Tesi sul tema "Zoonosis Virales"

Le virus de l'influenza aviaire hautement pathogène (IAHP) de sous-type H5N1 a causé de nombreuses pertes humaines, animales et économiques à travers le monde, notamment en Asie du Sud-Est. Son potentiel pandémique est une source d'inquiétude majeure en santé publique. Au Cambodge, l'infection est enzootique, et a causé la mort de 16 personnes depuis sa première détection en 2004 dans le pays, dont 8 pour la seule année 2011. Bien que l'hypothèse de la transmission directe hôte-hôte (animal-animal ou animal-homme) soit privilégiée, de récentes études semblent clairement incriminer certains éléments constitutifs de l'environnement dans le cycle de transmission du virus. Cependant, peu de données sont actuellement disponibles sur le sujet. Le travail de cette thèse a consisté en grande partie à apporter quelques réponses aux nombreuses questions soulevées. Des méthodes de détection du virus H5N1 dans l'environnement ont été mises au point, validées, et utilisées pour la détection de virus dans des prélèvements environnementaux collectés sur des sites d'épizooties au Cambodge. Le rôle de passereaux, capturés pour la réalisation de certains rituels bouddhistes en Asie, dans la dissémination du virus aux populations aviaires et humaines, a également été étudié. En parallèle, des données importantes du mode d'évolution du virus H5N1 au sein d'hôtes aviaires, jusqu'alors inexistantes, ont été apportées par l'étude des quasi-espèces du virus. L'ensemble des résultats rassemblés dans cette thèse souligne l'importance du rôle de l'environnement dans la dissémination et la transmission du virus IAHP H5N1
The Highly Pathogenic Avian Influenza (HPAI) virus, subtype H5N1, has caused important human, animal and economical and losses in all countries affected, especially in Southeast Asia. Its pandemic potential is a major public health concern. In Cambodia, the infection is enzootic, and has caused 16 human fatalities since its first detection in the country in 2004, out of which 8 occurred in 2011. Although the hypothesis of direct host-to-host (animal-to-animal or animal-to-human) transmission is commonly accepted, recent studies clearly identified some environmental components as sources for avian and/or human contamination with H5N1 virus. Nonetheless, only few data are currently available on this topic. The work presented in this thesis aimed at better describing the role of the environment in the transmission cycle of the H5N1 virus. H5N1 virus detection methods in the environment were designed, validated and used for the detection of virus in environmental samples collected during epizootic outbreaks in Cambodia. The role of the Merit Release Birds, used during some common Buddhist rituals in Asia, in the dissemination of the virus to avian and human populations was also studied. In parallel, important and novel data regarding the evolution of the H5N1 virus within avian hosts were provided by quasi-species studies. The findings described in this thesis emphasize the relevance of the role of the environment in the dissemination and transmission of the HPAI H5N1 virus

O sequenciamento de alto desempenho, pela redução dos custos nos últimos anos, vem sendo cada vez mais utilizado para prospectar e identificar vírus. Estes métodos são extremamente mais sensíveis que outros métodos moleculares, e capazes de sequenciar genomas virais sem conhecimento prévio, clonagem ou isolamento. Neste estudo, utilizamos o sequenciamento de alto desempenho para conhecer, e caracterizar genomas completos de arbovírus isolados nas Américas, incluindo a prospecção de vírus em amostras de pequenos mamíferos do estado de São Paulo, Brasil. Assim, sequenciamos e caracterizamos 44 Bunyavirales, 35 no gênero Orthobunyavirus, família Peribunyaviridae, oito no gênero Phlebovirus, família Phenuiviridae, e um orthonairovírus, família Nairoviridae. Entre os Bunyavirales identificamos uma provável nova estratégia de codificação da proteína não estrutural do segmento pequeno, e ainda identificados sete vírus que são reassortants naturais. Caracterizamos o genoma completo do vesiculovírus Piry, determinando sua relação filogenética com arbovírus pertencentes ao gênero Vesiculovirus, família Rhabdoviridae. Prospectamos novos vírus, os quais incluímos em três famílias, Parvoviridae, Anelloviridae e Hepeviridae. Na família Parvoviridae, identificamos 20 chapparvovírus endógenos e exógenos, oriundos de grande diversidade de hospedeiros vertebrados e invertebrados, e que representam uma nova subfamília, a Chapparvovirinae. Também, descrevemos onze novas espécies de Anelloviridae em roedores silvestres e marsupiais, fornecendo importantes informações sobre a diversidade, a taxonomia, e ainda ampliamos a gama de hospedeiros de anellovírus conhecidos. Por fim, identificamos e caracterizamos uma nova espécie de Orthohepevirus de roedores Sigmodontinae, nomeada \"Orthohepevirus E\". Acreditamos que estamos a fornecer relevantes informações sobre genômica, epidemiologia molecular, evolução e taxonomia de 45 arbovírus americanos, bem como sobre 13 novas espécies virais encontradas em pequenos mamíferos. Tais informações deverão dar subsídios para múltiplos futuros estudos visando compreender a importância destes novos vírus e a desenvolver métodos diagnósticos.
In last years, high-throughput sequencing (HTS) has been cost-effective and increasingly used for prospection and identification of viruses. These methods are extremely more sensitive than other molecular methods and are capable of sequencing viral genomes without prior knowledge, cloning or isolation. In this study, we used HTS approach to identify and characterize complete genomes of arbovirus isolated in the Americas, as well as viral prospection in samples of small mammals from São Paulo State, Brazil. Thus, we sequenced and characterized 44 viruses from Bunyavirales order, including 35 in Orthobunyavirus genus, family Peribunyaviridae, eight in Phlebovirus genus, family Phenuiviridae, and one in Orthonairovirus genus, family Nairoviridae. Among the Bunyavirales we identified a novel putative strategy for encoding the non-structural protein of the small segment, as well as we identified seven viruses that are natural reassortants. Also, we characterized the complete genome of the Piry vesiculovirus, determining its phylogenetic relationship with arboviruses belonging to the Vesiculovirus genus, family Rhabdoviridae. On the other hand, we have prospected novel viruses, which included in three families, Parvoviridae, Anelloviridae, and Hepeviridae. In the Parvoviridae family, we identified 20 endogenous and exogenous chapparvoviruses from a broad diversity of vertebrate and invertebrate hosts, representing a new subfamily, the Chapparvovirinae. Also, we have described eleven new species of Anelloviridae in wild rodents and marsupials, providing important information on diversity, taxonomy and even broadening the range of known anelloviruses hosts. Finally, we identified and characterized a novel species of orthohepevirus in Sigmodontinae rodent, named \"Orthohepevirus E\". We believe that we are providing relevant relevant on genomics, molecular epidemiology, evolution and taxonomy of 45 American arboviruses, as well as on 13 new viral species found in small mammals. Thus, these informations should provide support for multiple future studies to understand the importance of these new viruses, as well as to develop diagnostic methods.

