Les phlébotomes sont les vecteurs reconnus de plusieurs arbovirus, en particulier du genre Phlebovirus, ainsi que de parasites du genre Leishmania. Les infections par les phlébovirus sont responsables chez l’homme de maladies décrites depuis longtemps, pourtant ils demeurent méconnus, avec en particulier un manque de données épidémiologiques et d’outils de diagnostic.Dans une première partie, des études de séroprévalence nous ont permis d’aborder l’impact en santé publique, dans le sud-est de la France, de deux phlébovirus connus pour leur pouvoir pathogène chez l’homme, Toscana virus (TOSV) et Sandfly fever Sicilian virus (SFSV). Pour ce dernier, des anticorps spécifiques (IgG) ont été détectés dans moins de 1% des sérums testés, ce qui suggère que SFSV joue un rôle mineur dans les pathologies humaines de cette région ; ces résultats sont corroborés par l’absence, durant ces dernières décennies, de cas documentés d’infection aigüe due à SFSV en Europe occidentale. Nous avons donc pu concentrer notre travail sur le deuxième groupe de phlébovirus d’intérêt chez l’homme, le groupe des Sandfly fever Naples virus, qui inclut notamment TOSV. Nous avons démontré l’existence d’un lien épidémiologique entre les infections à Leishmania infantum et celles à TOSV, certainement dû au fait que ces pathogènes sont transmis par un vecteur commun (Phlebotomus perniciosus). Les analyses statistiques ont montré que les personnes exposées aux infections à TOSV ont plus de chance d’être aussi infectées par les parasites leishmanies (et vice versa). En admettant que ce lien épidémiologique entre leishmanioses et infections à TOSV est représenté par l’exposition à la piqûre d’un vecteur commun, cette étude confirme l’implication de Phlebotomus perniciosus en tant que vecteur principal de TOSV dans le sud de la France. Cette étude suggère également que certaines données épidémiologiques disponibles pour la leishmaniose pourraient être utilisées pour décrypter l’épidémiologie des infections à TOSV.La deuxième partie de cette thèse est consacrée à la détection, l’isolement et la caractérisation de virus, déjà connus ou inconnus, dans les populations de phlébotomes en France et en Afrique du Nord. Pour atteindre cet objectif, nous avons dû développer une plateforme d’analyse à au débit, adaptée pour la découverte de virus dans les phlébotomes, qui permette de traiter un grand nombre d’échantillons à faible coût. Cette plateforme a récemment été complétée par un outil de Next Generation Sequencing, afin de réaliser la caractérisation génétique complète des virus isolés et découverts. Au total, 12 576 phlébotomes ont été capturés au cours de 12 campagnes de capture menées en France, en Tunisie et en Algérie. Au sein d’une même zone géographique, la découverte de plusieurs nouveaux phlébovirus, ainsi que leur taux d’infection observé dans les populations de phlébotomes, ont démontré que la diversité de phlebovirus est bien plus importante qu’attendue.Dans la troisième partie de cette thèse, une étude de séroprévalence a été menée sur des sérums humains en utilisant des tests comparatifs de neutralisation de virus. Cette étude nous a permis d’exclure le virus Punique, récemment découvert, de la liste des principales menaces en santé publique au nord de la Tunisie, et de confirmer que TOSV est le principal phlebovirus pathogène ayant un impact en santé publique dans cette région du pays. Cette méthode de neutralisation est capable d’identifier précisément, parmi des virus génétiquement proches, le virus contre lequel les anticorps présents dans le sang ont été produits, ce qui permet de déterminer la capacité de chacun de ces virus à jouer un rôle en santé publique. / Sandflies are vectors of various arthropod-borne viruses, in particular viruses within the genus Phlebovirus, family Bunyaviridae, and of parasites in the genus Leishmania. Human diseases caused by infection with sandfly-borne phleboviruses are known for a long time, but they remain neglected due to the lack of epidemiological knowledge and of diagnostic tools.The first part consisted of seroprevalence studies in human sera to address the public health impact in south-eastern France of two recognized sandfly-borne phleboviruses, namely Toscana virus (TOSV) and Sandfly fever Sicilian virus (SFSV). Concerning the latter, specific IgG were detected in less than 1% of tested sera, suggesting that SFSV play a minor role in human disease in the region; this finding was corroborated with the lack of documented case of acute infection due to SFSV in Western Europe during the last decade. This pleaded for focusing on the other group of sandfly fever viruses known for their human interest, namely the group of Sandfly fever Naples virus that includes TOSV. We demonstrated an existing epidemiological relationship between Leishmania infantum and TOSV infections, presumably through the transmission by the common arthropod vector (Phlebotomus perniciosus). Statistical analysis showed that persons exposed to TOSV infection are at greater risk of being infected with