Background: The mosquito Aedes aegypti has long been a vector for human illness in the Southeastern United States. In the past, it has been responsible for outbreaks of dengue, chikungunya, and yellow fever and, very recently, the Zika virus that has been introduced to the region. Multiple studies have modeled the geographic distribution of Ae. aegypti as a function of climate factors; however, this ignores the importance of humans to the anthropophilic biter. Furthermore, Ae. aegypti thrives in areas where humans have created standing water sites, such as water storage containers and trash. As models are developed to examine the potential impact of climate change, it becomes increasingly important to include the most comprehensive set of predictors possible. Results: This study uses Maxent, a species distribution model, to evaluate the effects of adding poverty and population density to climate-only models. Performance was evaluated through model fit statistics, such as AUC, omission, and commission, as well as individual variable contributions and response curves. Models which included both population density and poverty exhibited better predictive power and produced more precise distribution maps. Furthermore, the two human population characteristics accounted for much of the model contribution-more so than climate variables. Conclusions: Modeling mosquito distributions without accounting for their dependence on local human populations may miss factors that are very important to niche realization and subsequent risk of infection for humans. Further research is needed to determine if additional human characteristics should be evaluated for model inclusion.

Diese Arbeit beschäftigt sich mit zwei Aspekten der serologischen Forschung am Zika-Virus (ZIKV). Im ersten Teil wurden ZIKV NS1 und ZIKV Equad in ihrer Eignung als Antigene für serologische Assays zur spezifischen Detektion der ZIKV Infektion in Abgrenzung zu Infektionen mit anderen Flaviviren untersucht. Der zweite Teil vergleicht zwei Methoden zur Expression von ZIKV NS1: Drosophila S2 Zellen und in E. coli Bakterien. ZIKV zog 2014-2016 bei einem Ausbruch in Brasilien große Aufmerksamkeit auf sich, da Zusammenhänge mit einerseits gehäuft auftretenden Mikrozephaliefällen bei Infektionen in der Schwangerschaft und andererseits schweren neurologischen Komplikationen wie dem Guillain-Barré-Syndrom hergestellt wurden. ZIKV ko-zirkuliert mit anderen Flaviviren wie dem West-Nil Virus (WNV) und dem Dengue Virus (DENV). Da viele Infektionen mit Flaviviren asymptomatisch verlaufen oder nur mit unspezifischen Symptomen einhergehen, ist eine sichere labordiagnostische Unterscheidung von hoher Relevanz. Die Flavivirus-Diagnostik basiert dabei hauptsächlich auf zwei Methoden: dem direkten Virusnachweis aus Blut oder Urin (bis zu 14 Tage nach Symptomauftritt möglich) mithilfe von PCR und dem serologischen Nachweis von Antikörpern. Hohe strukturelle Ähnlichkeiten zwischen verschiedenen Flaviviren wie ZIKV und DENV, welche zum Auftreten kreuzreaktiver Antikörper führen, sorgen dafür, dass die spezifische Serologie bisher eine Herausforderung darstellt. Ein häufig verwendetes Antigen in der serologischen Flavivirus-Diagnostik ist das Envelope-Protein (E-Protein). Dieses ist bei der ZIKV-Diagnostik aufgrund der strukturellen Ähnlichkeit mit den E-Proteinen anderer Flaviviren Ziel kreuzreaktiver Antikörper. Besonders eine Struktur, die Fusion-Loop Sequenz, ist hoch konserviert. Das Auftreten kreuzreaktiver Antikörper erschwert eine spezifische Diagnostik. Um die mangelhafte Spezifität der serologischen Tests zu verbessern, wurden zwei Antigene entwickelt. Ein Antigen basiert auf dem Nicht-Struktur-Protein 1 (NS1), ein anderes auf einer E-Protein-Mutante mit vier Aminosäureninsertionen in der Fusion-Loop-Sequenz. ZIKV NS1 zeigte bisher in zwei Studien durch Huzly et al. (2016) und Steinhagen et