Organisation guidelines recommend that cART should be initiated in all adults and children living with HIV regardless of disease stage or CD4 count, thereby escalating the demand for HIV viral load testing. Potential solutions to expand implementation and scale up of viral load testing in low and middle income countries are whole blood testing through point of care (POC) viral load assays or dried blood spots (DBS) collected at the health facility. Utilization of whole blood instead of plasma would simplify sample collection, storage and transportation requirements and be cost effective. However, the paucity of studies comparing whole blood HIV viral load across different test platforms, especially in the correct classification of virological failure, has resulted in the lack of a standardised programmatic approach to whole blood viral load testing. Methods: We evaluated four HIV whole blood viral load test methods namely Alere q HIV-1/2 POC, Abbott RealTime HIV-1 DBS original and updated protocols, and Roche CAP/CTM DBS free virus elution (FVE) protocol, against the standard of care, plasma viral load, on 299 samples across the viral load spectrum from South African patients on cART. Virological failure was defined at >1000 copies/ml. Proportions of correct classification of virological failure and overall correlation with plasma were used for evaluating each method's performance. Results: Alere q, Abbott original and updated, and Roche FVE correctly classified virological failure in 61%, 89%, 87% and 76% of all samples tested respectively. The performance varied across plasma viral load categories. Alere q showed good correlation above plasma viral load of 1000 copies/ml, with correct classification of virological failure in 100% of samples. However, below the plasma threshold of 1000 copies/ml, Alere q demonstrated significant over-quantification, resulting in reduced specificity and upward misclassification of virological failure in 39% of all samples tested. Abbott original and updated also had good sensitivity of 98% and 91% respectively and the best overall correlation with plasma (r² = 0.76 and 0.72 respectively), but there was upward misclassification in 10% and 8% of samples tested respectively. Roche FVE had the best specificity of 99% but with significantly reduced sensitivity of 53%, especially between 1000–10,000 copies/ml of plasma, resulting in downward misclassification in 24% of all samples tested. Greatest variability between the different testing methods was seen when plasma viral load was 40-1000 copies/ml. Correlation was best for all whole blood viral load assays at >10,000 copies/ml. Conclusion: The key finding highlighted by this study is the great variability between the different whole blood test methods. Various factors influence the ability to quantify whole blood HIV viral load such as input volume used in each assay vary, sample treatment/processing (DBS versus fresh blood samples versus FVE), extraction (RNA selective, total nucleic acid extraction), amplification target and detection methods are different for each of the platforms tested. Based on our study, Alere q and Abbott DBS need to raise their whole blood threshold for virological failure in order to reduce upward misclassification and Roche FVE needs to achieve better sensitivity around its limit of detection. Receiver operating characteristic curve analysis can be used to determine the optimum threshold of virological failure for each assay.

The United States Environmental Protection Agency (USEPA) has established the Total Coliform Rule as an indication of health risks associated with microbial contamination of drinking and ground water. In addition, the fecal coliform test is used as an indicator to reflect the suitability of use by consumers of class A biosolids. However, numerous studies have shown that bacterial indicators are not predictive of enteric viruses, such as human adenovirus (HAdV), which are much more resistant to treatment methods than bacteria. Enteric viral contamination of estuarine waters and locally-harvested shellfish as a result of receiving effluent from wastewater treatment plants, as well as run-off from agricultural land treated with biosolids, can have serious implications for human health. Preliminary results suggest that HAdV is present in biosolids, wastewater effluent, estuarine waters receiving effluent and shellfish harvested from these receiving waters. The density, persistence and infectivity of human adenovirus in these environmental matrices are not known. The focus of this research was to address the presence, persistence and viability of HAdV in all four matrices. Presence and density of the virus was established through the use of a nested polymerase chain reaction (PCR) and quantitative PCR (qPCR). HAdV DNA was detected in 21 of the 26 biosolid samples and 21 of the 24 effluent samples assayed. The treatment method employed in the processing of the samples appeared to have an effect on the detection and concentration of HAdV DNA. Persistence of HAdV DNA in estuarine water was addressed in an in situ study using seeded microcosms containing either sterile river water or unfiltered river water under various environmental conditions during the spring, summer and fall. Unfiltered river water collected during the summer had the greatest deleterious effect on HAdV DNA persistence. HAdV DNA was most persistent, under all environmental treatments, during the fall. An in vitro study of sterile river water confirmed that temperature, not salinity, had a greater effect on HAdV DNA degradation. Laboratory tank studies revealed that oysters are capable of filtering and retaining HAdV from contaminated water. In each of the three tank studies conducted, HAdV DNA was detected in tissue samples from oysters exposed to seeded river water for 18 hours. It was also established that the oysters could depurate the virus, in an open system, in as little as three days. Integrated cell culture (ICC) - qPCR was used to determine the viability of detected viral particles. No direct correlation between the detection of HAdV DNA and the presence of viable viruses was found. Frequently, samples that contained HAdV DNA failed to produce viable virions. Current research corroborates these results, suggesting the detection and persistence of viral DNA is not sufficient evidence to support the assumption of viability.