Leishmania parasite (and vice versa). Assuming that epidemiological link between leishmaniasis and TOSV infection may be represented by the exposure to the bite of a common vector, this study confirms the involvement of Phlebotomus perniciosus as the major vector of TOSV in the South of France. This study also suggests that some of the epidemiological data available on Leishmaniasis may be used to decipher the epidemiology of TOSV infections..The second part of this thesis was dedicated to detection, isolation and characterization of existing and/or new phleboviruses in sandfly populations in France and in North Africa. To achieve this aim, we had to set up a high-throughput cost-effective platform amenable to virus discovery in sandflies; this sandfly-processing platform has been recently docked to a Next Generation Sequencing platform for full genetic characterization of newly isolated and discovered viruses. A total of 12,576 sandflies were trapped during 12 campaigns conducted in France, Tunisia and Algeria. The discovery of several new phleboviruses and their observed frequency in sandfly populations has clearly demonstrated that within a given geographic area, virus diversity is much higher than previously believed.In the third part of this thesis, a seroprevalence study based on comparative virus neutralization tests was performed on human sera and allowed to exclude the newly described Punique virus from the list of major public health threats in northern Tunisia, and to confirm that TOSV is the dominant phleboviral pathogen with an impact on public health in this part of the country. This neutralization method is suitable to identify precisely the virus against which antibodues were elicited, allowing to discriminate among closely related phleboviruses, and to determine their propensity to play a role in public health.

Parmi les phlébovirus (famille des Bunyaviridae, genre Phlebovirus), ceux qui sont transmis par les phlébotomes de l'Ancien Monde sont largement distribués dans le bassin méditerranéen. Les infections humaines causées certains de ces phlébovirus sont connues depuis longtemps, mais elles restent tout de même négligées en médecine en raison de l'absence de données épidémiologiques solides (problème des réactions croisées) et d'outils de diagnostic rapides et fiables.La première partie de cette thèse a été consacrée à l'optimisation d'un test de neutralisation du virus pour étudier la séroprévalence de 5 virus, et leur capacité respective à infecter les humains et les animaux.La deuxième partie visait à mesurer la séroprévalence de phlébovirus appartenant aux 3 complexes antigéniques transmis par les phlébotomes dans le bassin méditerranéen (Sandfly fever Naples, Sandfly fever Sicilian et Salehabad). Ces études ont été menées sur des sérums de chiens et de chats en Tunisie, Portugal, Grèce/Chypre.La troisième partie a montré la capacité de virus récemment découverts dans le serocomplexe Salehabad (Adana et Medjerda valley virus) à infecter l'homme et les animaux traduisant un potentiel pathogène à explorer par des études spécifiques.La dernière partie a démontré la présence du virus Toscana en Kabylie (Algérie du Nord), et l'exposition extrêmement élevée des populations humaines vivant dans la région, avec des prévalence 10 fois plus élevées que dans les régions les plus à risque du sud-est de la France. / Sandfly-borne phleboviruses, transmitted by phlebotomine sandflies and belonging to the genus Phlebovirus within the Bunyaviridae family are widely distributed in Mediterranean basin. Human diseases caused by infection with phleboviruses are known for a long time, but they are still neglected due to the lack of epidemiological knowledge and of diagnostic tools.The first part of this thesis was dedicated to optimize a comparative virus neutralisation test to study the seroprevalence of selected phleboviruses and to assess the capacity of each virus to infect humans and animals. The second part aimed to estimate the epidemiology of phlebovirus serocomplexes (Naples, Sicilian and Salehabad) in Mediterranean basin. In order to update the presence of these viruses and their capacity to infect animals, several serologic studies were carried out on animal blood samples in Tunisia, Portugal, Greece and Cyprus. The results demonstrated that the phleboviruses belonging to 3 distinct groups are widely circulating and capable to infect non human vertebrate at different rates in studied countries.The third part showed the capacity of newly discovered viruses (Adana and Medjerda valley viruses) belonging to Salehabad serocomplex to infect human and animal at low and high rates, respectively. These findings suggest the medical and veterinary importance of these viruses. The last part of this thesis, confirm the circulation of Toscana virus by seroprevelance study which was carried out in local population in north Algeria where Toscana virus was isolated recently. The high rate of circulate suggests that Toscana virus is heavily affecting sandfly-exposed people in Algeria.

