Viruses in the Flavivirus genus (e.g., Zika, yellow fever, dengue, West Nile, and Japanese encephalitis viruses) are arthropod-borne, globally distributed, and can cause a range of neurological or hemorrhagic diseases. The ongoing epidemics of flaviviral disease consistently demonstrate the need for new vaccines capable of outbreak control. However, safe, effective, and easy-to-produce vaccines remain relatively elusive due to limitations of conventional vaccine development that make it difficult to balance safety and efficacy. Insect-specific flaviviruses (ISFVs) are emerging as a novel method to overcome this challenge. Herein, we develop a new flavivirus vaccine platform based on the novel insect-specific flavivirus called Aripo virus, which we used to create a preclinical Zika virus (ZIKV) vaccine named Aripo/Zika virus (ARPV/ZIKV). ARPV/ZIKV is a live recombinant virus consisting of the ZIKV pre-membrane and envelope protein genes expressed on an Aripo virus backbone. In this work, we quantify the safety and efficacy of ARPV/ZIKV in multiple murine models, and begin to elucidate the mechanisms of humoral and cell-mediated immune induction for this new platform. Overall, the vaccine showed no evidence of pathogenicity in immunocompromised or suckling mice, and demonstrated a complete inability to replicate in various vertebrate cell lines. Despite this lack of replication, a single dose of live, unadjuvanted ARPV/ZIKV completely prevented ZIKV disease in mice and prevented in utero ZIKV transmission in gravid mice. Direct protection post-ZIKV challenge appears to be primarily mediated by neutralizing antibodies based on passive transfer, adoptive transfer, and T-cell depletion studies. However, vaccination studies in Rag1 KO, Tcra KO, and muMt- mice demonstrate the critical role of T-cell responses in developing immunity post-vaccination. In summary, ARPV/ZIKV is a promising vaccine candidate that induces robust adaptive immune responses, and this success is a positive indication of ARPV's potential as a new resource for flavivirus vaccine development. This body of work contributes to the rapidly expanding field of insect-specific virus-based vaccines and generates new insights into their optimization. Ultimately, this work may help protect the health of millions of people worldwide that are currently at risk of flavivirus infection. / MPH / Arthropod-borne viruses (especially flaviviruses such as Zika virus (ZIKV), yellow fever virus, West Nile virus) represent a major global health threat and a significant burden on human life. Vaccination is a critical tool for controlling the often unpredictable outbreaks of flavivirus diseases. However, licensed flavivirus vaccines remain relatively elusive. This is, in part, because the same characteristics of traditional live-attenuated vaccines that make them highly effective can also reduce their safety. Insect-specific flaviviruses (ISFVs) are emerging as a novel method to overcome this challenge. ISFVs only replicate in insects and thus are safe in humans. They do not cause disease in vertebrates, eliminating the need for the chemical or physical inactivation methods required by traditional whole inactivated vaccines and which can result in reduced efficacy. Herein, we develop a new flavivirus vaccine platform based on a novel ISFV called Aripo virus (ARPV). As proof of concept, we used ARPV to create a preclinical ZIKV vaccine named Aripo/Zika virus (ARPV/ZIKV). ARPV/ZIKV expresses immune system-stimulating ZIKV structural proteins on its virus particle. However, it remains highly safe because the genetic material from ARPV makes it incompatible for replication in human cells. Here, we demonstrate the safety and protective ability of ARPV/ZIKV, and begin to elucidate its mechanisms of protection. Overall, ARPV/ZIKV shows promise as a ZIKV vaccine candidate, which supports the potential of ARPV as a platform for new flavivirus vaccines and the potential to protect the health of the millions of people currently at risk of flavivirus infection.

Intervirology has decided to publish the ensuing letter by Madani et al. [1] and a response by Alzahrani et al. [2] concerning a prior publication [3] . As the letter by Madani et al. was obviously not accepted by EID, Intervirology feels that it is fair and appropriate to provide the authors the opportunity to discuss what they see as substantial errors in the work of Alzahrani et al. [3] . Taxonomy is apparently a matter that raises emotions. Disagreements between researchers on designation of a new virus have occurred in the past, one only has to remember the issue on HTLV-III versus LAV between renowned groups of researches that was eventually cleared.

Emerging viruses represent a significant threat to human health. Understanding the drivers of emergence, such as viral evolution, is a critical avenue to combat these pathogens. One specific group of emerging pathogens of interest is flaviviruses. Flaviviruses are arthropod-borne viruses (arbovirus) in the family Flaviviridae. The medically relevant flaviviruses can be divided into two groups – tick-borne and mosquito-borne. Included within the mosquito-borne flaviviruses group are dengue viruses 1-4 (DENV 1-4), which causes 400 million infections annually, and Zika virus (ZIKV), which caused over 128 million infections from 2013-2018. These viruses, which are cocirculating, share high sequence similarity in key antigenic regions. Because of these similarities, pre-existing immunity to DENV has been correlated with altered pathogenesis of subsequent ZIKV infections. Despite this, there has been little analysis of the effects of pre-existing DENV immunity on the evolution of subsequent flavivirus infection, despite being characterized for many other viruses. Given that mutation that could arise from cross-reactive immune selection could alter pathology or transmissibility, it is critical to assess the role of cross-reactive immune selection as an evolutionary driver. However, this line of research has historically been difficult due to the inherent toxicity of flavivirus infectious clones in bacteria. To mitigate the toxic nature of flavivirus clones, we developed several entirely in vitro workflows using a combination of rolling circle amplification (RCA) and replication cycle reaction (RCR). We demonstrated that RCA was a comparable substitute to traditional plasmid propagation using an alphavirus infection clone. We further demonstrated that RCR could be used to generate infectious clones by producing infectious clones of DENV2 and SARS-CoV-2, as well as demonstrating it could be used to introduce mutations into infectious clones by producing a D614G SARS-CoV-2 mutations. With this technology in place, we used in vitro directed evolution system, where we passaged ZIKV in convalescent patient serum to assess the role of cross-reactive immune selection as an evolutionary driver. After passaging, we performed next-generation sequencing to assess the impacts of cross-reactive immune selection on the viral populations and to identify mutations that arose post-passaging. We observed that ZIKV passaged in convalescent DENV serum had reduced diversity and divergence in the premembrane region. Within the convalescent DENV passaged population, we identified two mutations of interest with the dominant antibody binding region – E-V335I and NS1-T139A. These mutations were then introduced using our in vitro workflows. The resulting mutant viruses were then assessed for their replicative fitness in mammalian cell culture and mosquito models and their sensitivity to neutralization. We observed that while both E-V355I and NS1-T139A have increased fitness in mammalian cells, they had reduced fitness in mosquitoes. These results align with the trade-off hypothesis, which states that in a multi-host system, adaptation to one host reduces fitness