Les zoonoses constituent plus des deux tiers des pathologies virales qui concernent l’homme. Le développement et la démocratisation des outils de métagénomique en font de bons outils d’inventaire et de surveillance de virus potentiellement émergents.Dans un premier temps j’ai développé et validé un protocole expérimental de purification des viromes à ARN qui permettait le maintien de l’infectivité des particules virales. Ce protocole a ensuite été appliqué pour caractériser les communautés virales d’arthropodes hématophages et de prélèvements de faune sauvage. J’ai par la suite réalisé l’inventaire des communautés virales de viande de singe fumée illégalement importée en France et confisquée par les douanes, qui a révélé la présence de nombreux bactériophages, dont certains pourraient infecter des bactéries potentiellement pathogènes pour l’homme.Enfin j’ai caractérisé les communautés virales de culicoïdes collectés au Sénégal, ce qui a permis de mettre en évidence la présence de nombreux virus géants à ADN infectant les amibes. Le séquençage des viromes à ARN a quant à lui révélé la présence d'un certain nombre d'arbovirus qui pourraient constituer un risque d’émergence pour la santé humaine. Du fait de nombreux facteurs intrinsèques et extérieurs à l’agent infectieux, la prédiction des futures émergences de virus zoonotiques est très compliquée voire utopique, mais elle reste un challenge crucial et d’actualité. La stratégie de réalisation d’inventaires des communautés virales présentes dans les différents acteurs des cycles de transmission zoonotique est un premier pas indispensable dans la connaissance des risques potentiels d’émergence en population humaine
Zoonoses are responsible of more than two thirds of human viral infections. The development of high-throughput sequencing tools and their application in metagenomics allow inventorying the viral communities of various reservoirs in order to detect the emergence of viruses before their infection to humans. In this context, I characterized the viral communities of simian bushmeat illegally imported into France and of Culicoides biting midges, recognized vectors of several viruses of human and veterinary medicine importance. I have first developed a protocol for the purification of RNA viromes which allowed maintaining the infectivity of viral particles. This protocol was subsequently applied to characterize viral communities of bloodsucking arthropods and wildlife samples. In a second part I realized the inventory of viral communities of smoked simian bushmeat illegally imported into France and confiscated by the French customs. This study revealed the presence of a wide diversity of bacteriophages, in which some of them could infect bacteria potentially pathogenic for humans.Finally I characterized the viral communities of Culicoides biting midges collected in Senegal, which revealed the presence of sequences related to several giant DNA viruses infecting amoeba. Sequencing of the RNA virome revealed the presence of several arboviruses that could constitute a risk of emergence of zoonoses for humans.The prediction of future emerging zoonotic viruses is very difficult, if not impossible. However the characterization of viral communities present in the different actors of zoonotic transmission cycle is a first step to evaluate potential risks of transmission to humans

