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Las enfermedades virales constituyen uno de los principales factores limitantes del cultivo de Lilium spp. Los virus más comunmente encontrados en Lilium spp. en diferentes países son Lily symptomless virus (LSV), Lily mottle virus (LMoV) y Cucumber mosaic virus (CMV). El objetivo de esta tesis fue evaluar la presencia de LSV, LMoV y CMV en Lilium en Argentina, y en el caso de que estos virus estuvieran presentes se planteó estimar su incidencia en diferentes híbridos cultivados y localidades de producción, así como evaluar diferentes alternativas para obtener plantas de sanidad controlada. Se trabajó con híbridos de Lilium longiflorum (Ll), Asiáticos (As), Orientales (Or), Longiflorum × Asiáticos (LA), Longiflorum × Orientales (LO) y Orientales × Trompeta (OT). Se llevó a cabo el reconocimiento de la sintomatología asociada a la infección viral y se identificaron áfidos posibles vectores de virus en parcelas de producción de Lilium. Se evaluaron plantas sintomáticas y asintomáticas mediante DAS-ELISA y RT-PCR. Para esto se utilizaron anticuerpos policlonales específicos contra las proteínas de la cápside de LSV, LMoV y CMV, así como primers degenerados y específicos, respectivamente. Se pudo detectar la presencia de los tres virus. Las secuencias del gen de la proteína de la cápside (CP) y de aminoácidos de la CP de LSV, LMoV y CMV, aislados desde plantas cultivadas en Argentina, presentaron una elevada similitud con las de otras cepas de los mismos virus registradas en el GenBank. Asimismo, se detectaron infecciones virales en bulbos provenientes de Holanda, lo cual indica una posible vía de entrada de los virus. La incidencia de virosis fue evaluada por medio de DAS-ELISA en plantas de bulbos provenientes de siete localidades de Argentina entre las latitudes 26° 56′ S y 43° 03′ S, y longitudes 65° 21′ O y 71° 29′ O. Los virus detectados en órden decreciente fueron LSV (42,1%), LMoV (35,6%) y CMV (20,0%) y se encontraron en infecciones simples o mixtas. La incidencia de virus varió entre los híbridos desde 36% (O ‘Montecristo’) a 94,7% (Ll ‘Avita’) en 2006, y desde 38,9% (OT ‘Yelloween’) a 82,1% (LO ‘Triumphator’) en 2007, con una incidencia total de 64,1% y 70,7% en 2006 y 2007, respectivamente. También se encontró una variación en la incidencia entre localidades. La mayor incidencia de virus (89,6 y 87,6% en 2006 y 2007, respectivamente) se observó en Bahía Blanca, y la menor incidencia fue detectada en Trevellin (47,4%) en 2006 y en Malargüe (48,6%) en 2007. La alta incidencia de virus en el cultivo de Lilium puede estar asociada a la utilización de bulbos infectados para la propagación y a la falta de medidas de control de vectores. Para ajustar un sistema de obtención de bulbos de sanidad controlada se utilizó material infectado con virus de híbridos cultivados en Argentina. Los bulbillos fueron producidos por scaling ex vitro o por multiplicación in vitro con o sin tratamiento de termoterapia y/o quimioterapia. Posteriormente, se extrajeron y cultivaron los ápices meristemáticos. La obtención de plantas sanas fue dependiente de los híbridos, de los virus presentes y de los tratamientos aplicados. La aplicación de termoterapia ex vitro en escamas de bulbos durante la diferenciación de bulbillos (scaling) y la obtención de