Interakce savčích endogennich retrovirů a jejich hostitelů

Endogenní retroviry (ERV) vznikají retrovirovou infekcí zárodečné linie a následným přenosem do dalších generací podle pravidel Mendelovy dědičnosti. Až na pár výjimek jsou všechny druhy savčích ERV evolučně staré a fixované v populaci svých hostitelských druhů. O některých skupinách retrovirů se předpokládálo, že nejsou schopny vytvářet endogenní kopie. Objevili jsme další příklad endogenního Lentiviru a první endogenní Deltaretrovirus. Obě tyto skupiny byly dříve považovány za neschopny vytvářet endogenní kopie. Endogenní lentiviry byly objeveny pouze nedávno a stale se považují za velmi vzácné. Toto jsou stále jen minoritní důkazy z kterých nemůžeme získat celkový obraz o průběhu virové endogenizace. Popsali jsme nový endogenní Lentivirus v genomu letuchy malajské (Galeopterus variegatus) a nazvali ho ELVgv (endogenous Lentivirus of G. variegatus). Na základě několika analýz jsme dokázali, že se jedná o nejstarší dosud objevený Lentivirus, a potvrdili jsme jeho přítomnost v jediném jiném současném druhu Dermopter - Cynocephalus volans. Endogenní deltaretroviry byly poslední retrovirovou skupinou bez nalezeného endogenního člena. Našli jsme zbytky endogenního Deltaretroviru v genomu netopýra létavce natalského (Miniopterus natalensis). Tato retrovirová sekvence byla přítomna v genomu pouze v jedné kopii....Endogenous retroviruses (ERVs) originate by germline infection and subsequent mendelian inheritance of their exogenous counterparts. With notable exceptions, all mammalian ERVs are evolutionarily old and fixed in the population of its host species. Some groups of retroviruses were believed not to be able to form endogenous copies. We discovered an additional endogenous Lentivirus and a first endogenous Deltaretrovirus. Both of these groups were previously considered unable to form endogenous copies. Endogenous lentiviruses were discovered only recently and are still quite rare. These are still just small pieces of evidence insufficient to give a broader picture about the history of virus endogenization. We described a novel endogenous Lentivirus in the genome of Malayan colugo (Galeopterus variegatus) denoted ELVgv (endogenous Lentivirus of G. variegatus). Based on several analyses we proved that this is the oldest Lentivirus discovered up to date and confirmed its presence in the only other extant species of Dermoptera - Cynocephalus volans. Endogenous deltaretroviruses were the last group without a single endogenous member. We detected the remnants of endogenous Deltaretrovirus in the genome of Natal Long-fingered bat (Miniopterus natalensis). However, this sequence was present in the genome only in one...Katedra genetiky a mikrobiologieDepartment of Genetics and MicrobiologyFaculty of SciencePřírodovědecká fakult

Endogenous retroviruses in primates

Numerous endogenous retroviruses (ERVs) are found in all mammalian genomes and represent retroviruses which have, by chance, integrated into the germline and are transmitted vertically from parents to offspring. In many non-human primates these insertions have not been well-studied. ERVs provide a snapshot of the retroviruses a host has been exposed to during its evolutionary history, including retroviruses which are no longer circulating. Accurate annotation and characterisation of ERV regions is an important step in interpreting the huge amount of genetic information available for increasing numbers of organisms. This project represents an extensive study into the diversity of ERVs in the genomes of primates and related ERVs in rodents, lagomorphs and tree shrews. The focus is on groups of ERVs for which previous analyses are patchy or outdated, particularly in terms of their evolutionary history and possible transmission routes. A pipeline has been developed to comprehensively and rapidly screen genomes for ERVs and phylogenetic analysis has been performed in order to characterise these ERVs. Laboratory study was used to complement the bioinformatics analysis. Almost 200,000 ERV fragments, many of which have not previously been characterised, were identified. A novel endogenous member of the lentivirus genus of retroviruses, which are rarely found in an endogenous form, was identified in the bushbaby Galago moholi. This ERV may represent an ancient ancestor of modern human immunodeficiency virus (HIV). Another retrovirus, gibbon ape leukaemia virus, previously thought to be a common pathogen in gibbons, was found to not exist in contemporary gibbons and a route through which a single cross species transmission event may have resulted in all known cases of this disease worldwide was identified. Endogenous epsilonretroviruses, usually considered to be viruses of fish and amphibians, were identified in all screened species of primates