in the other hosts. When we assessed the neutralization sensitivity of the mutants, we observed that while NS1-T193A was resistant to neutralization, E-V355I was more sensitive to neutralization. These results indicate that neutralization escape is not necessary for enhanced post-passaging in convalescent DENV serum. Our findings demonstrate that cross-reactive immune selection can generate several mutations with altered fitness in mammalian cells and mosquitos. This research is significant for both highlighting novel technologies to facilitate molecular virology and demonstrating that cross-reactive immune selection has the potential to alter the evolutionary trajectory of flaviviruses. This work provides critical information to understand how flaviviruses are evolving and emerging, and therefore critical information to address their threat to human health. / Doctor of Philosophy / Emerging viruses represent a significant threat to human health. We must understand what drives these viruses to adapt and evolve to respond to these threats. One virus family of extreme importance is the genera flavivirus. Flaviviruses are arthropod-borne viruses (arbovirus) that can be spread by the bites of ticks and mosquitoes. Included in the mosquito-borne flavivirus are dengue virus 1-4 (DENV1-4), which accounts for 400 million new infections annually, and Zika virus (ZIKV), which caused more than 128 million infection from 2013-2018. In addition to co-circulating, DENV 1-4 and ZIKV share several key similarities in their protein structures, which results in pre-existing DENV immunity effect how subsequent infections behave. The effect of pre-existing immunity on the evolution of these viruses has not been well established, despite similar studies being performed for other viruses. Given that the mutations that could arise from immune-driven evolution could alter disease severity or transmissibility, the impacts of immune-driven evolution must be characterized. However, the current tools available to perform this research are suboptimal, as the toxicity of flavivirus genomes hampers out ability to perform bacterial cloning, which has historically been necessary to develop and modify infectious clones. To mitigate the toxicity, we developed a "bacteria-free" workflow using emerging technologies like rolling circle amplification (RCA) and replication cycle reaction (RCR). With the technology in place, we propagated several generations of ZIKV or DENV in the presence of serum from human patients with a history of DENV infections. We then sequenced the viruses and identified mutations that arose during passaging. The mutations were then inserted using our bacteria-free workflow into infectious clones. The resulting viruses were assessed for their ability to replicate in mammalian cells, their ability to infect mosquitos, and their sensitivity to patient serum. We found that exposing ZIKV to serum from patients with pre-existing immunity to DENV can result in ZIKV developing several mutations. These mutations make the virus more effective at infecting mammalian cells and less effective at infecting mosquitos. This research is significant as it highlights novel technologies to aid researchers, and it demonstrates that pre-existing immunity has the potential to alter the evolutionary trajectory of flaviviruses. This information is critical in understanding flavivirus evolution and their emergence and therefore is critical to addressing their threat to human health.

Flaviviruses are a genus of vector-transmitted viruses that are nearly globally distributed, and flavivirus infections can result in life threatening diseases. Many flaviviruses such as Dengue, West Nile, yellow fever and Zika viruses are globally distributed. Zika virus (ZIKV) is a single strand positive-sense RNA virus, and its disease has been linked to Guillain Barré Syndrome (i.e., a debilitating autoimmune disorder that affects the nerves) in adults and congenital birth defects including microcephaly (i.e., a neurodevelopmental disorder due to impaired neural cell proliferation) in newborns. Insect-specific flaviviruses (ISFVs) are understudied given their apathogenic characteristics to humans and animals. However, given their close genetic relationship to vertebrate infectious flaviviruses, ISFVs can serve as a delivery system (i.e., vector) for flavivirus antigenic proteins. Aripo virus (ARPV) is a recently discovered ISFV isolated in Trinidad. We developed a chimeric Zika vaccine, Aripo-Zika, by substituting the pre-membrane and envelope genes of ZIKV into the ARPV genome. Here, we explored (i) the efficacy of Aripo-Zika (AZ) vaccination by evaluating passive transfer of maternal antibodies, (ii) the optimal dosage regimen, (iii) anti-vector immunity to the ARPV backbone, and (iv) the effects of boosters on vaccine efficacy. We also evaluated AZ safety via a co-infection study. Our results show a near linear relationship between increased dose and immunogenicity, with 1011 genome copies being the most effective minimum dose administered. Inclusion of boosters further increased the immunogenicity of AZ. Additionally, prior immunization with AZ showed minimal effects on subsequent immunization with an ARPV-West Nile virus (AWN) vaccine candidate, confirming the applicability of the ARPV backbone to multiple flavivirus vaccine candidates. In vitro co-infection of ZIKV with ARPV, and ZIKV with AZ in African green monkey kidney cells (i.e., Vero-76) indicated ARPV and AZ remain incapable of replication in vertebrate cells, even in the presence of active ZIKV replication. Altogether, our data suggests that the ARPV platform is a safe and effective strategy for the development of flavivirus vaccines. / Master of Science in Life Sciences / Vaccines are one of the best tools available since their initial conception. Vaccines have collectively increased human lifespan and reduced the burden of disease in humans and animals worldwide. Vaccine research aims to create vaccines that have a perfect balance of safety and efficacy. The goal is to produce a vaccine that can generate a strong immune response against the virus(es) of interest, while causing the least harm or side effects from the vaccine. Insect-specific viruses are viruses that infect insect cells, but are unable to replicate in humans or other vertebrate cells. The Auguste Lab has created a chimeric vaccine using the genome of an insect-specific virus called Aripo-Zika virus (AZ) that is genetically related to Zika virus. A person vaccinated with AZ is expected to develop an immune response against Zika but would not have any disease or side effects associated with a Zika infection or virus replication. In order to determine if this vaccine would be safe and effective enough to advance to clinical trials in humans, we must first determine if it is safe in smaller animal models. My studies have five central aims. First, determine the lowest dose of AZ that can be given and still be protective against Zika disease in mouse models. Second, determine if boosters are necessary and if they increase protection. Third, determine if immunity derived from vaccination can be passed down from mother to pups. Fourth, determine if Zika virus and AZ can co-exist in the same cell line without AZ replication occurring. Lastly, determine if mice can be vaccinated with AZ and subsequently with another similar Aripo virus-based vaccine (i.e., Aripo-West Nile) without changing the effectiveness of the subsequent immunization. Our results showed that AZ is able to be passed from mother to pup, 1011 genome copies is the minimum protective dose, and boosters can increase the effectiveness of AZ. We also found that AZ does not replicate in vertebrate cells when it co-exists with ZIKV and subsequent vaccination with Aripo-West Nile does not seem affect the effectiveness of either vaccine.