meristemas desde dichos bulbillos se propone como una innovación para un protocolo exitoso de obtención de bulbos de Lilium spp. de sanidad controlada. / Viral diseases are one of the main limiting factors in lily production. The viruses most commonly found in lilies worldwide are Lily symptomless virus (LSV), Lily mottle virus (LMoV) and Cucumber mosaic virus (CMV). The aim of this thesis was to evaluate the presence of LSV, LMoV and CMV in Lilium in Argentina, and when present, to estimate their incidence in different hybrids and localities, and also to evaluate different alternatives to produce virus-free plants. Hybrids evaluated belong to six groups: Lilium longiflorum (Ll), Asiatics (As), Orientals (Or), Longiflorum × Asiatics (LA), Longiflorum × Orientals (LO) and Orientals × Trumpet (OT). Different symptoms associated to viral infection were recognized and aphid species with potential capacity to transmit viruses were identified in Lilium production plots. Symptomatic and asymptomatic plants were evaluated by DAS-ELISA and RT-PCR using polyclonal antiserum against the capsid protein of LSV, LMoV and CMV, and with degenerate and specific primers, respectively. The gene nucleotide and amino acid sequences of the coat protein of LSV, LMoV and CMV were highly similar to those from other isolates of the same viruses registered in the GenBank. Infections were detected in bulbs imported from the Netherlands, which indicates a possible way of entry of viruses into our country. Virus incidence was evaluated by DAS-ELISA in bulbs coming from seven Argentinean localities. The areas surveyed were between latitude 26° 56′ S and 43° 03′ S, and longitude 65° 21′ W and 71° 29′ W. Virus infection in decreasing order were LSV (42.1%), LMoV (35.6%), and CMV (20.0%) and single or mixed infections were detected. Virus infection varied among hybrids from 36.0% (Oriental ‘Montecristo’) to 94.7% (Lilium longiflorum ‘Avita’) in 2006, and from 38.9% (OT ‘Yelloween’) to 82.1% (LO ‘Triumphator’) in 2007, with an overall infection incidence of 64.1 and 70.7% in 2006 and 2007, respectively. A variation in virus infection incidence among localities was also detected. The highest virus infection incidence (89.6 and 87.6% in 2006 and 2007, respectively) was observed in Bahía Blanca (38° 44′ S, 62° 16′ W). The lowest virus infection incidences, were 47.4% in Trevellin (43° 03′ S, 71° 29′ W) in 2006, and 48.6% in Malargüe (35° 28′ S, 69° 35′ W) in 2007. The high occurrence of viruses infecting lily crops in Argentina could be due to the use of infected bulbs during propagation and also to the absence of preventive virus vector control measures. The starting material to set a system for obtaining virusfree plants were infected hybrids grown in Argentina. For bulblet production, scales or scale sections were cultured ex vitro or in vitro, with or without thermotherapy and /or chemotherapy. Then, meristem-tips were cultured in vitro. Different percentages of virus-free plants were obtained depending on the hybrid, the treatment and the viruses present in infected tissues. The application of thermotherapy to bulb scales during the differentiation of bulblets (scaling) and the extraction of meristem-tips from these bulblets is an innovative proposal for a successful protocol for obtaining virus- free Lilium spp plants.