Endogenous retroviruses in primates

Numerous endogenous retroviruses (ERVs) are found in all mammalian genomes and represent retroviruses which have, by chance, integrated into the germline and are transmitted vertically from parents to offspring. In many non-human primates these insertions have not been well-studied. ERVs provide a snapshot of the retroviruses a host has been exposed to during its evolutionary history, including retroviruses which are no longer circulating. Accurate annotation and characterisation of ERV regions is an important step in interpreting the huge amount of genetic information available for increasing numbers of organisms. This project represents an extensive study into the diversity of ERVs in the genomes of primates and related ERVs in rodents, lagomorphs and tree shrews. The focus is on groups of ERVs for which previous analyses are patchy or outdated, particularly in terms of their evolutionary history and possible transmission routes. A pipeline has been developed to comprehensively and rapidly screen genomes for ERVs and phylogenetic analysis has been performed in order to characterise these ERVs. Laboratory study was used to complement the bioinformatics analysis. Almost 200,000 ERV fragments, many of which have not previously been characterised, were identified. A novel endogenous member of the lentivirus genus of retroviruses, which are rarely found in an endogenous form, was identified in the bushbaby Galago moholi. This ERV may represent an ancient ancestor of modern human immunodeficiency virus (HIV). Another retrovirus, gibbon ape leukaemia virus, previously thought to be a common pathogen in gibbons, was found to not exist in contemporary gibbons and a route through which a single cross species transmission event may have resulted in all known cases of this disease worldwide was identified. Endogenous epsilonretroviruses, usually considered to be viruses of fish and amphibians, were identified in all screened species of primates

Investigating the development and evolution of drug resistance in the HIV-1 pol gene

The prognosis of those infected with HIV-1 has improved significantly since the introduction of highly active antiretroviral therapy (HAART). This has led to complete suppression of HIV-1 replication and reduction of viraemia to undetectable levels. Initially HAART comprised of three classes of drugs, namely nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs) and protease inhibitors (PIs), which target two important viral enzymes. Until recently, patients developing highly drug-resistant HIV-1 have had limited therapy options. This has changed in the last few years with the development and approval for use of second-generation NNRTIs and PIs, and three new classes of drugs including integrase strand transfer inhibitors (INSTIs). This means that patients undergoing INSTI-containing salvage therapy are taking drugs targeting all three HIV-1 pol genes; protease (PR), RT and IN. However, little data is available on the evolution, interaction and linkage of drug resistance mutations throughout the pol gene. In this study, we developed a single genome sequencing assay of the full-length HIV-1 pol gene and used it to investigate the development and linkage of drug resistance mutations in sequential samples from two patients failing INSTI-containing salvage therapy. Different phylogenetic methods were used to explore the evolution and dynamics of drug resistance mutations in the full-length HIV-1 pol gene. Furthermore, we examined the effect of co-evolved PR and RT on the susceptibility of patient-derived viruses to INSTIs and viral replicative fitness. Our data indicate that the development of drug resistance mutations in IN is complex and is a fine balance between attaining high levels of drug resistance and decent replicative fitness. This is to a degree influenced by mutations in other regions of the HIV-1 pol gene. Taken together, the data suggests that larger regions of patient-derived HIV-1 genome should be examined in order to get a good understanding of HIV-1 drug susceptibility

Links between inadequate immune responses to viral infections and disease outcome in humans