Made available in DSpace on 2016-01-26T12:51:28Z (GMT). No. of bitstreams: 1 adrianomondini_tese.pdf: 12162182 bytes, checksum: fe0a4d238020f614624084ebb47e1011 (MD5) Previous issue date: 2010-03-05 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Dengue belongs to the Flavivirus genus and is the most common arboviral infection worldwide. It can be caused by four antigenically different serotypes (DENV 1-4). These serotypes are transmitted mainly by the bite of Aedes aegypti mosquitoes. The vector is widely associated with human activity and the influence of organized social space favors the interaction among vector, virus and man, making populated areas sources of dengue dispersion. In this study, we performed a molecular, spatial and temporal study of DENV transmission through positive samples of blood and infected mosquitoes captured in São José do Rio Preto/SP in a period of four years. Material and Methods: Serum samples of patients presenting dengue like symptoms and pools of mosquitoes had their viral RNA extracted and were tested by Multiplex- RT-PCR with Flavivirus generic primers based on non-structural protein (NS5) in the first round, followed by Nested assays with species-specific primers for the identification of DENV 1-3, yellow fever virus, Saint Louis encephalitis virus (SLEV) among others. Positive samples were analyzed spatially and phylogenetically. Results and Discussion: We analyzed 613 blood samples for four years: 199 in 2006, 94 in 2007, 313 in 2008 and 10 in 2009. The positivity was high in 2006 and 2007, with 106 and 51 infected patients, respectively. The major dengue serotype circulating during the 2006 and 2007 epidemics was DENV-3 and few cases of DENV-2, which is an indication of its recent introduction in the municipality. We also reported the first outbreak of SLEV in Brazil in 2006. Among DENV patients in 2008, only seven were infected by DENV-3 and 90 were infected by DENV-2, suggesting the reemergence of this serotype. We detected the circulation of DENV-1 in two Abstract xxv patients in 2008 and in four patients in 2009. Nearly 1200 mosquitoes were captured from December 2007 to March 2008. We have captured 814 Aedes aegypti mosquitoes, which were divided in 463 pools. Only 3.67% of them were positive for DENV-3 and DENV-2. Pools containing only male mosquitoes were positive for DENV, indicating the presence of transovarial transmission. We obtained sequences from 82 patients among 174 blood samples. We were able to geo-code 46 sequences. The alignment generated a 399-nucleotide long dataset with 134 taxa. The phylogenetic analysis indicated that all samples were of DENV-3 and related to strains circulating on the isle of Martinique in 2000 2001. Sixty DENV-3 from São José do Rio Preto formed a monophyletic group (lineage 1), closely related to the remaining 22 isolates (lineage 2). We assumed that these lineages appeared before 2006 in different occasions. The possibility of inferring the spatio-temporal dynamics from genetic data has been generally little explored, and it may shed light on DENV circulation. The use of both geographic and temporally structured phylogenetic data provided a detailed view on the spread of at least two dengue viral strains in a populated urban area. / Dengue pertence ao gênero Flavivirus e é a infecção por arbovírus mais comum no mundo todo. Pode ser causada por quatro sorotipos antigenicamente distintos (DENV 1-4). Estes sorotipos são transmitidos pela picada do mosquito Aedes aegypti. O vetor está amplamente associado a atividade humana e a influencia do espaço urbano favorece a interação entre o vetor, o vírus e o homem, tornando áreas populosas, grandes centros de dispersão do dengue. Neste estudo, foi realizada um estudo molecular, espacial e temporal da transmissão de DENV através de amostras positivas de sangue e de mosquitos infectados capturados em São José do Rio Preto/SP, num período de quatro anos. Materiais e métodos: Soro de pacientes apresentando sintomas de dengue e pools de mosquitos tiveram seu RNA viral extraído e foram testados por Multiplex-RT-PCR, com primers genéricos de Flavivirus baseados na proteína não estrutural 5 (NS5) numa primeiro ciclo,seguida por ensaios Nested com primers específicos para DENV, para o vírus da febre amarela, para o vírus da encefalite de Saint Louis, entre outros. As amostras positivas foram analisadas espacial e filogeneticamente. Resultados e discussão: Analisamos 613 amostras de soro durante 4 anos: 199 em 2006; 94 em 2007; 313 em 2008 e 10 em 2009. A positividade foi alta em 2006 e 2007, com 106 e 51 pacientes infectados, respectivamente. O principal sorotipo circulante durante as epidemias de 2006-2007 foi DENV-3 e poucos casos de DENV-2, o que pode ser a indicação de sua recente introdução no município. Nós também descrevemos a primeira epidemia de SLEV no Brasil em 2006. Dentre os pacientes com DENV em 2008, apenas sete estavam infectados com DENV-3 e 90 com DENV-2, sugerindo a reemergência do sorotipo. Nós Resumo xxiii detectamos a circulação de DENV-1 em dois pacientes em 2009 e em quatro pacientes em 2009. Aproximadamente 1200 mosquitos foram capturados entre Dezembro 2007 e Março de 2008. Capturamos 814 mosquitos Aedes aegypti, que foram divididos em 463 pools. Apenas 3,67% deles foram positivos para DENV-2 e DENV-3. Pools contendo apenas machos foram positivos para DENV, indicando a presença de transmissão transovariana. Nós obtivemos sequências de 82 pacientes dentre 174 amostras de sangue. Nós fomos capazes de geocodificar 46 sequências. O alinhamento gerou gerou nucleotídeos com 399 bp com 134 taxa. A análise filogenética indicou que todas as amostras foram de DENV-3 e estavam relacionadas às cepas circulantes na ilha da Martinica em 2000-2001. Sessenta pacientes com DENV- 3 de São José do Rio Preto formaram um grupo monofilético (linhagem 1), intimamente relacionado com os outros 22 isolados (linhagem 2). Nós assumimos que estas linhagens apareceram antes de 2006 em ocasiões diferentes. A possibilidade de inferir a dinâmica espaço-temporal através de dados genéticos é relativamente pouco explorada e pode esclarecer acirculação de DENV. O uso de dados filogenéticos estruturadosgeograficamente e temporalmente forneceu uma visão detalhada na dispersão de, pelo menos, duas cepas virais distintas numa área urbana.