A rapid coliphage detection assay (RCDA), based on the phage-induced release of b-galactosidase from cells of Escherichia coli (Ijzerman, M., J.O. Falkinham III and C. Hagedorn. (1993) [A liquid, colorimetric presence-absence coliphage detection method. J. Virol. Meth. 45:229-234] was modified to reduce the number of steps required to perform the assay, remove the need for specialized media and buffers, reduce the volumes required, and simplify growth and reaction conditions. Tolerances of the assay were defined at each step of the assay. The number of steps has been reduced from 12 to 7. The b-galactosidase reaction buffer was eliminated. Culture volumes were reduced from 25 ml to 5 ml and reaction volumes were reduced from 10 ml to 0.5 ml. Optimal growth conditions were 37 o C with orbital shaking at 200 rpm, a one hour subculture time and an incubation of subculture with water sample for two hours. Color development occurred at 37 o C in 30 minutes. The changes and modifications of the assay increased the ease of its performance without sacrificing the ability of the assay to detect as few as two phage particles per sample. By understanding the tolerances of the assay, technical support representatives of companies producing kits modeled after the assay will be prepared to answer questions from customers concerning possible kit failures or user error. / Master of Science

Il a récemment été démontré que la nucléocapside du virus de l’hépatite C (VHC) subit l’ajout post-traductionnel d’un groupement acyl gras sur le résidu C172. Cette modification, appelée palmitoylation, est requise pour la production de virions infectieux. Chez les eucaryotes, la palmitoylation est catalysée par les enzymes de la famille DHHC. Notre objectif était d’identifier lesquelles des 23 protéines DHHC présentes chez les mammifère possèdent une activité palmitoyl-acyl transferase (PAT) sur la nucléocapside du VHC. Nous avons d’abord étudié l’expression et la localisation des protéines DHHC dans les hépatocytes humaines. Nous avons ensuite mesuré la variation du niveau de palmitoylation de la nucléocapside lorsque chaque DHHC candidate est surexprimée ou réprimée. Ce criblage a mené à l’identification de 5 enzymes qui exercent une activité PAT sur la nucléocapside du VHC : DHHC 1, 2, 3, 6 et 7. Leur co-localisation avec le substrat viral a été confirmée. / As previously demonstrated, a fatty acid group is post-translationnally added on the residue C172 of Hepatitis C virus (HCV) core protein and this modification, termed palmitoylation, is required for viral assembly. In eukaryotes, palmitoylation is performed by a family of enzymes sharing a closely related DHHC motif. The purpose of this study was to identify which of the 23 mammalian DHHCs could perform palmitoyl acyltransferase activity on the HCV core protein. First, we characterized the expression and localization of DHHC enzymes in human hepatocytes. Then we evaluated the variation in core palmitoylation levels when each cellular DHHC proteins was over-expressed or repressed. This screen identified five enzymes with PAT activity on HCV core protein; DHHC 1, 2, 3, 6 and 7. Their co-localization with the viral protein was also demonstrated. These findings pave the way for future studies on the role of core palmitoylation during the HCV infection.

Le virus de l'hépatite C infecte plus de 3% de la population mondiale et il n'existe aucun vaccin disponible à ce jour afin de prévenir l'infection. La cirrhose et la stéatose sont des manifestations pathologiques de l'infection chronique auxquelles s'ajoute également le développement de tumeurs chez certains patients. En utilisant des peptides marqués, des protéines recombinantes et des capsides produites in vivo, j'ai tenté de montrer que la protéine de la capside du VHC pénètre dans les cellules humaines, sous forme libre ou encapsidée. Les résultats démontrent que les deux formes de la protéine permettent l'entrée dans la cellule. Il semble également que le premier domaine basique (a.a. 5-24) soit impliqué dans l'entrée de la protéine libre. Le mécanisme d'entrée est inconnu. Cette entrée alternative permettrait au VHC d'élargir son tropisme cellulaire et de contribuer au développement des pathologies et à l'établissement de la persistance chez l'hôte.

Peritoneal carcinomatosis (PC), the intra-abdominal dissemination of malignancy, is equated with a 5-year survival of 15%, depending on the source. Appendiceal PC is a challenge to treat as cancer cells are embedded in copious amounts of mucin and are difficult to target. Oncolytic viruses (OVs) preferentially replicate and lyse cancer cells and present a targeted, novel strategy for PC. The hypothesis of this study is that appendiceal PC will show variable susceptibility to OVs and that protein expression in these tumours will predict OV replication efficiency. Human appendiceal PC infected ex-vivo with 4 different OVs displayed variable infectivity and replication by fluorescence microscopy and plaque assay. Immunohistochemistry analysis revealed differential expression of IRF3, pERK and TK in tumour compared to normal appendix. No correlation of protein expression with viral replication was observed. Personalizing OV therapy will be critical in the optimization of future care of patients treated with this modality.

Peritoneal carcinomatosis (PC), the intra-abdominal dissemination of malignancy, is equated with a 5-year survival of 15%, depending on the source. Appendiceal PC is a challenge to treat as cancer cells are embedded in copious amounts of mucin and are difficult to target. Oncolytic viruses (OVs) preferentially replicate and lyse cancer cells and present a targeted, novel strategy for PC. The hypothesis of this study is that appendiceal PC will show variable susceptibility to OVs and that protein expression in these tumours will predict OV replication efficiency. Human appendiceal PC infected ex-vivo with 4 different OVs displayed variable infectivity and replication by fluorescence microscopy and plaque assay. Immunohistochemistry analysis revealed differential expression of IRF3, pERK and TK in tumour compared to normal appendix. No correlation of protein expression with viral replication was observed. Personalizing OV therapy will be critical in the optimization of future care of patients treated with this modality.