Deux pandémies virales touchent aujourd’hui des millions d’individus : la pandémie du Coronavirus Disease 2019 (COVID-19), causée par le Syndrome respiratoire aigu sévère coronavirus 2 (SRAS-CoV-2) et celle du Syndrome d’Immunodéficience Acquise (SIDA) causée par le Virus de l’Immunodéficience Humaine (VIH). Ces deux maladies diffèrent par leur physiopathologie, dont une meilleure compréhension a permis le développement de traitements efficaces. Pourtant, ces deux pandémies persistent. Une infection par SARS-CoV-2 peut être mortelle en raison d’une réponse immunitaire exacerbée et potentiellement retardée. Le VIH à l’inverse échappe continuellement à la réponse immunitaire, l’affaiblissant au cours des années jusqu’au développement du SIDA, ouvrant la porte à des maladies opportunistes mortelles. L’intérêt global de cette thèse était d’étudier comment la réponse immunitaire échoue pour ces deux infections. L’objectif de la première étude était de trouver un biomarqueur sanguin robuste et fiable pour prédire le risque de mortalité des patients hospitalisés pour la COVID-19. Nous avons mesuré la quantité d’ARN viral, de cytokines et de marqueurs de dommages tissulaires, ainsi que la réponse humorale contre le virus dans des échantillons de plasma de 279 patients à travers trois cohortes. Nous avons trouvé que l’ARN viral mesuré environ 11 jours après le début des symptômes, et ajusté pour l’âge et le sexe, peut prédire la mortalité dans les 60 jours suivant le début des symptômes. Nous avons également trouvé que des fortes concentrations de cytokines inflammatoires et de marqueurs de dommages tissulaires, ainsi qu’un faible niveau d’anticorps liant le Region Binding Domain (RBD) de la protéine Spike de SARS-CoV-2, sont aussi associées à la mortalité. Dans un deuxième projet, nous nous sommes servis d’outils de réduction de dimensionnalités combinant les facteurs associés à la mortalité, permettant une stratification des patients en quatre groupes basés uniquement sur leur profil immuno-virologique plasmatique. Un seul de ces groupes est lié à une plus grande mortalité. Mis ensemble, nos travaux permettent une meilleure compréhension de l’hétérogénéité des patients hospitalisés pour la COVID-19, incluant l’identification des patients à haut risque de mortalité. Ces données pourraient servir à cibler les traitements thérapeutiques selon la réponse immunologique du patient. Notre troisième étude portait sur l’étude de la dysfonction des lymphocytes T CD4+ spécifiques du VIH. Ceux-ci sont affaiblis par l’infection au VIH et perdent leur capacité à combattre l’infection. Notre objectif était de caractériser la réponse au blocage du point de contrôle immunitaire (BPCI - immunothérapie qui renverse partiellement la dysfonction des lymphocytes T) PD-1 parmi les divers types des lymphocytes T CD4+. Nous avons d’abord comparé l’état de dysfonction des lymphocytes chez deux cohortes de personnes vivant avec le VIH (PVVIH) non-traitées. Pour l’une des deux cohortes, la virémie est contrôlée par le système immunitaire (cohorte dite de « Contrôleurs Élites »), à l’inverse de la seconde cohorte (dite « Virémique »). Les lymphocytes T CD4+ des personnes virémiques perdent leur activité antivirale et ont une forte expression de points de contrôles immunitaires. Au contraire, les contrôleurs élites ont une charge virale indétectable en l’absence de thérapie antirétrovirale (TAR), et des lymphocytes T CD4+ relativement fonctionnels. En réponse au BPCI, les lymphocytes T CD4+ spécifiques du VIH démontrent une plus grande réponse chez les PVVIH virémiques, via l’augmentation du nombre de cellules produisant des cytokines. Chez ces individus, toutes les fonctions mesurées augmentent, à l’exception des cytokines associées aux cellules T CD4+ folliculaires auxiliaires qui sont impliquées dans l’amorçage des réponses immunologiques des lymphocytes B. Ces données montrent qu’il existe une réponse spécifique des sous-types de lymphocytes T CD4+ aux BPCI. Ce projet démontre l’avantage du blocage du ligand de PD-1 (PD-L1) sur les effets hétérogènes au niveau unicellulaire, soulignant l’importance de considérer les T CD4+ dans les analyses futures des essais cliniques évaluant le bénéfice des BPCI. Mis ensemble, cette thèse permet une meilleure caractérisation de la réponse immunologique contre un virus à infection aiguë et, dans un second temps, un autre à infection chronique. Ces études permettent une meilleure compréhension de l’hétérogénéité dans la réponse immunologique des personnes infectées