Call number: LD2668 .T4 1965 A22 / Master of Science

Healthcare providers encounter a wide range of hazards on the job, including exposure to infectious diseases. Protecting them from occupational infectious disease is very important. Healthcare workers use personal protective equipment (PPE) as a measure to decrease the risk of getting infected during patient care. For high-risk diseases like Ebola, Tyvek suits are coverall suits that protect the body and reduce the risk of body fluid exposure. However, a person removing a contaminated suit may also be exposed to virus. Previous studies have shown that enveloped viruses can survive on different types of surfaces, so the objective of this study is to determine the inactivation of bacteriophage Φ6, a surrogate for enveloped human virus, on the surface of Tyvek suits at two different relative humidity levels, 40% and 60% at 22°C. The results showed the inactivation rate of virus was higher at 60% RH than 40% RH. There was ~3log10 (99.9%) reduction of virus inactivation after 6 hours at 40% but ~3log10 (99.9%) inactivation took 9 hours at 60%. This suggests that enveloped viruses can survive on the surface of Tyvek suits for more than 6 hours, and should be considered a potential risk for contamination when they are taken off after use.

BACKGROUND:Tomato leaf curl Sudan virus (ToLCSDV) is a single-stranded DNA begomovirus of tomato that causes downward leaf curl, yellowing, and stunting. Leaf curl disease results in significant yield reduction in tomato crops in the Nile Basin. ToLCSDV symptoms resemble those caused by Tomato yellow leaf curl virus, a distinct and widespread begomovirus originating in the Middle East. In this study, tomato samples exhibiting leaf curl symptoms were collected from Gezira, Sudan. The associated viral genome was molecularly characterized, analyzed phylogenetically, and an infectious clone for one isolate was constructed.FINDINGS:The complete genomes for five newly discovered variants of ToLCSDV, ranging in size from 2765 to 2767-bp, were cloned and sequenced, and subjected to pairwise and phylogenetic analyses. Pairwise analysis indicated that the five Gezira isolates shared 97-100% nucleotide identity with each other. Further, these variants of ToLCSDV shared their highest nucleotide identity at 96-98%, 91-95%, 91-92%, and 91-92% with the Shambat, Gezira, Oman and Yemen strains of ToLCSDV, respectively. Based on the high maximum nucleotide identities shared between these ToLCSDV variants from Gezira and other previously recognized members of this taxonomic group, they are considered isolates of the Shambat strain of ToLCSDV. Analysis of the complete genome sequence for these new variants revealed that they were naturally occurring recombinants between two previously reported strains of ToLCSDV. Finally, a dimeric clone constructed from one representative ToLCSV genome from Gezira was shown to be infectious following inoculation to tomato and N. benthamiana plants.CONCLUSION:Five new, naturally occurring recombinant begomovirus variants (>96% shared nt identity) were identified in tomato plants from Gezira in Sudan, and shown to be isolates of the Shambat strain of ToLCSDV. The cloned viral genome was infectious in N. benthamiana and tomato plants, and symptoms in tomato closely resembled those observed in field infected tomato plants, indicating the virus is the causal agent of the leaf curl disease. The symptoms that developed in tomato seedlings closely resembled those observed in field infected tomato plants, indicating that ToLCSDV is the causal agent of the leaf curl disease in Gezira.