Les norovirus (NoVs) responsables de la majorité des gastroentérites virales se divisent en 7 génogroupes GI à GVII. Les génogroupes GI, GIV, GVI et GVII regroupent les souches humaines, le génogroupe GII les souches humaines et porcines et les génogroupes GIII et GV respectivement les souches bovines et murines. La fixation des NoVs humains sur les antigènes tissulaires de groupe sanguin (HBGAs) constitue la première étape de l'infection. Des NoVs porcins et bovins dont les séquences sont très voisines des séquences des NoVs humains ont été identifiés suggérant que ces animaux pourraient constituer un réservoir pour l’homme, d'autant que ces animaux présentent des HBGAs à la surface de leurs cellules épithéliales. En effet, il est admis que la présence de récepteurs partagés par différentes espèces augmente le risque de transmission inter-espèce, Nous nous sommes donc proposés d'étudier les récepteurs sur les cellules humaines, porcines, bovines et de l’huître, des NoVs humains GI. 1 et GII. 4, des NoV bovins GIII. 2 et NoV porcins GII. 11. Nous avons ainsi caractérisé l’expression des HBGAs le long du tube digestif des bovins, du porc et de l’huître, et étudié la fixation des NoVs humains et animaux sur les tissus de ces animaux. Nous avons caractérisé chez le bovin le ligand cellulaire du NoV GIII. 2 aboutissant à la conclusion que l’homme ne peut être infecté par ce virus du fait de la perte totale de l'expression de ce ligand. Nous avons aussi démontré que certaines souches humaines peuvent trouver leur ligand chez le porc et le bovin soulignant la possibilité de co-infection de ces espèces qui pourraient être à l'origine de l'émergence de nouvelles souches zoonotiques
Noroviruses (NoVs), most common etiological agent of acute viral gastroenteritis outbreaks worldwide, are divided into 7 genogroups GI to GVII. Genogroups GI, GIV, GVI and GVII contain human strains, the genogroup GII human and porcine strains, and genogroups GIII and GV bovine and mouse respectively. Binding of NoVs to human blood group antigens (HBGAs) constitute the first step of infection. Pigs and cattle noroviruses whose sequences are very similar to human noroviruses sequences have been identified, suggesting that these animals might constitute a reservoir for humans, as these animals present HBGAs on the surface of their epithelial cells. Indeed, it is known that the presence of receptors shared by different species increases the risk of cross-species transmission. We therefore proposed to study the binding of human NoVs GI. 1 and GII. 4, bovine NoV GIII. 2 and porcine NoV GII. 11 on human, pig, bovine and oysters tissues. We have characterized along the digestive tract of cattle, pig and oyster the expression of HBGAs and we studied the binding of human, porcine and bovine noroviruses on human and animal tissues. We have characterized the cellular ligand of the bovine noroviruses GIII. 2 leading to the conclusion that humans cannot be infected by this virus because of the total loss of expression of this ligand. We also demonstrated that some human strains can find their ligand in pigs and cattle suggesting the possibility of co-infection of these species that could be responsible for the emergence of new zoonotic strains

Les SIV infectant les chimpanzés et les gorilles sont les précurseurs des virus de l'immunodéficience humaine de type 1. Les quatre groupes du VIH-1 sont le résultat de quatre transmissions virales des grands singes à l'Homme. Des méthodes non invasives ont permis d'identifier le réservoir des VIH-1 M et N dans deux communautés de chimpanzés (Ptt) au Cameroun et de montrer que les gorilles (Ggg) sont infectés par un SIV proche des VIH-1 O et P. Si le SIVgor n'a jamais été détecté chez les chimpanzés, la phylogénie montre que les Ptt ont transmis ce virus aux gorilles. Par une méthode pluridisciplinaire, nous avons étudié les caractéristiques de l'infection SIVgor en milieu naturel. Nous avons prospecté 13 sites au Cameroun et 2 en RCA. Au total, 2120 fèces de gorilles et 442 de chimpanzés ont été collectées. L'infection SIVgor a été détectée dans 3 sites Camerounais et les prévalences varient entre 3,2% et 4,6%, résultats plus faibles que ceux retrouvés chez les chimpanzés. Nous avons ensuite montré que plusieurs groupes sociaux de Ggg dont les domaines vitaux se chevauchent sont infectés et que les prévalences SIV dans les groupes peuvent dépasser 25%. Les virus touchant les gorilles du même groupe sont génétiquement proches montrant des liens épidémiologiques. Enfin, un suivi de l'infection réalisé de 2004 à 2009 sur un site a permis de découvrir un foyer d'infection, 2 cas de séroconversions et de retrouver une femelle gorille infectée à 5 ans d'intervalle. Dans ce site, la prévalence SIV est stable et le nombre de femelles infectées est plus important que le nombre de mâles. La structure sociale des gorilles et leur comportement peuvent alors expliquer en partie la répartition et la prévalence du SIVgor, ainsi que les différences avec l'infection chez les chimpanzés.Cette étude multidisciplinaire montre la faisabilité du suivi de l'infection SIV chez les gorilles en milieu naturel. Si le SIVgor est pathogène, le suivi pourra s'avérer essentiel chez cette espèce menacée d'extinction
SIV infecting chimpanzees and gorillas are the precursors of the Human Immunodeficiency Virus type 1. The four groups of HIV-1 are the results of four different viral transmissions from apes to humans. Using non invasive methods we discovered the reservoir of HIV-1 M and N in two communities of chimpanzees (Ptt) in Cameroon and found that Gorillas (Ggg) are infected by a SIV close to HIV-1 O and P. While SIVgor has not yet been detected in chimpanzees, phylogeny shows that Ptt transmitted this virus to Ggg. Using a multidisciplinary approach, we studied the characteristics of the infection in wild living gorillas. We prospected 13 sites in Cameroon and 3 in CAR. 2120 fecal samples of gorillas and 442 of chimpanzees were collected. SIVgor infection was detected in 3 sites in Cameroon and the prevalence ranges from 3.2% to 4.6%, lower than in chimpanzees. Several social groups of gorillas with overlapping home-ranges were infected and the prevalence within group could exceed 25%. Viruses of the same group are genetically close, showing epidemiologic links. In a follow up study between 2004 and 2009 on one site, we discovered a focus of infection with 2 cases of seroconvertion and we re-sampled one infected female 5 years after. In this site, the prevalence of SIVgor is stable and the number of infected females is higher than the males. The social structure of gorillas and their behavior can partly explain for the repartition and prevalence of SIVgor, as well as the differences with the infection in chimpanzees. This multidisciplinary study proves the feasibility of a follow up study in wild living gorillas. If SIVgor turns out to be pathogenic, a follow up will be essential for this endangered species

El hombre excreta una gran cantidad de virus a través de las heces y la orina, que llegan al medio acuático y se dispersan. Su gran estabilidad hace que en muchas ocasiones los procesos de depuración actualmente aplicados no consigan eliminarlos completamente. La ingestión de agua o alimentos contaminados puede provocar una gran diversidad de enfermedades, incluso con dosis infecciosas muy bajas.

