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The release of modified mosquitoes to suppress/replace vectors constitutes a promising tool for vector control and disease prevention. Evidence regarding these innovative modification techniques is scarce and disperse. This work conducted a systematic review, gathering and analysing research articles from PubMed and Biblioteca Virtual em Saúde databases whose results report efficacy and non-target effects of using modified insects for disease prevention, until 2016. More than 1500 publications were screened and 349 were analysed. Only 12/3.4% articles reported field-based evidence and 41/11.7% covered modification strategies’ post-release efficacy. Variability in the effective results (90/25.7%) questioned its reproducibility in different settings. We also found publications reporting reversal outcomes 38/10.9%, (e.g. post-release increase of vector population). Ecological effects were also reported, such as horizontal transfer events (54/15.5%), and worsening pathogenesis induced by natural wolbachia (10/2.9%). Present work revealed promising outcomes of modifying strategies. However, it also revealed a need for field-based evidence mainly regarding epidemiologic and long-term impact. It pointed out some eventual irreversible and important effects that must not be ignored when considering open-field releases, and that may constitute constraints to generate the missing field evidence. Present work constitutes a baseline of knowledge, offering also a methodological approach that may facilitate future updates. publishersversion published

Background: Mosquito-borne viruses—such as Zika, chikungunya, dengue fever, and yellow fever, among others—are of global importance. Although vaccine development for prevention of mosquito-borne arbovirus infections has been a focus, mitigation strategies continue to rely on vector control. However, vector control has failed to prevent recent epidemics and arrest expanding geographic distribution of key arboviruses, such as dengue. As a consequence, there has been increasing necessity to further optimize current strategies within integrated approaches and advance development of alternative, innovative strategies for the control of mosquito-borne arboviruses. Methods and findings: This review, intended as a general overview, is one of a series being generated by the Worldwide Insecticide resistance Network (WIN). The alternative strategies discussed reflect those that are currently under evaluation for public health value by the World Health Organization (WHO) and represent strategies of focus by globally recognized public health stakeholders as potential insecticide resistance (IR)-mitigating strategies. Conditions where these alternative strategies could offer greatest public health value in consideration of mitigating IR will be dependent on the anticipated mechanism of action. Arguably, the most pressing need for endorsement of the strategies described here will be the epidemiological evidence of a public health impact. Conclusions: As the burden of mosquito-borne arboviruses, predominately those transmitted by Aedes aegypti and A. albopictus, continues to grow at a global scale, new vector-control tools and integrated strategies will be required to meet public health demands. Decisions regarding implementation of alternative strategies will depend on key ecoepidemiological parameters that each is intended to optimally impact toward driving down arbovirus transmission. publishersversion published

PMID: 25767886 WOS:000352199400001 The ability to effectively modify behaviours is increasingly relevant to attain and maintain a good health status. Current behaviour-change models and theories present two main approaches for (healthier) decision-making: one analytical/logical, and one experiential/emotional/intuitive. Therefore, to achieve an integral and dynamic understanding of the public perceptions both approaches should be considered: community surveys should measure cognitive understanding of health-risk contexts, and also explore how past experiences affect this understanding. In 2011, community perceptions regarding domestic source reduction were assessed in Madeira Island͘. After Madeira’s first dengue outbreak (2012) a unique opportunity to compare perceptions before and after the outbreak-experience occurred. This was the aim of this study, which constituted the first report on the effect of an outbreak experience on community perceptions regarding a specific vector-borne disease. A cross-sectional survey was performed within female residents at the most aegypti-infested areas. Perceptions regarding domestic source reduction were assessed according to the Essential Perception (EP)-analysis tool. A matching process paired individuals from studies performed before and after the outbreak, ensuring homogeneity in six determinant variables. After the outbreak, there were more female residents who assimilated the concepts considered to be essential to understand the proposed behaviour. Nevertheless, no significant difference was observed in the number of female residents who achieved the defined ‘minimal understanding’’. Moreover, most of the population (95.5%) still believed at least in one of the identified myths. After the outbreak some myths disappeared and others appeared. The present study quantified and explored how the experience of an outbreak influenced the perception regarding a dengue-preventive behaviour. The outbreak experience surprisingly led to the appearance of new myths within the population, apart from the expected increase of relevant concepts’ assimilation. Monitoring public perceptions is therefore crucial to make preventing dengue campaigns updated and worthy. publishersversion published