al. (2016) eine hohe Spezifität. Das modifizierte E-Protein konnte bereits bei WNV und DENV die serologische Spezifität verbessern. Diese beiden Antigene wurden in der vorliegenden Arbeit mit einem Panel aus Seren von Infektionen mit ZIKV, DENV, WNV und Tick-borne encephalitis Virus (TBEV) getestet. Als Referenzgruppe diente eine Reihe Flavivirus-negativer Seren. Nach Klonierung, Synthese und Aufreinigung des NS1 Proteins wurde dieses sowie das ZIKV Equad mit den Seren in IgM und IgG Assays getestet. Aus den Ergebnissen dieser Tests wurden für beide Proteine Sensitivität und Spezifität ermittelt, anschließend wurden beide Antigene verglichen. Die Ergebnisse zeigen, dass ZIKV NS1 statistisch signifikant im IgM- und IgG-Assay zwischen ZIKV und anderen Flavivirus-positiven Seren unterscheiden kann (p<0,05). Diese Unterscheidung war bei ZIKV Equad im IgG Assay zwischen ZIKV- und DENV-positiven Seren statistisch signifikant. Nach Ermittlung eines ROC-Cut-Offs war ZIKV Equad dem ZIKV NS1 in Hinblick auf Sensitivität (IgM: NS1 100%, Equad 86%; IgG: NS1, Equad 100%) und Spezifität (IgM: NS1 90%, Equad 85%; IgG: NS1 97% Equad 73%) jedoch unterlegen. Die Spezifität von ZIKV NS1 zu DENV positiven Seren ist mit 89% bereits gut. Dennoch zeigt sich anhand der hohen Signale zweier DENV positiver Seren, dass auch ZIKV NS1 Ziel kreuzreaktiver Antikörper ist. Diese Beobachtung wird durch die Studien von Chang et al. (2017) und Freire et al. (2017) bestärkt, welche ZIKV NS1 mit bioinformatischen Methoden untersuchten und so durch Vergleich mit DENV NS1 auf das Vorhandensein kreuzreaktiver Epitope schlossen. Für ZIKV Equad konnte von Rockstroh et al. (2018) durch einen Kompetitionsassay mit DENV Equad eine deutliche Verbesserung der Spezifität erreicht werden, die eine nahezu sichere Abgrenzung zu DENV ermöglicht. Daraus folgend könnte ein analog durchgeführter Kompetitionsassay mit ZIKV und DENV NS1 ebenfalls eine Verbesserung der Spezifität bringen. Im zweiten Teil der Arbeit wurden zwei Verfahren zur Synthese des ZIKV NS1 Proteins verglichen. Das im ersten Teil verwendete Protein wurde in Drosophila S2 Zellkulturen hergestellt. Eine sehr weit verbreitete Methode stellt die Synthese in E. coli Bakterien dar. Diese hat den Vorteil, dass mit wenig Aufwand, kostengünstig und in kurzen Zeitabschnitten eine große Proteinmenge synthetisiert werden kann. Sie hat im Vergleich zu eukaryontischen Zellkulturen wie der S2 Zelllinie den Nachteil, dass E.coli das Protein nicht in löslicher, sondern hauptsächlich in aggregierter Form produziert. Das Protein sammelt sich im Zellinneren an. Da das Protein bei der Extraktion denaturiert werden muss, ist im Anschluss eine Rückfaltung notwendig, um es in die korrekte Konformation zu überführen. Die richtige Faltung der Proteine ist in der serologischen Diagnostik besonders wichtig, da viele Antikörper strukturelle Epitope erkennen. Ziel dieser Arbeit war es daher, einen Vergleich und eine Bewertung der beiden unterschiedlich hergestellten Proteine in Hinblick auf Sensitivität und Spezifität mit einem Panel an ZIKV- und DENV-positiven Seren sowie negativen Seren durchzuführen. Daraus lässt sich ableiten, ob eine Proteinsynthese in E. coli äquivalente Sensitivtäten und Spezifitäten ergeben. Die Ergebnisse zeigen, dass grundsätzlich auch mit dem E. coli synthetisierten NS1 Protein eine spezifische und sensitive ZIKV Diagnostik möglich ist. Die Spezifität in Abgrenzung zu DENV-positiven Seren war allerdings niedriger als bei ZIKV NS1, welches in Insektenzellen synthetisiert wurde. Außerdem ließ sich eine stärkere Hintergrundbindung der negativen Seren messen. Möglicherweise lässt sich dies aber durch eine weitere Aufreinigung durch Größenausschlusschromatographie verringern. Zusammengefasst ergibt sich, dass die ZIKV NS1 Synthese in Drosophila S2 Zellen im Hinblick auf die Spezifität der Synthese in E. coli überlegen ist. Aufgrund