En este estudio se ha desarrollado una metodología para la recuperación de partículas víricas a partir de muestras de agua, suspensiones fecales y moluscos bivalvos, basados en la elución de los virus a partir del material particulado, utilizando tapón glicina a pH 9,5-10 y concentración por ultracentrifugación o ultrafiltración. Dicha metodología fue aplicada para evaluar la contaminación vírica del medio acuático del área metropolitana de Barcelona, que representa una población de aproximadamente dos millones de personas.

Realizou-se um estudo sobre a ocorrência da raiva bovina em 17 municípios que fazem parte da Unidade Regional de Supervisão de Rondonópolis do Instituto de Defesa Agropecuária do Estado de Mato Grosso - INDEA/MT. O objetivo do trabalho foi analisar a situação epidemiológica da doença com a finalidade de repensar as ações de atenção e vigilância epidemiológica. Foram analisados 70 Formulários de Investigação de Doenças (Inicial) (Form-in) da Coordenadoria de Controle das Doenças dos Animais do INDEA/MT, período correspondente de janeiro de 2003 a dezembro de 2007. Informações complementares foram obtidas junto ao Conselho Regional de Medicina Veterinária do Estado de Mato Grosso e banco de dados do IBGE. Verificou-se que a doença apresentou de forma endêmica entre os municípios, com pequena variação anual no número de casos. O maior porcentual dos casos ocorreu no mês de janeiro e julho, em animais com idade entre quatro a 12 meses e em propriedades com efetivo bovino superior a 500 cabeças no rebanho. Além disso, não existiu correlação entre densidade de bovinos com casos de raiva. A partir dos resultados, conclui-se a importância da manutenção das atividades de educação sanitária, o credenciamento ou incremento do diagnóstico laboratorial de raiva no estado, melhora das atividades de cadastramento e monitoramento dos abrigos e refúgios de morcegos hematófagos e o combate aos morcegos hematófagos, além da vacinação do gado bovino nas regiões endêmicas da raiva.
Study on the occurrence of bovine rabies was carried out in seventeen municipalities that are part of the Unidade Regional de Supervisão (Regional Unit of Supervision) of the Rondonópolis belonging to the Instituto de Defesa Agropecuária of the state of Mato Grosso. The objective of the study was to assess the epidemiological situation of the disease in order to rethink the actions of care and epidemiological surveillance. Seventy forms named as Formulários de Investigação de Doenças (Inicial) (Form-in), pertaining to the Coordenadoria de Controle das Doenças dos Animais of the INDEA/MT were analyzed, corresponding to the period of January 2003 to December 2007. Additional information was obtained from the Conselho Regional de Medicina Veterinária of the state of Mato Grosso and database of the IBGE. It was found that the disease had been endemic among the municipalities, with small annual variation in the number of cases. The higher percentages of cases occurred in January and July, in four to twelve year-old animals, in properties with the number of cattle greater than five hundred animals in the herd. Furthemore, there was no correlation between the cattle density and the occurrence of rabies. From these results, we conclude that it is important to keep maintaining the activities of health education, the accreditation or incrementation of laboratorial diagnosis of rabies in the state, amelioration of the activities of registration, monitoring of shelters and refuges of the vampire bats and bats control, besides the cattle vaccination in rabies endemic regions.

Every year thousands of cases of neurological disease go undiagnosed largely due to the vast number of potential causes, especially neglected are those thought to be of viral origin. The purpose of this study was to investigate the potentially novel causes of undiagnosed neurological disease in horses. An arbitrarily primed PCR was developed which allowed for the identification of unknown agents from cell culture and as well as tissue specimens. Shuni virus (SHUV) was identified in a cell culture isolate from a horse that had displayed severe neurological signs. This little known orthobunyavirus, had not been well studied since its discovery in the 1960’s and thus the focus became to further elucidate the role SHUV may play in neurological disease in South Africa. Two SHUV specific assays were developed and employed in a five year epidemiologic study. 497 horses and 143 other animals submitted to our surveillance program with febrile and neurological disease were screened for the presence of SHUV. 13 SHUV cases were identified, nine in horses and four in wildlife species. In horses symptoms ranged from mild febrile illness to severe neurological disease, where 45% of animals either died or were euthanized on humane grounds. All wildlife cases presented with paralysis, all cases proved fatal. A genome was amplified and characterised and SHUV’s (SAE 18/09) relationship to the prototype SHUV isolate and the Simbu serogroup fully clarified. Of significance was the finding that the prototype isolate’s sequence differed from SAE 18/09 at one of the M segment cleavage sites, such changes are known to affect pathogenicity. Finally due to the zoonotic potential of SHUV, a serological survey was conducted on veterinarians, to determine whether they may be at increased level of exposure due to occupational risk. WNV was used in comparison as zoonotic transmission of this virus had been documented and multiple studies conducted to analyse its sero-prevalance. 12.5% of veterinarians were found to have neutralizing antibodies to WNV and 4% to SHUV, these values correlate with what is seen in equine studies (WNV 8.7% - SHUV 1.9%), highlighting the zoonotic potential of these pathogens.
Thesis (PhD)--University of Pretoria, 2014.
Medical Virology
PhD
Unrestricted