Trabalho Final de Mestrado Integrado, Ciências Farmacêuticas, Universidade de Lisboa, Faculdade de Farmácia, 2014 Submitted by Repositório FFUL (repositorio@ff.ul.pt) on 2019-06-19T14:52:39Z No. of bitstreams: 1 MICF_Goncalo_Seixas.pdf: 1206389 bytes, checksum: b5b88b6d7dd8417675ab00f18cc85db5 (MD5) Made available in DSpace on 2019-06-19T14:52:58Z (GMT). No. of bitstreams: 1 MICF_Goncalo_Seixas.pdf: 1206389 bytes, checksum: b5b88b6d7dd8417675ab00f18cc85db5 (MD5) Previous issue date: 2014

Aedes (Stegomyia) aegypti (Linnaeus, 1762) é o principal vector da febre amarela e da dengue assim como um agente de incomodidade. Esta espécie invasora está actualmente presente na ilha da Madeira (Portugal). Foi detectada pela primeira vez em Outubro de 2005, mas a origem geográfica da população de mosquitos é desconhecida. Apesar das medidas de controlo vectorial implementadas na região, Ae. aegypti continua a expandir-se. Isto pode ser devido à presença de resistência aos insecticidas aplicados no controlo vectorial desta espécie. Assim, os objectivos deste estudo foram: (i) avaliar o nível de sensibilidade da população de Ae. aegypti aos insecticidas; (ii) caracterizar possíveis mecanismos de resistência envolvidos e; (iii) estabelecer a sua origem geográfica através do uso de marcadores de ADN mitocondrial (COI e ND4). De acordo com os resultados dos bioensaios da OMS, Ae. aegypti da ilha da Madeira apresenta alta susceptibilidade ao malatião, com taxas de mortalidade das fêmeas expostas de 99.0%. No entanto, a população mostrou um alto nível de resistência ao DDT e permetrina, com valores de mortalidade de 29.4% e 33.3%, respectivamente. Observou-se também uma marcada resistência à deltametrina, com uma taxa de mortalidade de 65.2%. Os ensaios de garrafa do CDC com alfa-cipermetrina e ciflutrina mostraram uma alta susceptibilidade de Ae. aegypti a estes insecticidas, com taxas de mortalidade de 100%. A sequenciação do gene do canal de sódio de Ae. aegypti, local alvo dos piretróides e DDT, revelou a presença de apenas uma mutação associada à resistência, V1016I (mutação kdr). A baixa frequência alélica (6%) desta mutação sugere que outros mecanismos podem estar presentes na resistência a estes insecticidas. Ambos os genes COI e ND4 apresentaram níveis baixos de variabilidade genética consistente com a recente introdução de Ae. aegypti na ilha. Observou-se dois haplótipos mitocondriais. A rede de haplótipos de ambos os genes sugere dois cenários para o estabelecimento de Ae. aegypti na região: i) ocorreram pelo menos duas introduções independentes correspondendo a cada haplótipo; ii) o estabelecimento de ambos os haplótipos ocorreu simultaneamente, no mesmo evento de colonização. Baseado na distribuição geográfica dos haplótipos e da mutação kdr observada na ilha, assim como a população humana migrante da Madeira, o Brasil e a Venezuela são os locais mais prováveis de origem da população de Ae. aegypti local. A frequência dos alelos kdr e análise filogeográfica suportam a hipótese de que a resistência a insecticidas detectada na população da ilha da Madeira já existia na população de Ae. aegypti que colonizou a região. Aedes (Stegomyia) aegypti (Linnaeus, 1762) is the primary vector of urban yellow fever and dengue viruses as well as a nuisance agent. This invasive species is currently present in Madeira Island (Portugal). It was first detected in October 2005 but the geographic origin of this mosquito population is unknown. Despite the vector control measures implemented in the region, Ae. aegypti, seems to be spreading. This may be due to the presence of resistance to insecticides. The aims of this study were: (i) to measure the susceptibility level of Ae. aegypti population to insecticides; (ii) to characterize possible resistance mechanisms involved and; (iii) to establish the geographical origin of Ae. aegypti present in Madeira Island through the use of mitochondrial DNA markers (COI and ND4).. According to WHO bioassay results, Ae. aegypti from Madeira island presents high susceptibility to malathion, with mortality rates of exposed females of 99.0%. However, the population showed a high resistance level to DDT and permethrin, with mortality values of 29.4% and 33.3%, respectively. There was also a marked resistance to deltamethrin, with a mortality rate of 65.2%. The CDC bottle assays with alpha-cypermethrin and cyfluthrin showed a high susceptibility of Ae. aegypti to these insecticides, with mortality rates of 100%. Sequencing of the DDT/pyrethoid insecticide target site at the voltage-gated sodium channel gene of Ae. aegypti identified the presence of only one resistance-associated mutation, V1016I (kdr-mutation). This mutation showed a reduced allele frequency (6%), which suggests the presence of other insecticide resistance mechanisms rather than kdr. Both COI and ND4 presented low levels of nucleotidic variability, consistent with a recent introduction of Ae. aegypti in the island. Two mitochondrial haplotypes were found. The haplotype network of the COI and ND4 genes suggest two scenarios for the establishment of Ae. aegypti in the region: i) two independent introductions corresponding to each haplotype; ii) the establishment of both haplotypes simultaneously at the same colonization event. Based on the geographic distribution of the haplotypes and the kdr-mutation detected in the island, and the origin of the migrant human population of Madeira, Brazil and Venezuela are the most likely source-places of the local Ae. aegypti. The frequency of the kdr alleles and the phylogeographic analysis support the assumption that the resistance to insecticides detected in the population of Madeira Island already exist in the individuals that colonized the region.