der geringen Anzahl untersuchter Seren ist die Beobachtung aber statistisch nicht signifikant und sollte mit einem größeren Serumpanel auch unter Einbeziehung von Seren mit Infektionen durch andere Flaviviren verifiziert werden.:1. Abkürzungsverzeichnis 5 2. Einleitung 7 2.1 Klinik & Historie 7 2.2 Epidemiologie der Flaviviren 8 2.3 Transmission 9 2.4 Struktur 9 2.5 Serologische Immunreaktion 11 2.6 Diagnostik 13 Direkter Virusnachweis 13 Serologischer Nachweis 13 2.7 Expression rekombinanter Proteine 14 3. Fragestellung 15 4. Material 17 4.1 Allgemeine Materialien: 17 4.2 Zellkulturmaterialen 17 4.3 Chemikalien 18 4.4 Kits 18 4.5 Puffer 19 4.6 Molekularbiologische Materialien 19 4.7 Antikörper 19 4.8 Geräte 20 4.9 DNA Sequenzen der Proteine 20 4.10 Primer 21 4.11 Verwendete Vektoren 21 4.12 Patient*innenproben 22 5. Methoden 23 5.1 Molekularbiologische Methoden 23 Klonierung 23 Transformation 26 Transfektion Drosophila S2 Zellen durch Calcium-Phosphat-Präzipitation 26 Analyse- und Messmethoden Molekularbiologie 28 Extraktions- und Reinigungsmethoden Molekularbiologie 28 5.2 Mikrobiologische Methoden 28 Bakterienkultivierung 28 Insektenzell-Kultivierung 28 Proteinexpression in Bakterienzellen 29 Proteinexpression in Insektenzellen 29 5.3 Proteinbiochemische Methoden 30 Aufreinigung der Insektenzell-exprimierten Proteine 30 Aufreinigung des E. coli-exprimierten ZIKV NS1 31 Proteinrückfaltung 32 Bicinchoninic Acid (BCA) Assay 32 Bradford Assay 33 Natriumdodecylsulfat-Polyacrylamidgelelektrophorese (SDS-PAGE) 34 Western Blot 36 5.4 Immunologische Methoden 37 Antikörperfärbung des Western Blots 37 Enzyme-Linked Immunosorbent Assay (ELISA) 37 5.5 Statistische Methoden 38 Mann-Whitney-Test 38 Bestimmung der Cut-Off Werte mithilfe einer Receiver-Operating Curve 39 Ermittlung von Sensitivität und Spezifität 39 6. Ergebnisse 40 6.1 Vergleich ZIKV Equad und ZIKV NS1 40 Insektenzell-Expression von ZIKV NS1 40 IgM Assays 48 IgG Assays 54 6.2 Vergleich der Expression von ZIKV NS1 in Insektenzellen und in E. coli 59 ZIKV NS1 Expression in E. coli 59 Vergleich der Rückfaltung des NS1 Protein vor und nach PBS Dialyse 64 Vergleich der NS1 Expression durch SDS PAGE 66 Vergleichender ELISA der ZIKV NS1 Insektenzell-Expression mit der E. coli-Expression 67 7. Diskussion 69 7.1 Vergleich ZIKV NS1 und ZIKV Equad 69 Ausblick 73 7.2 Vergleich der ZIKV NS1 Expressionsmethoden 73 Ausblick 74 8. Zusammenfassung der Arbeit 75 9. Erklärung über die eigenständige Abfassung der Arbeit 78 10. Danksagung 79 11. Abbildungsverzeichnis 80 12. Tabellenverzeichnis 81 13. Quellenverzeichnis 82

Through the SARS-CoV-2 pandemic, it has become clear that the development of antivirals is essential for the health and wellbeing of the population. In this study, novel active site protease inhibitors against SARS-CoV-2 were tested for their inhibitory activity against the viral 3-Chymotrypsin like protease through the means of FRET based enzymatic assays. Additionally, Compound 104 targeting the NS2B-NS3 protease was tested against Zika virus through yield reduction assays as a means to assess whether these assays are suitable for the assessment of peptide hybrid compounds in Zika virus.Novel compounds against SARS-CoV-2 were screened and five of the selected six active compounds were found to inhibit the viral protease at a half-maximal inhibitory concentration (IC50) of below 0.075 µM.In Zika virus, the yield reduction assay was assessed and it was found that under the conditions tested, this assay is not suitable for the assessment of peptide hybrid compounds in Zika virus.The active novel compounds against SARS-CoV-2 should be taken for further assessment in cell based assays as the next step of development. Compound 104 should be assessed under different experimental conditions to identify whether different conditions can make this assay suitable for the intended use.