The thesis deals with the occurrence of viral pathogens on grape - Chardonnay clones. Monitored and evaluated clones were 8, 95, 96 (foreign) on rootstocks 1103 Paulsen, SO4, Kober 5 BB and 110 Richter and VP-155/6-VP 161/6, PO-158/7 and PO-160 / 1 (Czech) on the rootstock Kober 5 BB. All plants have a controlled origin. The experiment was conducted in 2013 on the test sites in the cadastral Perná. At the beginning of vegetation were recorded values on 1 herbaceous plant -- sprouting and not-sprouting buds. During vegetation were the plants observed. From the monitored plants were harvested grapes and following parameters were checked: number and weight of the grapes, weight of berries and the stem. Furthermore, before leaf the leaves were sampled for subsequent ELISA test for viral diseases Grapevine fanleaf virus, Arabis mosaic virus, Grapevine fleck virus, Grapevine leafroll-associated virus 1 and 3, Grapevine virus A. All values were evaluated by statistical program Statistica 10.

Les phlébovirus présentent sont présents dans toutes les régions du globe. Certains phlébovirus transmis par phlébotomes provoquent une maladie fébrile et des infections du système nerveux central. Depuis, de plus en plus de données montrent que la péninsule des Balkans joue un rôle majeur dans l'émergence de maladies à transmission vectorielle. Au début de ce travail, on comptait un nombre très limité de phlébovirus identifiés et isolés dans cette région. Une étude intégrée et transdisciplinaire en vue d'un inventaire des virus circulant dans pays des Balkans. (i) Un total de 3,850 phlébotomes sont été recueillis dans sept pays des Balkans en 2014 et 2015. Ils ont été testés pour la présence d'ARN viral et inoculé sur des cellules VERO afin d'isoler le virus détecté; (ii) des études de séroprévalence utilisant des tests de neutralisation ont été effectuées sur des échantillons de bovins et de moutons pour évaluer à deux agents pathogènes humains : le virus Toscana (TOSV) et le virus Sandfly fever Sicilian virus (SFSV). Nos résultats se composent de (i) la découverte et le séquençage de 3 nouveaux phlébovirus appartenant à 2 espèces différentes, (ii) la première identification du genotype B de TOSV en Croatie, (iii) la preuve de la co-circulation de deux genotypes (B et C) de TOSV, (iv) des taux d'anticorps neutralisants qui sont beaucoup plus élevés chez les bovins et les moutons pour le SFSV que pour TOSV. En conclusion, les résultats obtenus au cours de ce travail démontrent qu’es les Balkans représentent une zone de très importante activité pour les phlebovirus et donc mérite une surveillance particulière à cause du risque d’émergence et de dissémination. / Phleboviruses have a worldwide distribution. In the areas where sand flies are present, some of the sandfly-borne phleboviruses cause febrile illness and central nervous system infections. Sandfly fever was first reported in the Balkan Peninsula at the end of the 19th century. Since there is accumulating data showing that the Balkan peninsula plays a major role in the emergence of vector-borne diseases. At the outset of this work, a very limited number of phleboviruses had been identified and isolated in this region. To fill this gap, an integrated and transdisciplinary study was designed aiming at an inventory of viruses circulating in Balkans and associated seroprevalence studies using domestic animals: (i) a total of 3,850 sandflies were collected in seven Balkan countries (Albania, Bosnia-Herzegovina, Croatia, Kosovo, Montenegro, Republic of Macedonia and Serbia) in 2014 and 2015. They were tested for the presence of viral RNA and inoculated on VERO cell for virus isolation; (ii) seroprevalence studies using neutralisation tests were performed on cattle and sheep samples to assess the level of exposure to two human pathogens, Toscana virus (TOSV) and Sandfly fever Sicilian virus (SFSV). Our results consist of (i) the discovery and sequencing of 3 novel phleboviruses belonging to 2 different species, (ii) the identification for the first time of TOSV lineage B in Croatia, (iii) evidence of co-circulation of two lineages (Lineage B and C) of TOSV, (iv) rates of neutralising antibodies that are much higher in cattle and sheep for SFSV than for TOSV. Together the findings obtained during this work demonstrate that the Balkan area is a hot spot for phleboviruses.