The search for alternative water disinfectants to those commonly used, such as chlorine, probably began when the disadvantages of those disinfectants became known. Soluble disinfectants have short half-lives and need to be replenished periodically which requires monitoring the determination of appropriate concentrations for waters being treated. This disadvantage may be balanced by the ability to alter dose concentrations of soluble disinfectants to meet changes in demands. Maintenance of a residual disinfectant concentration which can act throughout a water distribution system is another advantage of soluble disinfectants. Disadvantages due to reaction of organic materials include the loss of disinfecting capability, health hazards related to the disinfectant and the potential loss of aesthetic water qualities such as taste, smell and color. The purpose of this study was to investigate different surface contact disinfectants which, while having an inflexible dose concentration capability and releasing no residual disinfectant concentration, do not require monitoring and do not significantly leach into the waters they contact. Powdered aluminum when mixed with a loamy sand reduces virus concentration 3-4 orders of magnitude better than controls while not significantly altering the pH or aluminum concentration of waters that pass through the soil columns. Labeled poliovirus 1 was found to adsorb onto the aluminum surface in batch experiments and undergo degradation or dissociation of the capsid proteins with release of viral fragments and clumps within 76 hours. Freon dispersion of the viral clumps showed the clumps to be non-infective. Polyacrylamide gel electrophoresis of poliovirus 1 incubated with aluminum for 76 hours suggested degradation or dissociation of viral capsid proteins 1, 2 and 3. Magnesium peroxide and magnesium oxide, while effectively inactivating viruses, significantly increase the pH of water which they contact. Polyhalex resin and I5 resin reduce virus concentration, but release functional groups into the surrrounding water. Contact disinfectants such as aluminum, may find point-of-use application for drinking water, use in septic tank leachfields or sewage treatment infiltration basins to reduce the threat of spreading potential disease-causing organisms.

Drosophila melanogaster has been extensively used as a model organism to study diverse facets of biology, including host-pathogen interactions and the basic biology of its pathogens. I have used the fruit fly as a model to study elementary aspects of Nora virus biology, such as the role of the different proteins encoded by the virus genome. Nora virus, an enteric virus transmitted via the feca-oral route, does not cause any obvious pathology in the fly, although the infection is persistent. Nora virus genome consists of a positive strand RNA that is translated in four open reading frames (ORF).  Since sequence homology studies did not yield much information about the different Nora virus proteins, I have used the cDNA clone of the virus to construct mutants to identify the specific function of each protein. My results have shown that, 1) The protein(s) encoded by ORF 1 are crucial for the replication of the virus genome. 2) The C-terminus of the ORF 1-encoded protein (VP1), is an inhibitor to the RNAi pathway. 3) The transmembrane domain in the N-terminus of the ORF2-encoded protein (VP2) is important for the formation of Nora virus virions. 4) The ORF 3-encoded protein (VP3) forms α-helical trimers and this protein is essential for the stability of Nora virus capsid.                                                     I have also performed RNA sequencing to investigate the transcriptional response of D. melanogaster in response to Nora virus infection and my results indicate that,                        5) The upregulation of genes related to cellular stress and protein synthesis and the downregulation of basal digestive machinery, together with the induction of upd3, implies major gut epithelium damage and subsequent regeneration.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Wenjun Ma / Influenza A virus (IAV) is a negative sense, single-stranded, segmented RNA virus with eight gene segments. It is an important respiratory pathogen which causes annual epidemics and occasional pandemics worldwide in humans and leads to considerable economic problems for the livestock industry. To control and prevent this significant disease, understanding the pathogenesis of IAVs is critical. Although some molecular mechanisms regarding virulence have been determined, IAV pathogenesis is not completely understood and is difficult to predict. The eight viral gene segments of IAV were thought to encode for 10 viral proteins. Since 2001, eight additional viral proteins have been identified, including PB1-F2, PB1-N40, PA-X, NS3, PA-N155, PA-N182, M42, and PB2-S1. However, the functions of these novel proteins in influenza virus replication as well as pathogenesis have not been fully elucidated. Although PB1-F2 protein is an important virulence factor of IAV, the effects of this protein on viral pathogenicity of swine influenza virus (SIV) remain unclear. In Chapter 2, we investigated the contribution of the PB1-F2 protein to viral pathogenicity of a virulent triple-reassortant (TR) H1N1 SIV in different hosts, pigs and mice. Our data indicate that PB1-F2 expression in virulent TR H1N1 SIV modulates virus replication and pathogenicity in the natural host, pigs, but not in mice. In addition, single amino acid (aa) substitution at position 66 (N/S) in the PB1-F2 has a critical role in virulence in mice but no effect was found in pigs. A novel IAV protein, PA-X consists of the N-terminal 191aa of PA protein and a unique C-terminal 41 (truncated form) or 61 (full-length form) aa residues encoded by +1 ribosomal frameshifting. Although several studies have demonstrated the PA-X protein as an important immune modulator and virulence factor, the impact of different expressions of PA-X protein including full-length, truncated or PA-X deficient forms on viral pathogenicity and host response remains unclear. In Chapter 3, we showed that expression of either truncated or full-length PA-X protein in 2009 human pandemic H1N1 (pH1N1) viruses suppresses host antiviral response by host shutoff activity which promotes viral growth and virulence in mice when compared to loss of PA-X expression. Furthermore, full-length PA-X expression displayed stronger impact on viral pathogenicity and host immune response compared to truncated PA-X expression. Taken together, our results provide new insights into the impact of PB1-F2 and PA-X proteins on virus replication, pathogenicity and modulation of host immune responses. This knowledge is important for better understanding of IAV pathogenesis.

