Papilloomiviiruse transkriptsiooni ja regulaatorvalgu E2 uurimine

Väitekirja elektrooniline versioon ei sisalda publikatsioonePapilloomiviirused on viiruste perekond, mis nakatab nii inimeste kui loomade epiteelrakke ja põhjustab papilloome ehk näsakasvajaid, mis võivad teatud tingimustes areneda halvaloomulisteks kasvajateks. Inimese papilloomiviiruseid on teada üle 200 tüübi ja nad jagatakse kaheks grupiks nende koespetsiifilisuse alusel: limaskesti nakatavad papilloomiviirused ja naharakke nakatavad papilloomiviirused. Naha papilloomiviirused on siiani pälvinud vähem tähelepanu, sest nende võimalik seos nahakasvajatega on tulnud ilmsiks alles viimasel kümnendil. Meie keskendusime oma töös naha papilloomiviirusele HPV5, mida on viimasel ajal hakatud seostama nahakasvajate tekkega, aga mida on siiani ikkagi suhteliselt vähe uuritud. Meie töö tulemusena valmis HPV5 transkriptsiooni kaart, mille iseloomustamiseks me kasutasime inimese sääreluu kasvaja rakuliini U2OS. Selle rakuliini abil õnnestus meil kirjeldada 14 erinevat viiruse mRNAd. Edasises töös keskendusime viirusvalgu E2 uurimisele, mis on põhiline viiruse elutsükli regulaator. E2 valgul on lisaks täispikale valgule veel kaks isovormi, valgu C-terminaalset osa sisaldav E2C ja alternatiivse splaissinguga saadud E8/E2. Me uurisime E2 paiknemist rakus ja leidsime, et lühike E8 valgu järjestus on piisav selleks, et valk suunata raku tuuma. Meile teadaolevalt on see järjestus lühim tuuma suunav järjestus, mis on siiani teada. Tänu proteoomika arengule on siiani leitud üle 200 E2 partnervalgu, millest enamuse funktsioon viiruse elutsüklis on siiani teadmata. Meie leidsime uue E2-ga seonduva rakulise valgu, milleks on tuumamüosiin 1. See on esimene müosiini perekonda kuuluv E2 partnervalk ja katsed näitasid, et tuumamüosiin 1 mõjutab HPV5 DNA paljunemist rakus.Cutaneous papillomaviruses infect human cutaneous epithelium and in most cases these infections pass without symptoms, but in some instances, they can cause lesions and induce squamous cell carcinomas. One of the most prevalent virus types detected in skin cancer is HPV5. We tried to bring through our work more understanding about the properties of these viruses and focused our attention to HPV5 and to the first part of its life-cycle in the cells. We wanted to characterize HPV5 more thoroughly at RNA level and managed to identify HPV transcription map. We also studied papillomavirus main regulator protein E2, which influences all main viral life events. We studied E2 localization in the cell and identified short nuclear targeting signal, that sends proteins to the nucleus. We also found new E2 interaction partner nuclear myosin 1, which influences HPV5 replication

High frequency of JCV DNA detection in prostate cancer tissues

BACKGROUND: Prostate cancer (PC) represents the most frequently diagnosed cancer in men. Exposure to infectious agents has been considered to induce prostatic inflammation and cancerous transformation. Controversial data exist concerning the role of the human polyomaviruses BK (BKV) and JC (JCV) in PC etiology. Therefore, a possible association between these polyomaviruses and PC was investigated. MATERIALS AND METHODS: Urine, blood and fresh prostatic tissue specimens were collected from 26 patients with PC. The presence of BKV and JCV, the possible non-coding control region (NCCR) variations and the genotyping analysis of viral protein 1 (VP1) of both viruses were assessed. RESULTS: Data showed a preferential viral re-activation in the urinary compartment and a statistically significant prevalence of JC viruria and of BKV in PC tissues. A BKV DDP-like NCCR sequence was isolated in two patients, whereas JCV NCCR was consistently of an archetypal structural organization. A prevalence of the European genotypes was observed for both viruses. CONCLUSION: Our data demonstrated the presence of JCV DNA in 14/24 (58.3%) cancerous prostatic tissue specimens, confirming the results obtained in a previous study, in which JCV has been defined as common inhabitant of the prostate, and opening the discussion about its potential role in PC