Yes / Coronavirus is the most dangerous virus in the world wide and it can easy spread between people, animals and plants because it is existing on one strand of RNA (Ribonucleic Acid) and it can duplicate faster than any virus. The source of coronavirus is still unknown, but some sources said that it came from seafood market and other sources said that it came from bat and snakes. It starts in Wuhan; China and every day the fatality increases. The symptoms are like a SARS-CoV (acute respiratory syndrome coronavirus)) and MERS-CoV (Middle East Respiratory Syndrome Coronavirus). By using nucleotide sequence of coronavirus from NCBI (National Center for Biotechnology Information) and some programs that ran on Matlab, the results show that there are some differences and similarities between coronavirus and other viruses such as Ebola, Flu-b, Hepatitis B, HIV and Zika especially for DEBs (distinct excluded blocks) program that shows at 5bp (base pair) there is a common with slightly difference between coronavirus “cgggg” and Ebola virus “cgtgg”. The aim from this study is to find a way to help doctors and scientists to stop spreading the coronavirus or to destroy it.

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.

Introdução: A Emergência de Saúde Pública de Importância Internacional foi declarada pela Organização Mundial da Saúde em fevereiro de 2016, em decorrência da notificação e resposta do governo brasileiro ao aumento da prevalência de microcefalia e outras alterações do sistema nervoso central. Esse evento é considerado uma das epidemias de maior complexidade e impacto da história da saúde pública. Objetivo: Descrever os principais marcos da epidemia de Zika vírus (ZIKAV) no Brasil, relacionando às ações de saúde pública adotadas e caracterizar as diferenças regionais com base nas taxas de incidência de ZIKAV em gestantes e de prevalência de microcefalia em casos registrados no Sistema de Informação de Agravos de Notificação e no formulário de Registro de Eventos de Saúde Pública, no período de janeiro de 2015 a novembro de 2016. Metodologia: Foi realizada a revisão da literatura nas principais bases de dados e também da literatura cinzenta (protocolos, portarias, manuais e informes) buscando elementos que fundamentaram as ações de saúde pública e fatos que marcaram o histórico desta epidemia no Brasil. Também foi realizada análise descritiva e comparativa das bases de dados do Sistema de Informação de Agravos de Notificação (SINAN) e do formulário de Registro de Eventos de Saúde Pública (RESP), no Brasil no período de janeiro de 2015 a novembro de 2016. Resultados: Em 22 de outubro de 2015, a Secretaria de Saúde de Pernambuco notificou o aumento na prevalência de microcefalia, no Estado. Em 11 de novembro foi declarada a emergência de saúde pública de importância nacional e em 1º de fevereiro de 2016, a Organização Mundial da Saúde declara emergência de saúde pública de importância internacional. Entre 2015 e 2016, foram notificadas 41.473 gestantes com quadro clínico compatível com ZIKAV e, no mesmo período, foram notificados e confirmados 1.950 casos de microcefalia. Destes, 70% foram confirmados por método de imagem. Observou-se que em 2015 a região mais afetada foi a nordeste e em 2016, apesar de não apresentar a mesma magnitude, observou-se uma possível segunda onda de casos de microcefalia a partir do mês de junho, principalmente na região centro-oeste, corroborando com a maior circulação de casos de ZIKAV no primeiro semestre. Limitações: O uso de dados secundários (oportunidade, completitude, representatividade, subnotificação etc), a indisponibilidade de testes laboratoriais para ZIKAV, principalmente no início da epidemia em 2015, o conhecimento limitado sobre a doença e suas consequências, apesar dos avanços nos últimos meses, a indisponibilidade de série histórica de microcefalia e outras anomalias congênitas para essa condição e o proxy de infecção pelo ZIKAV: casos negativos de dengue e chikungunya e microcefalia relacionada à infecção. Conclusões: Conclui-se que o desencadeamento da resposta em suas quatro fases operacionais foi oportuno, apesar das limitações do conhecimento; fundamentou-se na Legislação e instrumentos próprios para resposta às ESP e na melhor evidência disponível em cada fase operacional. Até o final de 2016, a magnitude da Síndrome Congênita Associada à Infecção pelo Vírus Zika vírus (SCZ) não apresentou o mesmo padrão observado em 2015, sendo que a região Nordeste apresentou maior magnitude somente na primeira onda (setembro/2015-abril/2016); Em 2016, a região Centro-Oeste apresentou a maior magnitude de casos de SCZ, seguida das regiões Sudeste e Norte. Esse padrão corrobora com o nexo causal entre infecção pelo ZIKAV na gestação e a manifestação da SCZ. Muitos avanços foram alcançados nos últimos dois anos. No entanto, ainda há importantes lacunas no conhecimento científico sobre o espectro clínico dessa nova doença e fatores relacionados à transmissão e endemicidade. / Introduction: On February 2016, The World Health Organization declared Public Health Emergency of International Concern (PHEIC). This action, due to the Brazilian notification and response, after the prevalence of microcephaly and other Central Nervous System disorders increase in Northeast Region. This event is one of the most complex epidemics of the Public Health history. Objective: Describe the sequence of events which occurred from January 2015 to November 2016 in Brazil, as a result of Zika virus