Die zunehmende Ausbreitung des Usutu-Virus in Europa als Ursache für fatale Ausbruchsgeschehen innerhalb der Avifauna, insbesondere unter Sperlingsvögeln (Passeriformes) und Eulenartigen (Strigiformes), stellt in Zusammenhang mit einem neuroinvasiven sowie zoonotischen Potential ein Risiko für die Veterinär- sowie Humanmedizin dar. Trotz dieser Relevanz stehen derzeit keine zugelassenen Therapeutika gegen eine Usutu-Virus-Infektion zur Verfügung. Auf Basis indikationsfremder Substanzen mit pharmakologischer Zulassung im Sinne des drug repositioning sowie auf Grundlage der Gegenregulation viral-induzierter Modulationen des Zellmetabolismus wurde die Identifikation antiviraler Therapieoptionen gegen das Usutu-Virus in vitro angestrebt.:Abkürzungsverzeichnis Abbildungsverzeichnis Tabellenverzeichnis 1 Einleitung 2 Literaturübersicht 2.1 Das Usutu-Virus 2.1.1 Ursprung, Klassifikation und Epidemiologie 2.1.1 Transmissionszyklus und Wirtstropismus 2.1.2 Aufbau des Virions und des Genoms 2.1.3 Veterinärmedizinische Relevanz und pathologische Ausprägung 2.1.4 Humanmedizinische Bedeutung als Zoonose-Erreger 2.1.5 Vergleichende Aspekte zum West-Nil-Virus 2.2 Antivirale Präventions- und Therapieoptionen 2.2.1 Möglichkeiten und Grenzen der Immunprophylaxe 2.2.2 Pharmaka mit potentiell antiviraler Wirkung gegen Flaviviren 2.2.3 Identifizierte Substanzen gegen das Usutu-Virus 2.3 Zelluläre Systeme als antivirale Zielobjekte 2.3.1 Das Interferon-System und seine antivirale Schutzfunktion 2.3.2 Viral-induzierte Modulation des Wirtszellmetabolismus 3 Zielstellungen der Dissertation 4 Material 4.1 Zelllinien 4.2 Viruslinien 4.3 Zellkulturmedien 4.4 Pharmakologische und andere Substanzen 4.5 Chemikalien 4.6 Lösungen und Puffer 4.7 Antikörper 4.8 Reagenzien und Kit-Systeme 4.9 Enzyme und Nukleotide 4.10 Primer 4.11 Verbrauchsmaterialen 4.12 Geräte 4.13 Datenbanken und Software 5 Methodik 5.1 Zellkultivierungstechnik und PBMC-Isolation 5.2 Isolation von Usutu-Virus-Linien aus Gewebeproben in Zellkultur 5.2.1 Aufbereitung aviären Organmaterials 5.2.2 Typisierung von in Deutschland zirkulierenden Usutu-Virus-Linien (2019, 2020) 5.2.3 Virusanzucht in verschiedenen Zellkulturen zur Virusstock-Generierung 5.3 Quantifizierung des extrazellulären Virustiters 5.4 Immunfluoreszenzanalyse 5.5 Präparation pharmakologischer und anderer Substanzen 5.6 Infektionsansätze mit dem Usutu-Virus und WNV 5.7 Durchflusszytometrische Analyse 5.8 Zytotoxizitätsstudien 5.9 Extrazelluläre Fluxanalyse mittels Agilent Seahorse XF-Technologie 5.10 Statistische Analyse 6 Ergebnisse 6.1 Isolation des Usutu-Virus in Zellkultur 6.2 Replikationsdynamik des Usutu-Virus in verschiedenen Zelllinien 6.3 Pharmakologisches Screening zur antiviralen Wirksamkeit gegen das Usutu-Virus 6.4 Identifikation und Charakterisierung der antiviralen Eigenschaften von Ivermectin 6.5 Wirkung von Ivermectin gegen Linie 2 des West-Nil-Virus in einer aviären Zelllinie 6.6 Metabolischer Phänotyp Usutu-Virus-infizierter Zelllinien 6.7 Antivirale Inhibition der Glykolyse durch 2-Deoxy-D-Glukose 6.8 Einfluss von exogenem Interferon auf den Wirtszellmetabolismus unter Infektion 6.9 Auswirkung der Interferon-Rezeptor-Defizienz auf den Metabolismus unter Infektion 7 Diskussion 7.1 Typisierung und Isolation des Usutu-Virus in Zellkultur 7.2 Charakterisierung der zelltypspezifischen Permissivität und Replikationskinetik 7.3 Identifikation antiviral wirksamer Substanzen gegen das Usutu-Virus 7.4 Usutu-Virus-induzierte Modulation des Metabolismus als antiviraler Ansatz 8 Ausblick 9 Zusammenfassung 10 Summary 11 Literaturverzeichnis 12 Anhang 12.1 Zusatzmaterial 12.2 Publikation 1 12.3 Publikation 2 12.4 Publikation 3 12.5 Weitere Veröffentlichungen 13 Danksagung / The emerge of Usutu virus (USUV) in Europe as a causative agent of fatal outbreaks in avifauna, notably in Passeriformes and Strigiformes, as well as its neuroinvasive and zoonotic potential emphasize a considerable risk in veterinary and human medicine. Despite its relevance, recently, no approved drugs against USUV infections are available. The identification of antiviral therapeutic options against USUV in vitro was addressed based on the analysis of approved drugs of other medical indications in terms of drug repositioning and the counteraction of viral-induced alterations of the cellular metabolism.:Abkürzungsverzeichnis Abbildungsverzeichnis Tabellenverzeichnis 1 Einleitung 2 Literaturübersicht 2.1 Das Usutu-Virus 2.1.1 Ursprung, Klassifikation und Epidemiologie 2.1.1 Transmissionszyklus und Wirtstropismus 2.1.2 Aufbau des Virions und des Genoms 2.1.3 Veterinärmedizinische Relevanz und pathologische Ausprägung 2.1.4 Humanmedizinische Bedeutung als Zoonose-Erreger 2.1.5 Vergleichende Aspekte zum West-Nil-Virus 2.2 Antivirale Präventions- und Therapieoptionen 2.2.1 Möglichkeiten und Grenzen der Immunprophylaxe 2.2.2 Pharmaka mit potentiell antiviraler Wirkung gegen Flaviviren 2.2.3 Identifizierte Substanzen gegen das Usutu-Virus 2.3 Zelluläre Systeme als antivirale Zielobjekte 2.3.1 Das Interferon-System und seine antivirale Schutzfunktion 2.3.2 Viral-induzierte Modulation des Wirtszellmetabolismus 3 Zielstellungen der Dissertation 4 Material 4.1 Zelllinien 4.2 Viruslinien 4.3 Zellkulturmedien 4.4 Pharmakologische und andere Substanzen 4.5 Chemikalien 4.6 Lösungen und Puffer 4.7 Antikörper 4.8 Reagenzien und Kit-Systeme 4.9 Enzyme und Nukleotide 4.10 Primer 4.11 Verbrauchsmaterialen 4.12 Geräte 4.13 Datenbanken und Software 5 Methodik 5.1 Zellkultivierungstechnik und PBMC-Isolation 5.2 Isolation von Usutu-Virus-Linien aus Gewebeproben in Zellkultur 5.2.1 Aufbereitung aviären Organmaterials 5.2.2 Typisierung von in Deutschland zirkulierenden Usutu-Virus-Linien (2019, 2020) 5.2.3 Virusanzucht in verschiedenen Zellkulturen zur Virusstock-Generierung 5.3 Quantifizierung des extrazellulären Virustiters 5.4 Immunfluoreszenzanalyse 5.5 Präparation pharmakologischer und anderer Substanzen 5.6 Infektionsansätze mit dem Usutu-Virus und WNV 5.7 Durchflusszytometrische Analyse 5.8 Zytotoxizitätsstudien 5.9 Extrazelluläre Fluxanalyse mittels Agilent Seahorse XF-Technologie 5.10 Statistische Analyse 6 Ergebnisse 6.1 Isolation des Usutu-Virus in Zellkultur 6.2 Replikationsdynamik des Usutu-Virus in verschiedenen Zelllinien 6.3 Pharmakologisches Screening zur antiviralen Wirksamkeit gegen das Usutu-Virus 6.4 Identifikation und Charakterisierung der antiviralen Eigenschaften von Ivermectin 6.5 Wirkung von Ivermectin gegen Linie 2 des West-Nil-Virus in einer aviären Zelllinie 6.6 Metabolischer Phänotyp Usutu-Virus-infizierter Zelllinien 6.7 Antivirale Inhibition der Glykolyse durch 2-Deoxy-D-Glukose 6.8 Einfluss von exogenem Interferon auf den Wirtszellmetabolismus unter Infektion 6.9 Auswirkung der Interferon-Rezeptor-Defizienz auf den Metabolismus unter Infektion 7 Diskussion 7.1 Typisierung und Isolation des Usutu-Virus in Zellkultur 7.2 Charakterisierung der zelltypspezifischen Permissivität und Replikationskinetik 7.3 Identifikation antiviral wirksamer Substanzen gegen das Usutu-Virus 7.4 Usutu-Virus-induzierte Modulation des Metabolismus als antiviraler Ansatz 8 Ausblick 9 Zusammenfassung 10 Summary 11 Literaturverzeichnis 12 Anhang 12.1 Zusatzmaterial 12.2 Publikation 1 12.3 Publikation 2 12.4 Publikation 3 12.5 Weitere Veröffentlichungen 13 Danksagung

Les Flavivirus transmis par les moustiques comme le virus du Nil occidental, le virus de la dengue, le virus de la fièvre jaune, le virus de l’encéphalite japonaise et le virus Zika constituent des préoccupations croissantes de santé publique. Ils se sont répandus dans le monde au cours des dernières décennies, et les épidémies sont devenues plus fréquentes et plus sévères. Chaque année, des millions de personnes sont infectées et environ 50 000 patients décèdent d’infections à Flavivirus. Malgré les nombreux efforts de recherche, il n’y a actuellement aucun médicament antiviral spécifique disponible, et des nouvelles stratégies antivirales sont indispensables. Comprendre comment les Flavivirus fonctionnent au niveau moléculaire aidera à découvrir des nouvelles cibles pour l'intervention thérapeutique. Les Flavivirus ont un génome d'ARN simple brin de polarité positive qui code pour trois protéines structurales et huit protéines non structurales. Seules deux des huit protéines non structurales ont des activités enzymatiques. NS3 possède un domaine protéase et un domaine hélicase, et NS5 a un domaine méthyl- et guanylyltransférase et un domaine ARN polymérase ARN-dépendante. Ensemble, ils répliquent le génome viral. Ici, nous caractérisons l'interaction entre NS3 et NS5 dans le complexe de réplication du virus du Nil occidental pendant la synthèse d’ARN de polarité positive. Un modèle d'interaction comprenant NS3, NS5 et l’ARN viral a été développé basé sur des structures cristallines connues ainsi que des activités enzymatiques des deux protéines individuelles, et ce modèle a été soumis à des simulations de dynamique moléculaire. Les interactions potentielles entre les protéines NS3 et NS5 ont été identifiées. Les résidus impliqués dans ces interactions ont été mutés dans un réplicon du virus du Nil occidental et les effets de ces mutations sur la réplication virale ont été évalués. Une région particulière à la surface de la protéine NS3 a été identifiée comme étant cruciale pour la réplication virale, très probablement parce qu'elle interagit avec NS5. Cette région pourrait être une cible attrayante pour la recherche de composés qui pourraient interférer avec l'interaction entre NS3 et NS5 et donc posséder un potentiel antiviral intéressant. / Abstract : Mosquito-borne Flaviviruses like West Nile virus, Dengue virus, Yellow Fever virus, Japanese encephalitis virus, and Zika virus are increasing public health concerns. They have spread globally during the past decades, and outbreaks have recently become more frequent and more severe. Every year, millions of people are infected, and approximately 50,000 patients die from Flavivirus infections. Despite extensive research efforts, there are currently no specific antiviral drugs available, and new antiviral strategies are greatly needed. Understanding how Flaviviruses work on a molecular level will help in uncovering new points for therapeutic intervention. Flaviviruses have a single-stranded RNA genome of positive polarity that encodes three structural and eight non-structural proteins. Only two of the eight non-structural proteins have enzymatic activities. NS3 has an N-terminal protease domain and a C-terminal helicase domain, and NS5 has an N-terminal capping enzyme domain and a C-terminal RNA-dependent RNA polymerase domain. Together, they replicate the viral genome. Here we characterize the NS3:NS5 interaction within the West Nile virus RNA replicase complex during positive strand synthesis. An interaction model including NS3, NS5 and viral RNA was developed based on the known crystal structures as well as enzymatic activities of the two individual proteins, and this model was subjected to molecular dynamics simulations. Potential interactions between the NS3 and NS5 proteins were identified. Residues involved in these interactions were mutated in a West Nile virus replicon, and the effects of these mutations on viral replication were evaluated. One particular region on the surface of the NS3 protein was identified to be crucial for viral replication, most likely because it mediates the interaction with NS5. This region might be an attractive target for the search of compounds that could interfere with the NS3:NS5 interaction and therefore possess an interesting antiviral potential.