Diversity of Skin Infections

The identification of infectious agents in cancer has been one of the most rewarding endeavors in cancer research. Currently about 20% of the global cancer burden is linked to an infection. A common characteristic of virus-induced cancer is an increased incidence in immunosuppressed patients, presumably because of impaired host control of virus. Yet non-melanoma skin cancer (NMSC), the cancer that increases most among the immunosuppressed, does not have an established link to infection. NMSC, including squamous cell carcinoma (SCC) and basal cell carcinoma, is the most common cancer among Caucasians. Ultraviolet radiation is an established risk factor. Human papillomaviruses (HPVs) have been established as the major cause of cervical cancer. Many NMSCs contain one or several cutaneous types of HPV. Exploration of a possible infectious etiology of NMSC requires an unbiased and comprehensive approach for detection of as many infections as possible in the tumor. We examined NMSCs and other presumably HPV-associated lesions for the presence of unidentified HPV types or other microorganisms, using a combination of multiple displacement amplification (MDA), which amplifies all DNA in a sample without any requirement of prior knowledge of the nucleotide sequence, degenerate “general HPV primers” PCR and high-throughput sequencing. The most common microbial DNA in NMSC was Staphylococcus aureus (S. aureus). We also identified sequences from at least 40 previously not described putative HPV types, of which three novel types (HPV 109, 112 and 114) and an HPV 88 isolate were cloned and completely sequenced. Prevalences and viral loads were investigated in skin and genital samples from different patient groups. S. aureus DNA was more commonly detected in SCC compared to healthy skin (odds ratio, 6.23; 95% confidence interval, 3.10 – 12.53). However, the study design could not determine the causality of the association. HPV 88, 109 and 112 were almost only found in their index patients, whereas HPV114 was found in 1.7% of the female genital samples. In summary, we find that there is a wide diversity of HPV types in the skin. The association of S. aureus with SCC raises the possibility of general susceptibility to infection in SCC. An association of NMSC with a specific infection remains to be found

Des algorithmes bioinformatiques pour la recherche des régions génomiques responsables d'une maladie

L'évolution des espèces est régie par les modifications stochastiques qui ont eu lieu au niveau du code génétique -l'ADN -composé d'une suite de petites molécules (les nucléotides). Selon l'ampleur de ces évènements, il y a d'abord des modifications à petite échelle, impliquant quelques nucléotides -les insertions, délétions et substitutions. Due à l'impossibilité actuelle de différencier les insertions des délétions, on les appelle communément indels. D'un autre coté, il ya des modifications à grande échelle -impliquant parfois des grandes régions génomiques ou des chromosomes. Les modifications à grande échelle les plus fréquentes sont: les duplications, translocations, inversions et délétions. Au cours de ce projet, nous avons développé une méthode de génomique comparée, capable de relier l'information épidémiologique, comme la carcinogenicité et l'invasivité des souches, aux séquences génomiques. Cette méthode permet de détecter des régions statistiquement significatives à analyser plus en détail par des biologistes, tout en étant capable de discriminer ce seuil à l'aide du calcul des p-values. Nous avons utilisé cette méthode dans l'étude du virus du papillome humain et de la bactérie Neisseria Meningitidis, bactérie responsable de la méningite. Pour le virus du papillome humain, notre méthode a été capable de détecter le domaine PDZ, une région du gène E6, qui est une condition sine qua non de la carcinogenicité du produit de ce gène. Au cours des analyses phylogénétiques de cette famille nous avons trouvé une corrélation statistiquement significative entre les événements à petite échelle et les données épidémiologiques. Par la suite nous avons proposé une séquence de tests pour orienter l'analyse statistique de cette corrélation. Nous avons également remarqué que la carcinogenicité est généralement monophylétique, donc issue d'un ancêtre commun. L'arbre phylogénétique inféré est le premier basé sur les génomes entiers, ce qui a permis d'étudier la variabilité des topologies de gènes par rapport à celle du génome. Pour la bactérie Neisseria Meningitidis nous avons montré qu'il est possible de syntoniser les fonctions de discrimination, pour établir la différence entre les régions responsables du maximum d'invasivité et celles qui ont un rôle structural dans ce processus, détection des structures moléculaires connues (i.e. les anses extra cellulaires, dans notre cas). Les résultats de nos travaux ont permis la mise à la disposition de la communauté internationale de deux bases de données, pour le VPH et le Neisseria, respectivement. Ces bases contiennent des régions candidates à être analysées en laboratoire par des biologistes. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : Algorithme pour la détection des régions carcinogènes, Événements évolutionnaires, Analyse de redondance, Arbre phylogénétique, Conception de vaccin, Mutations, Invasivité, Neisseria Meningitidis, Virus du papillome humain

Human papillomavirus (HPV69/HPV73) coinfection associated with simultaneous squamous cell carcinoma of the anus and presumed lung metastasis