Nous proposons de contribuer par une approche géographique à l’étude du phénomène d’émergence de maladies virales en Afrique forestière équatoriale en nous appuyant sur les origines probables de l’infection à VIH-sida. Le Virus d’Immunodéficience Humaine (VIH), responsable de la pandémie actuelle de sida, est d’origine zoonotique. Né de la recombinaison des formes simiennes de rétrovirus, le virus humain (VIH) est issu du passage de la barrière inter-espèces des agents viraux portés par les primates (Virus d’Immunodéficience Simienne - VIS) et qui ont été sélectionnés au fil des temps et à l’occasion d’expositions multiples et prolongées aux organismes humains.L’objet de ce travail est la lecture du processus d’émergence des maladies virales comme un système dans lequel interagissent dynamiques spatiales, aspects humains et paramètres environnementaux. Il s’agit d’explorer la piste des origines de l’émergence du VIH-sida sous le prisme d’une étude géographique : les habitudes migratoires, territoriales, domestiques des populations forestières de l’est Cameroun peuvent créer des situations périlleuses en termes d’exposition, de diffusion et de propagation des maladies virales
Through a geographical approach, we propose to contribute to the study of viral disease emergence in the equatorial African forestry by focusing on the probable original causes of the HIV-Aids virus. The Human immunodeficiency virus, responsible for the current Aids pandemic has zoonotic origins. Born from the recombination of several forms of simian retroviruses, the human virus (HIV) comes from the crossing of the inter-species barrier by viral agents carried by the primates (Simian Immunodeficiency Virus - VIS) and which have over time been selected and following multiple exposures and extended to the human organisms.The subject of this work is treating the viral diseases emergence process as a system within which spatial dynamics, human aspects and environmental parameters interact. This requires exploring the origins of the emergence of HIV-aids, through the prism of a geographical study: migration, territorial, and domestic patterns of east Cameroonian forestry can create perilous situations in terms of exposure, diffusion and propagation of viral diseases

Thesis (PhD)-- Stellenbosch University, 2013.
ENGLISH ABSTRACT: The emergence and re-emergence of viral human pathogens from wildlife sources in the recent past has led to increased studies and surveillance of wildlife for potentially zoonotic agents in order to gain a better understanding of the pathogens, their sources as well as events that may lead to viral emergence. Of the >1407 known human pathogens, 13% are classified as emerging or re-emerging, and 58% as zoonotic; 37% of the (re-)emerging and 19% of the zoonotic pathogens are RNA viruses, accounting for the majority of recently emerged infectious diseases with a zoonotic origin, such as HIV, Ebola, Hendra, Nipah, Influenza and SARS. This study focusses on potentially zoonotic viruses hosted by rodents (Muridae family), shrews (order previously known as Insectivora/Soricomorpha, now reclassified as Eulipotyphla) and bats (order Chiroptera). Rodents and bats represent the largest (~40%) and second largest (~25%) mammalian orders and both occur on every continent except Antarctica. Together, the three mammalian orders investigated represent the most relevant potential sources of new zoonoses. In this study I investigated the occurrence of astroviruses, arenaviruses, coronaviruses and hantaviruses in South African small mammal species belonging to the orders mentioned above. These viruses have either been implicated in recent emerging zoonotic events or are considered to have the potential to cause cross-species transmissions resulting in a zoonotic event. In the first part of the study specimens collected from various bat, rodent and shrew species were screened for viral sequences by broadly reactive PCRs; positive samples were characterised by sequencing and sequence analysis. A separate part of the study focussed on hantavirus disease in humans: a seroprevalance survey was conducted to determine the presence of hantavirus antibodies in the local population. Additionally, acutely ill patients with potential hantavirus disease were tested in an attempt to identify possible acute infections and define clinical hantavirus disease in South Africa. Screening of rodent and shrew specimens resulted in the identification of eight novel arenavirus sequences. Seven of the sequences are related to Merino Walk virus, a recently identified South African arenavirus, and the eighth sequence represents a novel lineage of Old World arenaviruses. Screening of bat specimens resulted in the identification of highly diverse novel astrovirus and coronavirus sequences in various South African bat species, including the identification of a viral sequence closely related to the recently emerged Middle East Respiratory Syndrome coronavirus. While the study did not identify hantavirus infections in any of the acutely ill patients, it found seroprevalences similar to those observed in Europe and West Africa. The results obtained highlight the importance of small mammals in the emergence of potential zoonoses and further reinforce the importance of viral surveillance of relevant wildlife species. Further in-depth studies of naturally infected reservoir host populations are required in order to gain a better understanding of virus-host dynamics and the events that lead to virus emergence.
German Research Foundation (DFG) (project number: KR1293/9-1/13-1)
The Polio Research Foundation and the NHLS Research
Harry Crossley Foundation, the Polio Research Foundation and Stellenbosch University for granting scholarships and bursaries for PhD.