Os Flavivirus são transmitidos por mosquitos (Diptera: Culicidae) que se refugiam em remanesctentes de Mata Atlântica. Essas áreas verdes correspondem às Unidades de Conservação e parques urbanos, que estão espalhados pela região metropolitana de São Paulo. Este estudo foi realizado com o intuito de conhecer as espécies de culicídeos que circulam na Área de Proteção Ambiental (APA) Capivari-Monos, na zona Sul do município de São Paulo, e no Parque Estadual da Cantareira (PEC), na zona Norte do mesmo município, e de investigar infecção natural por Flavivirus na fauna de culicídeos amostrada. Também foi proposto relacionar a variedade, a quantidade e identidade dos Flavivirus detectados com os padrões de riqueza, abundância e diversidade das assembleias de mosquitos. Foram realizadas 14 coletas, mensalmente, em quatro pontos de coleta na APA e três no PEC, todos com diferentes níveis de intervenção antrópica, no período de março de 2016 a abril de 2017. Armadilhas automáticas luminosastipo CDC (com atração de CO2 e ácido lático) foram instaladas na copa das árvores e no nível do solo. O esforço amostral foi equivalente para os todos os pontos, sendo que foram instaladas duas armadilhas em cada ponto (uma na copa e outra no solo), com 18 horas de coleta, permitindo amostragem de culicídeos de hábitos diurnos, crepusculares e noturnos. Os espécimes foram transportados com vida para o Laboratório de Saúde Pública da Faculdade de Saúde Pública da Universidade de São Paulo, criopreservados a temperatura -70ºC, identificados morfologicamente e agrupados em pools (com até 10 indivíduos). Os pools foram submetidos à técnica de isolamento viral em cultura de células (C6/36), seguida do teste de imunofluorescência indireta. Os pools positivos foram submetidos à reação de RT-qPCR e, posteriormente, sequenciados. Duas árvores de similaridade foram construídas para confirmação dos Flavivirus. No total, 1216 exemplares de culicídeos foram amostrados (13 gêneros), cuja riqueza foi de 42 táxons. A APA registrou a maior abundância (878 espécimes) e maior riqueza (37 táxons). A Cachoeira foi o ponto de coleta na APA que amostrou a maior riqueza e abundância, contudo, com a mais baixa diversidade. Entretanto, a Borracharia obteve alta riqueza, baixa abundância e a maior diversidade. O PEC amostrou 338 indivíduos e a riqueza foi de 23 táxons. Dentre os pontos do PEC, a Trilha do Pinheirinho amostrou a maior riqueza e abundância. An. (Ker.) cruzii, Cx. (Cux.) sp, Cx. (Mel.) vaxus, Li. durhami, Wy. (Prl.) confusa e Wy. (Pho.) theobaldi foram detectadas com infecção natural por Flavivirus. O sequenciamento revelou infecção por ZIKV em An. (Ker.) cruzii, Li. durhami e Wy. (Prl.) confusa, e infecção por DENV-2 em Cx. (Cux.) sp e Cx. (Mel.) vaxus. Concluiu-se que a riqueza, abundância e diversidade estão relacionadas entre si e, juntas, influenciaram na detecção de espécies de culicídeos naturalmente infectadas por Flavivirus, sendo que estes foram detectados em espécies provenientes de pontos de coleta cuja riqueza e abundância foram altas, e a diversidade baixa. A quantidade e a variedade dos Flavivirus também foram influenciadas por esses três fatores, para ocorrer na natureza. Não foi possível correlacionar a identidade dos Flavivirus com os três fatores uma vez que as espécies detectadas com infecção natural por esses vírus não são apontadas como potenciais vetoras. Além disso, a abundância e a diversidade pareceram ter uma relação inversa entre si. / Flaviviruses are transmitted by mosquitoes (Diptera: Culicidae) that take refuge in remnants of the Atlantic Forest. These green areas correspond to Conservation Units and urban parks which are spread throughout the metropolitan region of São Paulo. This study was carried out in order to identify the Culicidae fauna that circulate in Capivari-Monos Environmental Protection Area (APA), located in the South area of the city of São Paulo, and in Cantareira State Park (PEC), North area of the same municipality and to investigate natural Flaviviruses infection in this sampled Culicidae fauna. It was also proposed to relate the variety, quantity and identity of the Flaviviruses detected with patterns of richness, abundance and diversity of mosquito assemblages. Fourteen collections were carried out monthly at four collection sites in the APA and three in the PEC, all sites with different levels of anthropogenic intervention, during March 2016 to April 2017. CDC automatic traps (with attraction of CO2 and lactic acid) were installed in the canopy and on ground. The sampling effort was equivalent for all the points, and two traps were installed at each point (one in the canopy and the other on ground), with 18 hours of sampling, allowing sampling culicidae of daytime, morning and evening twilight, and nightlyl habits. The specimens were carried alive to the Public Health Laboratory of the School of Public Health of the University of São Paulo, were cryopreserved at a -70ºC temperature, identified morphologically and grouped in pools (with up to 10 individuals). The pools were submitted to the virus isolation technique in cell culture tissue (C6 / 36), followed by the indirect immunofluorescence test. The positive pools were submitted to the RT-qPCR reaction and, subsequently, sequenced. Two similarity trees were made only to confirm Flaviviruses infection. In total, 1216 specimens of culicidae were sampled (13 genera), and the richness was 42 taxa. In addition to APA recorded the highest abundance (878 specimens) and