outbreaks and the related congenital syndrome; to characterize the main elements of the Brazilian National response to the epidemics describing the course of the dual epidemics of Zika virus (ZIKV) infection during pregnancy and microcephaly, from the registered cases at Brazilian National Notifiable Diseases Information System (SINAN) and Public Health Events Registry (RESP) forms up to the first anniversary of this declaration in Brazil. Methods: To obtain a comprehensive chronologic description, of the main epidemiologic events and of the Brazilian response, we conducted a literature review and used third party (gray literature), and fundamental elements registered at the Brazilian National Notifiable Diseases Information System (SINAN) and Public Health Events Registry (RESP) from January 2015 up to November 12th 2016. In order to describe the Brazilian response, we divided in four phases the operational response to the emergency in Brazil. Results: On October 22nd 2015 the Pernambuco Health Secretary notified the prevalence of Microcephaly increase. On November 11th 2016 the Ministry of Health declared Public Health Emergency of National Concern. On February 1st 2016 the World Health Organization declared (PHEIC) Public Health Emergency of International Concern, 41,473 pregnant women with some clinical signs, compatible with Zika virus, were notified between 2015 and 2016. In the same period, 1,950 cases of Microcephaly were reported and confirmed. From the reported cases, 70% cases were confirmed by imaging method. The Northeast Region was the most affected in 2015 and in 2016, although it did not present the same magnitude, a potential second wave of Microcephaly cases were observed, mainly in the Central Western Region. Limitations: Secondary data (opportunity, completeness, representativeness, underreporting etc.), the unavailability of laboratory tests for ZIKAV were used, mainly at the beginning of the epidemic in 2015. The limited knowledge about the disease and its consequences, despite of advanced months at the time being, combined with the unavailability of a historical series of Microcephaly and other congenital anomalies for this condition and the proxy of infection by ZIKAV: negative cases of Dengue and Chikungunya and Microcephaly related to infection. Conclusion: It was concluded that the triggering of the response, in its four operational phases, was timely despite of the knowledge limitations; it was based on the Legislation and its own instruments to respond to PHEIC, and on the most update existing evidences of the disease (self-limiting), its diagnostic and therapeutic method. To date, the magnitude of congenital syndrome associated with ZIKAV infection (SCZ) in 2016 did not follow the same pattern observed in 2015, and the Northeast Region was the region with the greatest impact of the SCZ epidemic during September 2015 thru April 2016, although with a very low expression in the end of the following year. In 2016, the pattern observed in the Central Western Region, and to a lesser extent in the Southeast and North Regions, corroborates the causal link between ZIKAV infection in pregnancy and the manifestation of Congenital Syndrome, and there are still important gaps as, scientific knowledge about the clinic aspect of this new disease and the related factors to the transmission and endemicity.

Introdução: A Emergência de Saúde Pública de Importância Internacional foi declarada pela Organização Mundial da Saúde em fevereiro de 2016, em decorrência da notificação e resposta do governo brasileiro ao aumento da prevalência de microcefalia e outras alterações do sistema nervoso central. Esse evento é considerado uma das epidemias de maior complexidade e impacto da história da saúde pública. Objetivo: Descrever os principais marcos da epidemia de Zika vírus (ZIKAV) no Brasil, relacionando às ações de saúde pública adotadas e caracterizar as diferenças regionais com base nas taxas de incidência de ZIKAV em gestantes e de prevalência de microcefalia em casos registrados no Sistema de Informação de Agravos de Notificação e no formulário de Registro de Eventos de Saúde Pública, no período de janeiro de 2015 a novembro de 2016. Metodologia: Foi realizada a revisão da literatura nas principais bases de dados e também da literatura cinzenta (protocolos, portarias, manuais e informes) buscando elementos que fundamentaram as ações de saúde pública e fatos que marcaram o histórico desta epidemia no Brasil. Também foi realizada análise descritiva e comparativa das bases de dados do Sistema de Informação de Agravos de Notificação (SINAN) e do formulário de Registro de Eventos de Saúde Pública (RESP), no Brasil no período de janeiro de 2015 a novembro de 2016. Resultados: Em 22 de outubro de 2015, a Secretaria de Saúde de Pernambuco notificou o aumento na prevalência de microcefalia, no Estado. Em 11 de novembro foi declarada a emergência de saúde pública de importância nacional e em 1º de fevereiro de 2016, a Organização Mundial da Saúde declara emergência de saúde pública de importância internacional. Entre 2015 e 2016, foram notificadas 41.473 gestantes com quadro clínico compatível com ZIKAV e, no mesmo período, foram notificados e confirmados 1.950 casos de microcefalia. Destes, 70% foram confirmados por método de imagem. Observou-se que em 2015 a região mais afetada foi a nordeste e em 2016, apesar de não apresentar a mesma magnitude, observou-se uma possível segunda onda de casos de microcefalia a partir