El presente trabajo tuvo como objetivo estandarizar y validar la técnica de RT-PCR en tiempo real para el diagnóstico del virus de la Necrosis Pancreática Infecciosa (IPNV) en la trucha arcoíris (Oncorhynchus mykiss) en el Perú. Se utilizaron muestras de riñón y bazo de truchas arcoíris provenientes de dos piscigranjas del departamento de Junín, tomándose 61 animales con signos clínicos de enfermedad y 60 animales aparentemente sanos. Todas las muestras fueron evaluadas mediante la técnica de Inmunofluorescencia Indirecta (IFI) para determinar la presencia del virus. La prueba de RT-PCR tiempo real se realizó utilizando un kit comercial en dos pasos y utilizando el fluoróforo Sybr Green I. Se utilizaron los primers WB1 y WB2 para identificar un segmento genómico específico de la proteína estructural VP2. Como controles positivos se utilizaron virus inactivado IPNV cepa Sp, así como 10 muestras inoculadas con IPNV; y como controles negativos, se utilizaron virus relacionados a IPNV como son Rotavirus A y el virus de la Bursitis Infecciosa del pollo; así como virus no relacionados. A su vez, se llevó a cabo la inoculación viral en muestras de tejido en diluciones decrecientes para determinar el grado de sensibilidad. Los resultados fueron determinados a partir de los valores del Ciclo Umbral (Ct) y temperatura de Melting (Tm) de los productos amplificados. La prueba fue capaz de detectar al virus hasta concentraciones de 102 UFP/ml. Las 121 muestras de campo resultaron negativas a IPNV; los animales enfermos evidenciaron etiologías de tipos bacterianos y no virales. Los resultados obtenidos determinaron una especificidad del 100% y una sensibilidad del 100%; a su vez, los valores predictivo positivo y predictivo negativo resultaron en 100%. La técnica de RT-PCR en tiempo real estandarizada en el laboratorio representa una técnica valiosa para el diagnóstico de IPNV. Palabras Clave: Virus de la Necrosis Pancreática Infecciosa, IPNV, RT-PCR, RT-PCR tiempo real / --- This study aimed to standardize and validate the real-time RT-PCR technique for diagnosis of Infectious Pancreatic Necrosis Virus (IPNV) in rainbow trout (Oncorhynchus mykiss) in Peru. Kidney and spleen samples of rainbow trout (n=121) from two fish farms of Junin were taken, 61 animals showed signs of disease and 60 were apparently healthy animals. All samples were assessed by indirect immunofluorescence technique (IIF) to determine presence of virus. Real-time RT-PCR was carried out using a two-step commercial kit. We used WB1 and WB2 primers for amplification of a specific genomic segment of the VP2 structural protein. IPNV Sp strain and 10 samples inoculated with IPNV were used as positive controls; whereas related viruses, such as rotavirus and infectious bursal disease virus from chickens, as well as unrelated viruses were all used as negative controls. Also, we inoculated decreasing dilutions of virus into tissue samples to determine the sensitivity degree of the test. Results were assesssed according to the values of the threshold cycle (Ct) and melting temperature (Tm) of the amplified products. The test was able to detect the virus in concentrations up to 102 PFU/ml. All 121 field samples were negative for IPNV; sick animals were apparently infected by bacterial agents. Real Time RT-PCR’s specificity and sensitivity were around 100%; moreover, the positive predictive value and negative predictive value was also set in 100%. Real time RT- PCR technique standardized in our laboratory is a valuable diagnostic tool for IPNV detection in field samples. Key Words: Infectious Pancreatic Necrosis Virus, IPNV, RT-PCR, real time RT-PCR