also highest richness (37 taxa). Cachoeira was the collection site in APA that showed the greatest richness and abundance as well, however, with the lowest diversity. In addition, Borracharia obtained high richness, low abundance and highest diversity. PEC sampled 338 specimens and the richness was 23 taxa. Among the collection sites of the PEC, Pinheirinho Trail showed the highest richness and also abundance. An. (Ker.) cruzii, Cx. (Cux.) sp, Cx. (Mel.) vaxus, Li. durhami, Wy. (Prl.) confusa and Wy. (Pho.) theobaldi were detected with natural Flaviviruses infection. The sequencing analyzes revealed ZIKV infection in An. (Ker.) cruzii, Li. durhami and Wy. (Prl.) confusa, and DENV-2 infection in Cx. (Cux.) sp and Cx. (Mel.) vaxus. It has concluded that the richness, abundance and also diversity are related to each other and, together, influenced the detection of species of culicidae naturally infected by Flaviviruses, which were detected in species from collection sites whose richness and abundance were high. About quantity and variety of Flaviviruses, these were also influenced by the three factors on nature. It was not possible to correlate the identity of the Flaviviruses with the three factors since the species detected with natural infection by these viruses are not indicated as potential vectors. Moreover, abundance and diversity appeared to have an inverse relation.

A Febre Amarela (FA) é uma doença viral infecciosa, não contagiosa que pode se manifestar desde um quadro febril até a forma clássica íctero-hemorrágica, cujo agente etiológico é o vírus da febre amarela (VFA). A doença tem avançando para as regiões Sudeste e Sul com um aumento de 4 vezes no número de vítimas fatais. O presente trabalho teve como objetivos avaliar a possível circulação do vírus da febre amarela silvestre, através da detecção de anticorpos IgM por meio da técnica de MAC-ELISA, e anticorpos IgG, pelas técnicas de Inibição da hemaglutinação e Neutralização; detecção do genoma viral utilizando a técnica de RT-PCR no soro de indivíduos assintomáticos; bem como avaliar a proteção vacinal, por meio da técnica de neutralização em soros de indivíduos das zonas rurais do município de Jacupiranga e Teodoro Sampaio. Um total de 238 soros foram coletados, 152 (Jacupiranga) e 86 (Teodoro Sampaio). Foram detectados um total 15,9% (38/238) de anticorpos IH contra o vírus selvagem e/ou vacinal. Destes 13,16% (5/38) anticorpos IH foram provenientes dos soros de Jacupiranga e 86,84 (33/38) de Teodoro Sampaio. Anticorpos neutralizantes foram detectados em 34% (13N/38IH) dos IH reativos, destes 15,38% (2/13) foram oriundos deJacupiranga e 84,62% (11/13). A detecçção de anticorpos neutralizantes nos indivíduos de Jacupiranga levantam a necessidade de pesquisas futuras na busca do vírus da febre amarela nos seus reservatórios selvagens e vetores. Não houve a detecção do genoma viral por RTPCR nas duas populações, bem como a detecção de anticorpos IgM específicos, por meio das reações de MACELISA, o que evidencia a não circulação recente deste vírus na população estudada. O grau de imunizaçao na população de Teodoro Sampaio, com histórico de vacinação, por meio das reações de IH e Neutralização foi baixo, uma vez que somente 42,30% da população apresentou anticorpos inibidores da hemaglutinação e o grau de proteção pela reação de neutralização foi de apenas 33,33%. Este trabalho aponta não só para a necessidade de monitoramento pós-vacinal em áreas endêmicas de febre amarela, com indicação de vacinação, como também a necessidade de estudos epidemiológicos, uma vez que se detectou reação monotípica no teste do IH, além de anticorpos neutralizantes para o vírus selvagem da febre amarela em um indivíduo residente em município de risco para a febre amarela. / Yellow fever (YF) is an non-contagious infection disease. The clinical signs can be a nonspecific fever or the classical ictero-hemorrhagic form as a result of the Yellow Fever virus (YFV) infection. Considering the recent spread of the disease to the South and Southeast of Brazilian regions and the increased number of fatal cases, the aim of this study was to evaluate the possible circulation of the YFV, using the IgM enzyme-linked immnunosorbent assay (MACELISA) and Reverse transcriptase polymerase chain reaction (RT-PCR), as well as, to conduct a serological inquire and evaluate the protective vaccine response, by the hemagglutination inhibition test (HI) and serum neutralization test (SN), in the rural population of Jacupiranga and Teodoro Sampaio, located in the southern region of Brazil. A total of 238 serum samples were tested, 152 from Jacupirang and 86 from Teodoro Sampaio. Of the serum collected, 15,9% (38/238) were positive by HI, using wild and vaccine strains of YF, and out of these 13,16% (5/38) were from Jacupiranga and 86,84% (33/38) were from Teodoro Sampaio. Neutralizing antibodies were detected in 34% of the HI positive samples (13SN/38HI) and out of these 15,38% (2/13) were from Jacupiranga and 84,62% (11/13) were from Teodoro Sampaio. None of the samples were positive by MACELISA and RT-PCR indicating no evidences of recent virus circulation in the population analyzed in this study. However, the YF neutralizing antibodies detection in samples from Jacupiranga indicates the necessity of further researches in order to detect the YFV in its wild reservoirs and vectors. On the other hand, considering that 42,30% of the Teodoro Sampaio samples were positive by HI and out of these only 33,33% had neutralizing antibodies to the YFV, our results also pointed out to the importance of pos-vaccination protective immunity surveys, in order to evaluate the efficiency of the vaccines in endemic areas.