do mês de junho, principalmente na região centro-oeste, corroborando com a maior circulação de casos de ZIKAV no primeiro semestre. Limitações: O uso de dados secundários (oportunidade, completitude, representatividade, subnotificação etc), a indisponibilidade de testes laboratoriais para ZIKAV, principalmente no início da epidemia em 2015, o conhecimento limitado sobre a doença e suas consequências, apesar dos avanços nos últimos meses, a indisponibilidade de série histórica de microcefalia e outras anomalias congênitas para essa condição e o proxy de infecção pelo ZIKAV: casos negativos de dengue e chikungunya e microcefalia relacionada à infecção. Conclusões: Conclui-se que o desencadeamento da resposta em suas quatro fases operacionais foi oportuno, apesar das limitações do conhecimento; fundamentou-se na Legislação e instrumentos próprios para resposta às ESP e na melhor evidência disponível em cada fase operacional. Até o final de 2016, a magnitude da Síndrome Congênita Associada à Infecção pelo Vírus Zika vírus (SCZ) não apresentou o mesmo padrão observado em 2015, sendo que a região Nordeste apresentou maior magnitude somente na primeira onda (setembro/2015-abril/2016); Em 2016, a região Centro-Oeste apresentou a maior magnitude de casos de SCZ, seguida das regiões Sudeste e Norte. Esse padrão corrobora com o nexo causal entre infecção pelo ZIKAV na gestação e a manifestação da SCZ. Muitos avanços foram alcançados nos últimos dois anos. No entanto, ainda há importantes lacunas no conhecimento científico sobre o espectro clínico dessa nova doença e fatores relacionados à transmissão e endemicidade. / Introduction: On February 2016, The World Health Organization declared Public Health Emergency of International Concern (PHEIC). This action, due to the Brazilian notification and response, after the prevalence of microcephaly and other Central Nervous System disorders increase in Northeast Region. This event is one of the most complex epidemics of the Public Health history. Objective: Describe the sequence of events which occurred from January 2015 to November 2016 in Brazil, as a result of Zika virus outbreaks and the related congenital syndrome; to characterize the main elements of the Brazilian National response to the epidemics describing the course of the dual epidemics of Zika virus (ZIKV) infection during pregnancy and microcephaly, from the registered cases at Brazilian National Notifiable Diseases Information System (SINAN) and Public Health Events Registry (RESP) forms up to the first anniversary of this declaration in Brazil. Methods: To obtain a comprehensive chronologic description, of the main epidemiologic events and of the Brazilian response, we conducted a literature review and used third party (gray literature), and fundamental elements registered at the Brazilian National Notifiable Diseases Information System (SINAN) and Public Health Events Registry (RESP) from January 2015 up to November 12th 2016. In order to describe the Brazilian response, we divided in four phases the operational response to the emergency in Brazil. Results: On October 22nd 2015 the Pernambuco Health Secretary notified the prevalence of Microcephaly increase. On November 11th 2016 the Ministry of Health declared Public Health Emergency of National Concern. On February 1st 2016 the World Health Organization declared (PHEIC) Public Health Emergency of International Concern, 41,473 pregnant women with some clinical signs, compatible with Zika virus, were notified between 2015 and 2016. In the same period, 1,950 cases of Microcephaly were reported and confirmed. From the reported cases, 70% cases were confirmed by imaging method. The Northeast Region was the most affected in 2015 and in 2016, although it did not present the same magnitude, a potential second wave of Microcephaly cases were observed, mainly in the Central Western Region. Limitations: Secondary data (opportunity, completeness, representativeness, underreporting etc.), the unavailability of laboratory tests for ZIKAV were used, mainly at the beginning of the epidemic in 2015. The limited knowledge about the disease and its consequences, despite of advanced months at the time being, combined with the unavailability of a historical series of Microcephaly and other congenital anomalies for this condition and the proxy of infection by ZIKAV: negative cases of Dengue and Chikungunya and Microcephaly related to infection. Conclusion: It was concluded that the triggering of the response, in its four operational phases, was timely despite of the knowledge limitations; it was based on the Legislation and its own instruments to respond to PHEIC, and on the most update existing evidences of the disease (self-limiting), its diagnostic and therapeutic method. To date, the magnitude of congenital syndrome associated with ZIKAV infection (SCZ) in 2016 did not follow the same pattern observed in 2015, and the Northeast Region was the region with the greatest impact of the SCZ epidemic during September 2015 thru April 2016, although with a very low expression in the end of the following year. In 2016, the pattern observed in the Central Western Region, and to a lesser extent in the Southeast and North Regions, corroborates the causal link between ZIKAV infection in pregnancy and the manifestation of Congenital Syndrome, and there are still important gaps as, scientific knowledge about the clinic aspect of this new disease and the related factors to the transmission and endemicity.