Rift Valley Fever (RVF) is an emerging infectious disease found in both livestock and humans. RVF is associated with high abortion and mortality rates in livestock and can be fatal in humans. As such, RVF is economically and socially significant to affected smallholder and subsistence farmers, those infected, and national livestock industries. However, Rift Valley Fever virus (RVFV) vaccines are not commercially available outside of endemic areas or for humans, and current vaccines are limited in their safety and efficacy. A plant-based, viral nanoparticle vaccine offers a more affordable alternative to conventional vaccines that is safe, rapidly producible, and easily scalable, better meeting the needs of impacted communities. This project focuses on assessing the potential of using a Nicotiana benthamiana plant expression system to generate recombinant tobacco mosaic virus (TMV) nanoparticles displaying RVFV glycoprotein epitopes. Eight TMV-RVFV glycoprotein constructs were designed. Five TMV-RVFV constructs were successfully cloned, and four recombinant TMV constructs were successfully expressed in planta. The antigenicity of these constructs was examined for their possible use in RVFV vaccine development.

La rage est une zoonose des mammifères qui provoque environ 59000 morts/an malgré l’existence d’une prophylaxie efficace réalisable en pré- ou post-exposition. L’OMS recommande une vaccination immédiate complémentée par l’instillation d’immunoglobulines antirabiques au site de morsure mais reste mortelle sans vaccination. Pourtant plusieurs approches antivirales ont été décrites dans la littérature comme le protocole de Milwaukee appliqué à une jeune fille déjà symptomatique qui a reçu un cocktail de drogues ciblant différentes étapes du cycle viral (amantadine, ribavirine, ketamine) après induction d’un coma thérapeutique. L’idée de développer des antiviraux à large spectre inhibant plusieurs pathogènes reste l’alternative la plus réaliste pour espérer trouver un antiviral efficace contre la rage. C’est dans cette optique que s’inscrit mon travail de thèse au sein de l’Unité des Stratégies Antivirales de l’Institut Pasteur. Ceci a permis l’identification de molécules ayant un potentiel inhibiteur contre le virus de la rage qu’elles soient (i) disponibles sur le marché à d’autres fins thérapeutiques ou (ii) candidates issues d’un criblage à haut débit dirigé originellement contre d’autres pathogènes. Je me suis principalement intéressée à l’action de 4 molécules : 1. Arbidol (Umefenovir), un inhibiteur de fusion à large spectre utilisé contre le virus Influenza en Russie et en Chine; 2/3. ABMA et DABMA, deux drogues issues d’un criblage à haut débit contre la ricine, possédant une similarité de structure avec l’amantadine utilisée dans le protocole de Milwaukee ; 4 la Ribavirine, elle aussi utilisée au cours du protocole, préconisée contre plusieurs infections virales et qui a déjà montré une efficacité contre le virus de la rage in vitro. Enfin, dans le cadre du développement d’antiviraux à large spectre, l’efficacité des ces drogues a aussi été évaluée contre d’autres virus à ARN négatif comme le virus de la vallée du Rift et l’hantavirus Tula
Rabies is a mammalian zoonosis that causes about 59,000 deaths / year despite the existence of effective prophylaxis feasible pre- or post-exposure. WHO recommends immediate vaccination supplemented by instillation of anti-rabies immunoglobulin at the bite site but remains fatal without vaccination. Yet several antiviral approaches have been described in the literature as the Milwaukee protocol applied to an already symptomatic girl who received a cocktail of drugs targeting different stages of the viral cycle (amantadine, ribavirin, ketamine) after induction of a therapeutic coma. The idea of developing broad-spectrum antivirals that inhibit several pathogens remains the most realistic alternative for hoping to find an effective antiviral against rabies. It is in this perspective that my PhD work is part of the Antiviral Strategies Unit of the Institut Pasteur. This allowed the identification of molecules with inhibitory potential against rabies virus whether they were (i) commercially available for other therapeutic purposes or (ii) candidates from a broadband screening originally designed against other pathogens. I was mainly interested in the action of 4 molecules: 1. Arbidol (Umefenovir), a broad-spectrum fusion inhibitor used against Influenza virus in Russia and China; 2/3. ABMA and DABMA, two high-throughput ricin screened drugs with similar structure to amantadine used in the Milwaukee protocol; 4 Ribavirin, also used during the protocol, recommended against several viral infections and has already shown efficacy against rabies virus in vitro. Finally, in the development of broad spectrum antivirals, the efficacy of these drugs has also been evaluated against other negative RNA viruses such as Rift Valley virus and Tula hantavirus