Microbiology and Immunology
Ph.D.
EBV is a human gammaherpesvirus that infects approximately 95% of the population worldwide and is associated with 1% of human cancer incidence. The EBV-genome-encoded latency membrane protein 1 (LMP1) is expressed in nearly all of EBV-associated malignancies and is the major EBV oncogene. Because LMP1 is essential for both establishing a latent EBV infection and promoting tumorigenesis, defining its role in these processes is critical in order to understand EBV-associated cancer development. Due to the importance of epigenetics in regulating EBV gene expression during latency, the goals of this dissertation were two-fold: to define the mechanisms of host epigenetic regulation of LMP1 expression and to establish how LMP1 alters the host epigenome. The host uses its epigenetic machinery to regulate EBV during latency. One mechanism is through the host protein CTCF, which acts as an insulator to prevent the intrusion of heterochromatin into euchromatic regions and prevent DNA methylation within the EBV genome. CTCF also participates in the three-dimensional organization of the EBV episome through the formation of a network of long-range interactions that, in turn, further regulate EBV gene expression. CTCF binds at several key regulatory regions in the EBV genome, including upstream latent gene promoters, with the strongest CTCF binding site in the EBV genome positioned at the LMP1 locus. However, the functional role of CTCF binding in regulating LMP1 has yet to be established. To define the role of CTCF binding at the LMP1 locus we utilized a genetically modified EBV bacmid that contained a site-directed deletion of the CTCF binding site at the LMP1 region. Using EBV-positive B cells we found that CTCF binding at the LMP1 locus is a key regulator of LMP1 expression. Loss of CTCF binding changed the epigenetic profile at the LMP1 locus resulting in the loss of euchromatic and a concomitant gain of heterochromatic histone modifications, as well as an associated increase in DNA methylation near the LMP1 promoter. These epigenetic changes mediated decreased LMP1 expression. Additionally, through chromosome conformation capture (3C) assays, we established that DNA loop formation between the LMP1 loci and the viral enhancer OriP is strictly dependent upon CTCF binding at LMP1. Taken together, these observations suggest an epigenetic mechanism by which the host, through CTCF, contributes to the regulation of LMP1 expression. LMP1 expression is also regulated by structural elements within the EBV genome called terminal repeats (TRs). The number of TRs within an EBV genome varies based upon the circularization of the viral genome, and LMP1 expression is inversely correlated with the number of TRs in epithelial cells. However, the mechanism by which TR number regulates LMP1 expression has yet to be elucidated. We hypothesized that TR number differentially regulates the epigenetic state of the LMP1 promoters to either promote or suppress their activity. By examining the epigenetic state at both LMP1 promoters in isogenic cell lines with different numbers of TRs, we identified differences in histone modifications and DNA methylation at the LMP1 promoters. Furthermore, decreasing the number of TRs eliminated a chromatin loop formed between the LMP1 loci and the viral enhancer OriP. This data suggests that the number of TRs regulates the epigenetic state of LMP1 and ultimately determines promoter usage, which is necessary for LMP1 expression. While the previous work focused on the host mechanisms that regulate LMP1 expression, we also explored if the reverse relationship existed, that is, if EBV hijacks the host epigenetic machinery as a means to alter host gene expression. LMP1 contributes to host cell proliferation and survival through the aberrant activation of biochemical signaling pathways that also modulate epigenetic regulation. Because the PARP1-mediated post-translational modification poly(ADP-ribosyl)ation (PARylation) regulates EBV latency, and LMP1 activates a PARP1 regulator MAPK/ERK, we hypothesized that LMP1 drives the induction of PARP1-mediated PARylation. PARP1 facilitates gene transcription by maintaining a euchromatic state, and as a result, the disruption in epigenetic regulation mediated by LMP1 through PARP1 may also induce changes in host gene expression, including genes that contribute to EBV-mediated tumorigenesis. A panel of EBV-positive cell lines were found to have higher PAR levels than EBV-negative B cells, establishing a relationship between latent EBV infection and cellular PARylation. Because expression of the EBV oncoprotein LMP1 was sufficient to induce cellular PARylation, we explored the model that disruption in cellular PARylation driven by LMP1 expression subsequently promotes epigenetic alterations to elicit changes in host gene expression. PARP inhibition resulted in the accumulation of the repressive histone mark H3K27me3 at a subset of LMP1-regulated genes through induction of EZH2 expression. Inhibition of PARP also suppressed the expression of LMP1-activated genes and LMP1-mediated cellular transformation, demonstrating an essential role for PARP activity in LMP1-induced gene expression and cellular transformation associated with LMP1. This dissertation reveals for the first time the importance of the host protein CTCF and the effect of the number of viral TRs in epigenetically regulating the expression of the oncogenic protein LMP1. This dissertation further establishes a mechanism by which EBV hijacks the host epigenetic machinery and identifies a novel role for LMP1 in driving the expression of tumor-promoting host genes by blocking the incorporation of an inhibitory epigenetic modification through the activation of PARP1, suggesting that PARP1 may be a target for treatment of EBV-associated malignancies.
Temple University--Theses

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.