Introdução: A Emergência de Saúde Pública de Importância Internacional foi declarada pela Organização Mundial da Saúde em fevereiro de 2016, em decorrência da notificação e resposta do governo brasileiro ao aumento da prevalência de microcefalia e outras alterações do sistema nervoso central. Esse evento é considerado uma das epidemias de maior complexidade e impacto da história da saúde pública. Objetivo: Descrever os principais marcos da epidemia de Zika vírus (ZIKAV) no Brasil, relacionando às ações de saúde pública adotadas e caracterizar as diferenças regionais com base nas taxas de incidência de ZIKAV em gestantes e de prevalência de microcefalia em casos registrados no Sistema de Informação de Agravos de Notificação e no formulário de Registro de Eventos de Saúde Pública, no período de janeiro de 2015 a novembro de 2016. Metodologia: Foi realizada a revisão da literatura nas principais bases de dados e também da literatura cinzenta (protocolos, portarias, manuais e informes) buscando elementos que fundamentaram as ações de saúde pública e fatos que marcaram o histórico desta epidemia no Brasil. Também foi realizada análise descritiva e comparativa das bases de dados do Sistema de Informação de Agravos de Notificação (SINAN) e do formulário de Registro de Eventos de Saúde Pública (RESP), no Brasil no período de janeiro de 2015 a novembro de 2016. Resultados: Em 22 de outubro de 2015, a Secretaria de Saúde de Pernambuco notificou o aumento na prevalência de microcefalia, no Estado. Em 11 de novembro foi declarada a emergência de saúde pública de importância nacional e em 1º de fevereiro de 2016, a Organização Mundial da Saúde declara emergência de saúde pública de importância internacional. Entre 2015 e 2016, foram notificadas 41.473 gestantes com quadro clínico compatível com ZIKAV e, no mesmo período, foram notificados e confirmados 1.950 casos de microcefalia. Destes, 70% foram confirmados por método de imagem. Observou-se que em 2015 a região mais afetada foi a nordeste e em 2016, apesar de não apresentar a mesma magnitude, observou-se uma possível segunda onda de casos de microcefalia a partir do mês de junho, principalmente na região centro-oeste, corroborando com a maior circulação de casos de ZIKAV no primeiro semestre. Limitações: O uso de dados secundários (oportunidade, completitude, representatividade, subnotificação etc), a indisponibilidade de testes laboratoriais para ZIKAV, principalmente no início da epidemia em 2015, o conhecimento limitado sobre a doença e suas consequências, apesar dos avanços nos últimos meses, a indisponibilidade de série histórica de microcefalia e outras anomalias congênitas para essa condição e o proxy de infecção pelo ZIKAV: casos negativos de dengue e chikungunya e microcefalia relacionada à infecção. Conclusões: Conclui-se que o desencadeamento da resposta em suas quatro fases operacionais foi oportuno, apesar das limitações do conhecimento; fundamentou-se na Legislação e instrumentos próprios para resposta às ESP e na melhor evidência disponível em cada fase operacional. Até o final de 2016, a magnitude da Síndrome Congênita Associada à Infecção pelo Vírus Zika vírus (SCZ) não apresentou o mesmo padrão observado em 2015, sendo que a região Nordeste apresentou maior magnitude somente na primeira onda (setembro/2015-abril/2016); Em 2016, a região Centro-Oeste apresentou a maior magnitude de casos de SCZ, seguida das regiões Sudeste e Norte. Esse padrão corrobora com o nexo causal entre infecção pelo ZIKAV na gestação e a manifestação da SCZ. Muitos avanços foram alcançados nos últimos dois anos. No entanto, ainda há importantes lacunas no conhecimento científico sobre o espectro clínico dessa nova doença e fatores relacionados à transmissão e endemicidade. / Introduction: On February 2016, The World Health Organization declared Public Health Emergency of International Concern (PHEIC). This action, due to the Brazilian notification and response, after the prevalence of microcephaly and other Central Nervous System disorders increase in Northeast Region. This event is one of the most complex epidemics of the Public Health history. Objective: Describe the sequence of events which occurred from January 2015 to November 2016 in Brazil, as a result of Zika virus outbreaks and the related congenital syndrome; to characterize the main elements of the Brazilian National response to the epidemics describing the course of the dual epidemics of Zika virus (ZIKV) infection during pregnancy and microcephaly, from the registered cases at Brazilian National