Depuis la découverte du virus de l'Immunodéficience humaine, un lentivirus, comme agent pathogène responsable de l'épidémie du SIDA en 1983, beaucoup de progrès sur le sujet ont été réalisés. Il existe deux types de virus différents pouvant infecter l'Homme, le HIV-1 et le HIV-2. Ces deux virus se regroupent en différents groupes et sous-types qui témoignent d'une grande diversité inter et intra individus (notions de quasi-espèces). La découverte de lentivirus infectant naturellement au moins quarante-cinq espèces de primates en Afrique sub-saharienne, a permis un enrichissement des connaissances sur les origines des épidémies lentivirales humaines. Aujourd'hui , il est clairement admis que l'origine des épidémies d'HIV-1 et HIV-2 sont le résultat de transmissions zoonotiques de virus de chimpanzés/gorilles et de mangabeys enfumées, respectivement. La mise en évidence de nombreux SIV circulant chez ces primates non-humains indique bien le risque potentiel de nouvelles zoonoses dans la population humaine exposée, cependant, il peut paraître surprenant que jusqu'à maintenant, deux lignées lentivirales seulement ont été capables de franchir cette barrière d'espèce. Pour pouvoir se répliquer dans les cellules d'un nouvel hôte, un lentivirus doit pouvoir contrecarrer les différents facteurs de restriction exprimés par les cellules cibles tout en exploitant au maximum la machinerie cellulaire. La famille de protéines TRIM5, APOBEC3 et les protéines Tetherin/Bst2 et SAMHD1 sont capables de bloquer une infection rétrovirale. Dans ce travail, le rôle des protéines TRIM5 a été étudié ainsi que celui d'autres protéines interagissant avec des capsides rétrovirales, dans un contexte de transmission inter-espèces de lentivirus de primates. L'étude de TRIM5α humain a montré que cette protéine n'était capable de bloquer aucune des infections par les lentivirus primates testés dans cette étude, ni par les autres SIV. Nous avons pu mettre en évidence que la dépendance de la liaison à la Cyclophiline A pour l'infection des différents SIV était variable en fonction de la capside testée. Ainsi, si cette interaction est largement répandue parmi les différentes lignées de SIV, elle n'est toutefois pas universelle. La sensibilité des SIV à la déplétion de nucléoporines qui sont connues pour affecter l'infection par HIV-1, était également variable pour différents SIV, et la même diversité a été observée concernant les déplétions de RanBP2 et Nup153. De plus, nous avons découvert une capside de SIV soumise à une forte restriction de son infection dans les cellules humaines, ce phénotype a été nommé Ref2.Il a été suggéré qu'il existait une possible corrélation entre des variations de la capside de HIV-2 et la progression vers le SIDA, nous avons donc élaboré une étude afin de déterminer si les protéines TRIM5 étaient impliquées dans ce phénotype. La conclusion est que TRIM5α humaine ne restreint fortement aucune des capsides de HIV-2 testées provenant d'une cohorte d'individus à “progression rapide“ ou “lente“ vers le SIDA. Cependant nous avons observé une capacité d'infection qui corrélait avec la pathogénicité. Il est intéressant de noter que toutes les capsides d'HIV-2 testées étaient dépendantes de la présence de Cyclophiline A pour leur infection. Toutes ces capsides étaient sensibles à la déplétion de RanBP2, et l'interaction est très probablement médiée par le motif C-ter de RanBP2 qui a une forte homologie avec la Cyclophiline A. En conclusion, il est très probable que des SIV infectant naturellement des singes puissent utiliser les mêmes protéines que HIV-1, pour un éventuel passage inter-espèces. TRIM5α ne semble pas être une barrière efficace aux différents SIV, et l'interaction avec la Cyclophiline A est probablement très conservée par les lentivirus primates
Ever since HIV has been discovered to be the pathogenic agent that causes AIDS in 1983, much progress has been made in the field. Two different viruses are now known to infect humans, HIV-1 and HIV-2. These two distinct viruses have many sub-types and clades representing a high diversity inter and intra-individuals (quasi-species). The finding of HIV simian counterparts, the Simian Immunodeficiency Viruses (SIVs), has broadened the knowledge of primate lentiviruses and to date forty-five species of non-human primates are known to be infected with SIVs in sub-saharan Africa. It is now clear that HIV-1 and HIV-2 epidemics are the result of zoonosis from chimpanzees/gorillas and sooty mangabeys, respectively. With such a big diversity of SIVs in the wild and a frequent contact of SIV infected monkey species with humans, it is interesting that so far, only two lineages breached the species barrier and infected human populations. To be able to correctly infect a cell, a lentivirus has to overcome the installed cellular barriers known as restriction factors while at the same time correctly exploiting the established host cellular machinery. Proteins such as TRIM5, APOBEC3, Tetherin/Bst2, SAMHD1 are able to restrict retroviral infections in certain conditions. In this thesis, it has been evaluated the role of TRIM5 proteins and other capsid interacting proteins with a scope to the eventuality of a cross-species transmission infection. The results showed that human TRIM5alpha does not restrict any of the primate lentiviruses tested, and so far, no primate lentivirus is known to be restricted by it. Cyclophilin A binding and dependence is variable depending on the SIV capsid; this interaction is widespread among the primate lentiviruses phylogenetic tree but not a universal phenotype. Different capsids from SIVs have been tested for the sensitivity to the depletion of nucleoporins that are known to be used by HIV-1 in its infection; it has been concluded that the same diversity applies to the interaction with RanBP2 and Nup153. Additionally, we identified a SIV capsid that is highly restricted in human cells; this phenotype was called Ref2. With the report of a possible correlation between HIV-2 capsid variations and different levels of progression to AIDS, we devised a study aiming to identify if TRIM5 proteins were involved in this phenotype. We concluded that human TRIM5alpha does not restrict any HIV-2 capsid obtained from a HIV-2 cohort, in which individuals were presenting different levels of progression to AIDS. However, we observed a different viral fitness that correlated with pathogenicity. Moreover, Cyclophilin A dependence seems ubiquitous among all of the tested HIV-2 capsids. All of these capsids are sensitive to RanBP2 depletion and the interaction is much likely mediated by RanBP2's C-terminal motif that shares a high homology with Cyclophilin A. Summing up, it is much likely that some SIVs that still circulate in the wild can hijack the same specific cellular co-factors as HIV-1 to produce a new epidemic in humans. TRIM5α does not seem to be a potent barrier to an eventual cross-species transmission from lower primates to humans, and Cyclophilin A interaction seems to play a major role to the infection of some SIVs