O Zika vírus (ZIKV) é um vírus RNA de fita única, pertencente à família Flaviviridae. É transmitido entre os humanos geralmente pelos mosquitos da espécie Aedes, mas transmissão via sexual, perinatal e por transfusão sanguínea também foram relatadas. Os sintomas aparecem em 20% dos indivíduos infectados e incluem febre, dor de cabeça, rash cutânea, conjuntivite, mialgia e artralgia. Em 2016, durante a grande epidemia do ZIKV pelas Américas, o interesse pelo seu diagnóstico rápido se intensificou, devido a relação do vírus com o aumento da incidência de casos da síndrome de Guillain-Barré em adultos e da microcefalia em recém nascidos de mulheres grávidas infectadas. De acordo com o CDC (Center for Disease Control and Prevention) o diagnóstico dos pacientes sintomáticos deve ser realizado através da detecção dos ácidos nucleicos do vírus por PCR (Polymerase chain reaction) em amostras pareadas de sangue e urina. Estudos recentes têm postulado que a saliva é uma alternativa importante para detecção do ZIKV. A saliva requer menor complexidade no processamento quando comparada ao sangue, simplificando a reação. A amplificação Isotérmica mediada por Loop (LAMP) é um teste sorológico de alta sensibilidade e especificidade para detectar rapidamente DNA ou RNA de patógenos, incluindo o ZIKV. O fato de não requerer ciclos térmicos como o PCR, faz do LAMP uma reação mais simples, rápida e mais econômica por exigir menos energia. O objetivo deste estudo foi de avaliar e comparar a eficácia da saliva e da urina em diagnosticar a infecção pelo Zika vírus em indivíduos na fase aguda da doença, através da detecção do RNA viral por meio do LAMP. Ao todo, 131 amostras (68 saliva e 63 urina) de 69 indivíduos brasileiros apresentando sinais e sintomas específicos e confirmados positivamente para o ZIKV através da análise do sangue por PCR, foram coletadas e analisadas por LAMP. A média de idade dos indivíduos foi de 34,7 (±13,6), sendo 46 (66,7%) do sexo feminino. Das 68 amostras de saliva analisadas por LAMP, 45 (66,2%) foram positivas para o ZIKV com o Tempo de positividade (Tp) médio de 13,5 minutos. Enquanto que das 63 amostras de urina, 25 (39,7%) foram positivas com o Tp médio de 15,8 minutos. A saliva pôde diagnosticar mais indivíduos (p=0.0042) e em menor Tp (p=0.0176) quando comparada à urina. A saliva demonstrou ser uma alternativa viável no diagnóstico da infecção do ZIKV, em indivíduos na fase aguda da doença, através do LAMP. Nossos achados contribuem para o conhecimento do comportamento do Zika vírus no organismo, uma vez que pouco se conhece em relação à excreção do ZIKV na saliva. / Zika virus (ZIKV) is a single-stranded RNA virus, member of the Flaviviridae family. It is transmitted among humans usually by Aedes mosquito species, but sexual transmission, perinatal and blood transfusion have also been reported. Symptoms appear in 20% of infected individuals and include fever, cutaneous rash, headache, conjunctivitis, myalgia and arthralgia. In 2016, during the Americas ZIKV outbreak, the interest in a rapid diagnosis intensified due to a sudden increase in cases of Guillain-Barré syndrome in adults and microcephaly in newborns of infected pregnant women related with ZIKV. According to CDC (Center for Disease Control and Prevention) the diagnosis of symptomatic patients should be done through nucleic acid detection by PCR (Polimerase Chain Reaction) in paired samples of blood and urine. Recent studies have reported that saliva can be an important alternative to detect ZIKV. Saliva requires less processing than blood, which greatly simplifies the assay process. Loop-mediated Isothermal Amplification (LAMP) is a molecular test with high sensibility and specificity for rapid detection of DNA or RNA of pathogens, including ZIKV. The fact that LAMP does not require thermal cycling makes the assay simple, fast and cost effective compared to PCR assay. The aim of this study was to evaluate the efficacy of saliva and urine to diagnose ZIKV infection in subjects during the acute phase, through ZIKV RNA detection by LAMP. A total of 131 samples (68 saliva and 63 urine) from 69 subjects in the acute phase of ZIKV infection and confirmed positive for ZIKV by blood analysis through PCR were collected and analyzed by LAMP. The mean age of the individuals was 34.7 (±13,6) years old, of whom 46 (66.7%) were females. From the 68 saliva samples, 45 (66.2%) were positive for ZIKV with an average time to positivity (Tp) of 13.5 minutes, and from the 63 urine samples, 25 (39.7%) were positive with an average Tp of 15.8 minutes. Saliva detected more samples (p=0.0042) and had faster Tp (p=0.0176) as compared to urine. Thus, saliva proved to be a feasible alternative for diagnosis of ZIKV infection during the acute phase by LAMP. The findings of this study can contribute to the knowledge of the Zika virus behavior in the human organism, since this issue is not totally understood.