Notifiable Diseases Information System (SINAN) and Public Health Events Registry (RESP) forms up to the first anniversary of this declaration in Brazil. Methods: To obtain a comprehensive chronologic description, of the main epidemiologic events and of the Brazilian response, we conducted a literature review and used third party (gray literature), and fundamental elements registered at the Brazilian National Notifiable Diseases Information System (SINAN) and Public Health Events Registry (RESP) from January 2015 up to November 12th 2016. In order to describe the Brazilian response, we divided in four phases the operational response to the emergency in Brazil. Results: On October 22nd 2015 the Pernambuco Health Secretary notified the prevalence of Microcephaly increase. On November 11th 2016 the Ministry of Health declared Public Health Emergency of National Concern. On February 1st 2016 the World Health Organization declared (PHEIC) Public Health Emergency of International Concern, 41,473 pregnant women with some clinical signs, compatible with Zika virus, were notified between 2015 and 2016. In the same period, 1,950 cases of Microcephaly were reported and confirmed. From the reported cases, 70% cases were confirmed by imaging method. The Northeast Region was the most affected in 2015 and in 2016, although it did not present the same magnitude, a potential second wave of Microcephaly cases were observed, mainly in the Central Western Region. Limitations: Secondary data (opportunity, completeness, representativeness, underreporting etc.), the unavailability of laboratory tests for ZIKAV were used, mainly at the beginning of the epidemic in 2015. The limited knowledge about the disease and its consequences, despite of advanced months at the time being, combined with the unavailability of a historical series of Microcephaly and other congenital anomalies for this condition and the proxy of infection by ZIKAV: negative cases of Dengue and Chikungunya and Microcephaly related to infection. Conclusion: It was concluded that the triggering of the response, in its four operational phases, was timely despite of the knowledge limitations; it was based on the Legislation and its own instruments to respond to PHEIC, and on the most update existing evidences of the disease (self-limiting), its diagnostic and therapeutic method. To date, the magnitude of congenital syndrome associated with ZIKAV infection (SCZ) in 2016 did not follow the same pattern observed in 2015, and the Northeast Region was the region with the greatest impact of the SCZ epidemic during September 2015 thru April 2016, although with a very low expression in the end of the following year. In 2016, the pattern observed in the Central Western Region, and to a lesser extent in the Southeast and North Regions, corroborates the causal link between ZIKAV infection in pregnancy and the manifestation of Congenital Syndrome, and there are still important gaps as, scientific knowledge about the clinic aspect of this new disease and the related factors to the transmission and endemicity.

Zikaviruset är ett virus som fått stor uppmärksamhet i framför allt Sydamerika från 2015 och framåt då allt fler fall uppmärksammats. Detta arbete har utförts som en litteraturstudie med mål att sammanfatta kunskapsläget kring Zikavirusets morfologi, spridning, historia, komplikationer, diagnostik samt rådande behandlingsmöjligheter. Som källor används information från Folkhälsomyndigheten, CDC, PAHO och WHO samt MeSH-sökningar via PubMed. Viruset tillhör familjen Flaviviridae. Liknande andra virus i samma grupp kan infektionen ge feber, makulopapulösa hudutslag, konjunktivit, ledvärk, huvudvärk och myalgi. Det beskrevs först redan på slutet av 1940-talet i Afrika och har sedan rapporterats ha spridit sig till Asien, Oceanien, Stilla havsöarna och nu senast med utbrott i Sydamerika. Virusinfektionen har blivit mycket omdiskuterad då allt mer bevis kunnat läggas fram för att den kan leda till Guillain-Barrés syndrom samt även utöva teratogena effekter med mikrocefali som följd. Man har kartlagt spridning framför allt via myggarten Aedes men bevis finns även för att sexuell spridning kan ske samt att sjukdomen förefaller även kunna spridas från mor till foster. Diagnostiken baseras på RT-PCR och serologiska tester. I nuläget finns ingen aktiv behandling. Sammanfattningsvis har Zikavirus spridit sig snabbt genom Syd- och latinamerika sista åren och visat sig utgöra ett hot mot folkhälsan i dessa områden varför ett framtagande av ett fungerande vaccin är önskvärt.