The general aim of the PhD project was to apply phylogenetic analysis to viral sequences obtained in different geographic areas at different times, in order to reconstruct the most probable places of origin and pathways of dispersion of infections. Viral population growth and evolution leave a measurable imprint on the genome of viruses over the course of years, months or even days and occur simultaneously with geographic dispersal (Holmes, 2008; Pybus & Rambaut, 2009). This interaction characterizes a spatial phylodynamic process that can be recovered from genomic data using phylogeographic analyses (Faria et al., 2011). The research activity has been focused on Pestivirus genus, that includes pathogens of livestock (Bovine viral diarrhea virus - BDV) and wildlife (Border disease virus - BDV), and on zoonotic emerging diseases, involving in their epidemiological cycle both livestock and wildlife (Crimean-Congo hemorrhagic fever - CCHF, Hepatitis E virus- HEV). Concerning BDV, since 2001 several outbreaks of disease have been reported in Pyrenean chamois in Spain, France and Andorra. These outbreaks have decimated several Pyrenean chamois populations, with mortalities ranging from 40% to 85%. The infection has become endemic in the Central and Eastern Pyrenees. The aim of this study was to clarify the origin and dispersion of the Pyrenean chamois BDV genetic variant by reconstructing the spatial and temporal dynamics of BDV 5’ UTR sequences of Pyrenean chamois, 10 novel sequences and 41 retrieved from public databases and Sheep BDV sequences (n=44) from Spain and France were also retrieved. The phylogenetic analysis was performed using a Bayesian Markov chain Monte Carlo (MCMC) method implemented in the BEAST v.1.74 package. The chamois clade originated from sheep BDV genotype 4, generating a founder effect due to intra-species spread and spatial dispersion. The time of the most recent common ancestor estimates for the chamois clade dated back to a time span between 1974 and 1996, with a mean estimation falling in 1988. The pathway of dispersion of isolates suggests a complex exchange between neighboring Pyrenean chamois populations, still going on such as Western direction. Genetic typing of bovine viral diarrhea virus (BVDV) has distinguished BVDV-1 and BVDV-2 species and an emerging putative third species (HoBi-like virus), recently detected in southern Italy, signaling the occurrence of natural infection in Europe. Recognizing the need to update the data on BVDV genetic variability in Italy for mounting local and European alerts, a wide collection of 5’ UTR sequences (n = 371) was selected to identify the frequency of genotypes and subtypes at the herd level. BVDV-1 had the highest frequency, followed by sporadic BVDV-2. No novel HoBi-like viruses were identified. Four distribution patterns of BVDV-1 subtypes were observed: highly prevalent subtypes with a wide temporal-spatial distribution (1b and 1e), low prevalent subtypes with a widespread geographic distribution (1a, 1d, 1g, 1h, and 1k) or a restricted geographic distribution (1f), and sporadic subtypes detected only in single herds (1c, 1j, and 1l). BVDV-1c, k, and l are reported for the first time in Italy. Italy is one of the countries with the highest genetic diversity of BVDV worldwide. Northern Italy ranked first for BVDV introduction, prevalence, and dispersion. Nevertheless, the presence of sporadic variants in other restricted areas suggests the risk of different routes of BVDV introduction. CCHF is a zoonosis mainly transmitted by ticks that causes sporadic cases and severe hemorrhagic fever of acute human disease with a mortality rate of 5-60% and it has recently emerged in the Balkans and eastern Mediterranean areas. In order to reconstruct the origin and pathway of the worldwide dispersion of the virus at global and regional (eastern European) level, we investigated the phylogeography of the infection by analysing 121 publicly available CCHFV S gene sequences including two recently characterised Albanian isolates. The spatial and temporal phylogeny was reconstructed using a Bayesian Markov chain Monte Carlo approach. CCHFV phylogeographic reconstruction suggests that the disease originated about one thousand years ago from a common ancestor probably located in Africa. The virus then spread to Asia in the XV century and entered Europe on at least two occasions: the first in the early 1800s and the second in the early 1900s. The most probable location for the origin of the European clade was Russia, but Turkey played a central role in spreading the virus throughout Europe. Our data suggest that the movement of wild and domestic ungulates from endemic areas probably represent the main cause of virus dissemination in Eastern Europe. Hepatitis E virus is classified into four genotypes that have different geographical and host distributions. The main cause of sporadic autochthonous type E acute hepatitis in developed countries is genotype 3, which has a worldwide distribution and widely infects pigs. The aim of this study was to make hypotheses concerning the origin and global dispersion routes of this genotype by reconstructing the spatial and temporal dynamics of 208 HEV genotype 3 ORF-2 sequences (retrieved from public databases) isolated in different geographical areas. The evolutionary rates, time of the most recent common ancestors (tMRCAs), epidemic growth and phylogeography of HEV-3 were co-estimated using a MCMC Bayesian method. On the basis of time-scaled phylogeographical reconstruction, we hypothesise that HEV-3, after originating in the early 1800s in Europe, reached Asia in the first decades of 1900, and then moved to America probably in the 1970s-1980s. Analysis of the skyline plot showed a sharp increase of the number of infections between the 1980s and 2005, suggesting the intervention of new and highly efficient routes of transmission, possibly related to changes in the pig industry.