El objetivo del presente estudio fue evaluar el grado de susceptibilidad, el efecto patológico y respuesta serológica de la paloma silvestre (especie Columba livia) frente a la inoculación experimental con una cepa de virus velogénico de la enfermedad de Newcastle. Se capturaron 28 aves adultas, 14 fueron inoculadas con una cepa velogénica viscerotrópica de la enfermedad de Newcastle, vía nasal y oral, mientras las restantes fueron criadas como grupo control. En las aves inoculadas se registró los signos clínicos y mortalidad. Se tomaron muestras de diferentes tejidos de aves que murieron así como del grupo control, para su análisis histopatológico. Se tomaron muestras de pulmón y tráquea e hisopado de cloaca para la recuperación viral, semanalmente durante seis semanas. El 64% de aves del grupo inoculado presentó signos clínicos y se obtuvo una mortalidad del 42.8%. Se presentaron estornudos a partir del cuarto día post inoculación. El erizamiento de plumas, aislamiento y letargia, se observó a partir del quinto día en aves que presentaron signos respiratorios y nerviosos. Al séptimo día se observó en el 42.8% opistótonos, tremores de cabeza y cuello que se exacerbaban al estímulo de ruidos. Al octavo día sólo en el 21 % de las aves mostraron diarrea. Los hallazgos a la necropsia consistieron en una congestión generalizada de los diferentes órganos y esplenomegalia. Las lesiones microscópicas más marcadas fueron edema, gliosis, manguito perivascular a mononucleares en cerebro y cerebelo, pérdida de cilios, infiltrado de linfocitos en tráquea, congestión interalveolar en pulmón, congestión en proventrículo, congestión e infiltración de linfocitos en intestinos y despoblamiento linfoide en bazo. El grupo inoculado mostró un incremento en sus títulos de anticuerpos a partir de la primera semana post inoculación llegando a su máximo nivel promedio geométrico de título (P.G.T) de 4.9 en la segunda semana para luego descender por completo a la quinta semana. Se logró la recuperación viral a partir de muestras de pulmón y tráquea durante las tres primeras semanas post inoculación. Se demostró que la especie Columba livia es susceptible a la inoculación experimental con una cepa velogénica del virus de la enfermedad de Newcastle. / -- This study was designed to asses the susceptibility, pathologic effect and serologic response of wild pigeons (sp. Columba livia) to Newcastle virus. A total of twenty eight adult wild pigeons were captured, fourteen were inoculated with a velogenic viscerotropic strain of Newcastle virus by oral and nasal route. The remaining pigeons were used as control group. The birds were observed to record clinical signs and mortality, also blood samples were collected for hemaglutination inhibition technique and tissue samples from lungs, tracheas and cloacal swabs were harvested for the viral recovery and histological studies. The 64 % of the inoculated group showed clinical signs and a mortality of 42.8 %. The clinical signs (sneezes, ruffled plumage, isolation and lethargy) started the fourth day post inoculation. The 43 % showed nervous signs: opisthotonos, tremors of head and neck that was exaggerated to the stimulus of noises and the 21 % showed diarrhea. In the necropsy were observed a widespread congestion and splenomegaly. The microscopic injuries were edema, gliosis, mononuclear perivascular cuffing in brain and cerebellum, loss ciliar, lymphoid infiltration in trachea, lungs congestion, proventricular congestion, and lymphocitic infiltration in intestines and lymphoid depletion in spleen. The inoculated group showed an increase in the antibody titers the first week post inoculation reaching the highest titer mean (P.G.T=4.9) in the second week after this they descended. The viral recovery was made upon lungs and tracheas tissues and from a cloacal swab in dead birds. It was showed that the specie Columba livia was susceptible to the experimental inoculation with a velogenic strain of Newcastle diasease virus.