Background: Human papillomaviruses (HPVs) have been linked to a variety of human cancers. As the landscape of HPV-related neoplasia continues to expand, uncommon and rare HPV genotypes have also started to emerge. Host-virus interplay is recognized as a key driver in HPV carcinogenesis, with host immune status, virus genetic variants and coinfection highly influencing the dynamics of malignant transformation. Immunosuppression and tissue tropism are also known to influence HPV pathogenesis. Methods: Herein, we present a case of a patient who, in the setting of HIV positivity, developed anal squamous cell carcinoma associated with HPV69 and later developed squamous cell carcinoma in the lungs, clinically presumed to be metastatic disease, associated with HPV73. Consensus PCR screening for HPV was performed by real-time PCR amplification of the L1 gene region, amplification of the E6 regions with High-Resolution Melting Curve Analysis followed by Sanger sequencing confirmation and phylogenetic analysis. Results: Sanger sequencing of the consensus PCR amplification product determined that the anal tissue sample was positive for HPV 69, and the lung tissue sample was positive for HPV 73. Conclusions: This case underscores the importance of recognizing the emerging role of these rare 'possibly carcinogenic' HPV types in human carcinogenesis. © 2020 by the authors. Licensee MDPI, Basel, Switzerland

Differential expression of microRNAs in bovine papillomavirus type 1 transformed equine cells

Bovine papillomavirus (BPV) types 1 and 2 play an important role in the pathogenesis of equine sarcoids (ES), the most common cutaneous tumour affecting horses. MicroRNAs (miRNAs), small non-coding RNAs that regulate essential biological and cellular processes, have been found dysregulated in a wide range of tumours. The aim of this study was to identify miRNAs associated with ES. Differential expression of miRNAs was assessed in control equine fibroblasts (EqPalFs) and EqPalFs transformed with the BPV-1 genome (S6-2 cells). Using a commercially available miRNA microarray, 492 mature miRNAs were interrogated. In total, 206 mature miRNAs were differentially expressed in EqPalFs compared with S6-2 cells. Aberrant expression of these miRNAs in S6-2 cells can be attributed to the presence of BPV-1 genomes. Furthermore, we confirm the presence of 124 miRNAs previously computationally predicted in the horse. Our data supports the involvement of miRNAs in the pathogenesis of ES

Human papillomavirus: pathogenesis and immunity

The involvement of human papilloma virus (HPV) in the aetiology and progression of cervical intraepithelial neoplasia (CIN) is still unresolved. This study was designed to assess the immunological responses to HPV types in patients presenting with varying degrees of CIN and in control groups, using in vitro measures of cellular and humoral responses.Lymphocyte proliferation assays (LPA) were performed using peripheral blood mononuclear cells (PBM) and various papillomavirus (PV) antigens. Twenty -five per cent (23/92) of patients with CIN responded to antigens derived from purified BPV, HPV -1 or HPV -2 with or without detergent disruption. The responses correlated with a past history of skin warts rather than cervical abnormalities, and the percentage of responders was similar to that in laboratory personnel (30 %) and lower than that in a group with recalcitrant common warts (50 %). Antigens specific to HPV -16 and HPV -18, in the form of bacterially expressed fusion proteins derived by the transcription and translation of the E6 and E4 open reading frames (ORF), occasionally produced specific positive responses, provided contaminating E.coli B galactosidase sequences had been removed during purification. Responses were low and suggested that the numbers of memory T cells specific to PV antigens were low and at the lower limit of detection of LPA. An indirect ELISA was developed to detect circulating IgG to PV antigens in colposcopy patients. Fifty per cent of patients had antibodies to disrupted HPV -1, HPV -2 or both, suggesting that a predominantly type- specific response was being detected. No correlation of immune responses with a degree of dysplasia or the presence of koilocytes in cervical biopsies was noted, but a high incidence of forgotten or inapparent past infection with cutaneous HPV types was found.In situ hybridisation (ISH) methods using non -radioactively labelled, cloned probes and synthetic oligonucleotide probes were developed for use on paraffin sections. Synthetic probes allowed a quicker, less destructive hybridisation protocol, with the sensitivity of detection being (y) improved by an anti -biotin -immunogold conjugated immunoglobulins -silver enhancement (IGSS) detection system. Double staining of PV antigen and nucleic acid on the same section was achieved. Synthetic oligonucleotides offer an exciting new tool for diagnostic virology, worthy of exploitation in many systems.Implantation of human foreskin infected with HPV -11 was shown to provide an animal model, albeit technically difficult, in which HPV could be produced, but a more practical technique of productive HPV infection in vitro is still required if the biology and pathogenesis of HPV infections is to be clarified further