qui, si prise en compte dans des essais cliniques, pourrait aider à expliquer la variété de l’efficacité des traitements.Viral infections are a major cause of disease in humans. Pandemics refer to virulent viruses that spread across more than one continent. In the last century, two major such pandemics have occurred with still-current repercussions: the acquired immunodeficiency syndrome (AIDS) pandemic caused by the Human Immunodeficiency Virus (HIV), and the Coronavirus Disease 2019 (COVID-19) pandemic by the severe acute respiratory coronavirus 2 (SARS-CoV-2). The diseases caused by both of these viruses are very different in their pathophysiology. A better elucidation of these diseases has already allowed researchers to develop therapeutic treatments against these infections; however, both pandemics caused by these viruses are ongoing. While SARS-CoV-2 proves to ultimately be fatal by an exacerbated and perhaps delayed immune response against the virus, HIV rather evades the host’s immune response, weakening it over time until inducing a severe immunocompromised state, opening the door for fatal opportunistic diseases. The overarching goal of this thesis was to study the failings of the immune response against each virus, and extract information useful to guide therapeutic practices. The objective of the first study was to find a robust and reproducible predictor of fatal outcome among patients hospitalized for their COVID-19. We profiled the plasma of a total of 279 patients across three independent cohorts to measure SARS-CoV-2 viral RNA, antibody responses against the virus and the quantities of inflammatory cytokines and markers of tissue damage. We found that plasma viral RNA could reproducibly predict fatal outcome on samples collected at 11 days after symptom onset, when adjusted for age and sex. Plasma vRNA’s predictive accuracy was maintained at earlier timepoints. We also found that low SARS-CoV-2-region-binding-domain (RBD)-specific IgG, low SARS-CoV-2-specific antibody-dependent cellular cytotoxicity, and elevated cytokines and injury markers were also strongly associated with mortality. In a second study using dimensionality reduction tools, we were able to separate our cohort in four distinct « patient clusters », based on their immunovirological plasma profile, with one cluster enriched in fatal outcomes. Our findings better characterize the heterogeneity of hospitalized COVID-19 cases, and may be useful in directing targeted therapeutic treatments. In our third study, our objective was to characterize the response of dysfunctional HIV-specific CD4+ T cells to immune checkpoint blockade (ICB) across multiple subsets. We first sought to compare the functional state of HIV-specific CD4+ T cells among two cohorts of HIV-infected untreated indivduals, based on their ability to spontaneously control viral replication. Elite controllers, who have no detectable viral load in the absence of anti-retroviral therapy (ART), had more functional HIV-specific CD4+ T cells than their viremic counterparts, as well as lower levels of dysfunction-related transcription factors and immune checkpoint expression. We then compared the response of HIV-specific CD4+ T cells to ICB and saw greater increase in functionality in the dysfunctional cells of viremic individuals. All functions assessed were increased except for B-cell helping T follicular-helper-associated functions, underlying subset-specific responses to ICB. This effect was largely lost once ART was initiated, suggesting that the use of ICB would be optimal right before the initiation of ART. Together, our results contribute to a better understanding of two pandemic-causing viral infections, and reveal key considerations for therapy

A Novel Bayesian Method for Detection of APOBEC3-Mediated Hypermutation and Its Application to Zoonotic Transmission of Simian Foamy Viruses

<div><p>Simian Foamy Virus (SFV) can be transmitted from non-human primates (NHP) to humans. However, there are no documented cases of human to human transmission, and significant differences exist between infection in NHP and human hosts. The mechanism for these between-host differences is not completely understood. In this paper we develop a new Bayesian approach to the detection of APOBEC3-mediated hypermutation, and use it to compare SFV sequences from human and NHP hosts living in close proximity in Bangladesh. We find that human APOBEC3G can induce genetic changes that may prevent SFV replication in infected humans in vivo.</p></div