Bovine papillomavirus type 2 infection in the pathogenesis of the Bovine Enzootic Hematuria : related oncological process

Tese de Doutoramento em Ciências Veterinárias, especialidade de Sanidade AnimalBovine Enzootic Hematuria (BEH) is a disease that affects cattle and water buffalos in specific regions of the globe, mainly associated with the chronic ingestion of bracken fern (Pteridium aquilinum) whose major characteristic is the development of urinary bladder tumors. One of the regions where this disease is endemic is the Azores Archipelago, Portugal. In these Atlantic islands bracken fern finds the appropriate conditions for continuous growth in the farmlands, and thus this toxic plant is frequently available for cattle consumption. The involvement of the Bovine papillomavirus type 2 (BPV2) in the genesis of this disease has been pointed out to be paramount, but some aspects involving the association between bracken fern and Papillomavirus remain unclear. This work aimed to study the role of this infectious agent in the oncological process observed in BEH, and is divided into seven chapters. In the first chapter, the major features, the implications and the etiology of this disease, with a special focus on ptaquiloside and BPV2, are reviewed. Additionally, urinary bladder tumors in man, and the genetic alterations associated to the disease, are briefly revised. The second chapter contains the specific objectives that were sought with the present work, and that resulted in the four experimental chapters that follow. In the third chapter, a detailed histopathological characterization of the urinary bladder lesions found in BEH-affected cattle from the Azores Archipelago which were used in the experimental work, is presented. Furthermore, an immunohistochemical study of the expression of cytokeratins in the epithelial neoplasms is also presented. Our results support previous findings that most urinary bladder BEH-associated lesions are neoplastic, and have epithelial origin. The decrease in the number of neoplastic cells expressing cytokeratin 7 in urinary bladder urothelial tumors was associated with increasing pathological grade and stage, whereas the decrease in the expression of cytokeratin 20 was only associated with increasing stage. The fourth chapter is dedicated to the prevalence and transcriptional activity of BPV2 in the urinary bladder lesions of BEH-affected cattle from the Azores Archipelago. The results obtained show that, though BPV2 is widely distributed within the bovine vii population of the Azores, the viral loads determined are very low, suggesting that BPV2 could be inactive, since no transcriptional activity was detected. In the fifth chapter a quantitative and qualitative gene expression study is presented, comparing the expression levels of cell cycle controlling genes and of a growth factor receptor gene in BPV2 positive and negative epithelial and endothelial bovine urinary bladder tumors of BEH-affected cattle. The expression of TP53, MDM2 and CCND1 genes was above normal, but no differences between BPV2 positive and negative epithelial and endothelial tumors were found. The expression of the EGFR gene was lower both in BPV2 positive epithelial and endothelial tumors when compared with BPV2 negative ones. This possible association between BPV2 infection and lower EGFR should be further investigated in future studies. The sixth chapter presents the preliminary in vitro study through which the effects of BPV2’s oncoproteins in a bovine cell line were assessed. Neoplastic transformation was not achieved, but changes in the cellular growth rate and gene expression patterns were observed, suggesting viral oncoprotein activities yet unknown. The main conclusions of this work are pointed out and discussed in the seventh chapter. The oncological process found BEH and its association with BPV2 are still a matter for further research. The results presented within this thesis provide a better insight into this subject but also open and support new questions worth investigating in future research.RESUMO - Infeção por Papilomavírus Bovino tipo 2 na patogenia do processo oncológico associado à Hematúria Enzoótica Bovina - A Hematúria Enzoótica Bovina (HEB) é uma doença que afeta bovinos e búfalos em regiões específicas do Globo particularmente relacionada com a ingestão do feto comum (Pteridium aquilinum), sendo um dos aspetos mais relevantes o desenvolvimento de tumores na bexiga dos animais afetados. Uma dessas regiões onde a HEB é endémica é o Arquipélago dos Açores, Portugal. O feto comum encontra nestas ilhas Atlânticas as condições apropriadas para o crescimento constante nas pastagens, fazendo com que esta planta tóxica esteja facilmente acessível aos animais criados em regime extensivo. A participação do Papilomavírus bovino tipo 2 (BPV2) também tem sido apontada como crucial na génese da HEB. Porém, alguns dos aspetos relacionados com a associação entre o feto comum e o Papilomavírus permanecem por esclarecer. O presente trabalho teve como objetivo estudar o papel deste agente infecioso no processo oncológico associado à HEB, estando dividido em sete capítulos..