Tomaz, F., Antunes, S., Parreira, R., Domingos, A., Seixas, G.,” Characterization of Hazara virus infection in Hyalomma lusitanicum tick cell line.” Poster ID: 185. National Congress of Microbiology and Biotechnology-MicroBiotec2. 23rd – 26th November 2021 (Online Web-conference) Crimean-Congo haemorrhagic fever virus (CCHFV) (Nairoviridae, Orthonairovirus) is a tick-borne virus that causes a severe disease with high mortality rates in humans and is classified as a biosafety level (BSL) 4 pathogen. This virus is mainly transmitted by Hyalomma spp. ticks and has been increasingly detected in Europe and other CCHFV-endemic areas. Since H. lusitanicum geographical distribution encompasses the Iberian Peninsula, where recent human cases of CCHFV disease have been detected, a more comprehensive understanding of the tick-virus interactions is needed to fully realize the role this vector plays in the (re)emergence of CCHFV. As showed in previous studies, Hazara virus (HAZV) is a valid surrogate model to study CCHFV under BSL2 conditions. The aim of this thesis was to establish and characterize the infection of three tick-derived cell lines - HLE/LULS42, HAECTVM9 and ISE6 - with HAZV to elucidate the dynamics of the infection (in vitro) in the vector. Tick cell lines were maintained in parallel with HAZV grown in Vero E6 and SW-13 cells. The three tick cell lines were infected with HAZV at different multiplicities of infection and had their supernatants retrieved at several time-points post infection and later processed for RNA extraction using tri-reagent. Viral RNA was quantified using an in-house designed quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) using a TaqMan probe targeting a region of the S segment of HAZV genome. Results obtained demonstrate that a persistent infection is formed in all three cell lines, without hampering the fitness of the tick cells. The viral titres obtained by RT-qPCR provided an image of the kinetics of the infection and show a peak of viral genomes at approximately 7-8 days post-infection. The data also shows that HLE/LULS42 – HAZV infected cells produce higher values of viral titre in all the three cell lines studied, displaying a difference in the kinetics of viral replication. Future studies are warranted in HLE/LULS42 tick cell line to study the molecular mechanisms underlying the viral persistence without damaging the host. Altogether, this work provides critical knowledge in understanding the vector-pathogen interactions and will guide future studies towards prevention and treatment of poorly studied viral threats. O vírus da febre hemorrágica da Crimeia-Congo (CCHFV) (Nairoviridae, Orthonairovirus) é um vírus transmitido por carraças responsável por uma grave doença com elevada taxa de mortalidade em humanos e é classificado como agente patogénico de nível de biossegurança (BSL) 4. Este vírus é transmitido principalmente por carraças do género Hyalomma spp. e a sua deteção na Europa e outras áreas endémicas de CCHFV tem aumentado. Visto que, a distribuição geográfica de H. lusitanicum abrange a Península Ibérica, onde foram detectados casos recentes de infeção por CCHFV, é necessária uma compreensão mais detalhada das interações entre a carraça e o vírus para melhor compreender o papel que este vetor desempenha na (re)emergência de CCHFV. Estudos prévios com o vírus Hazara (HAZV), validaram este vírus como um modelo para estudar CCHFV em condições BSL2. Esta tese teve como objectivos, estabelecer e caracterizar a infecção por HAZ, em três linhagens celulares distintas de carraças - HLE/LULS42, HAECTVM9 e ISE6 – de modo a elucidar a dinâmica de infecção (in vitro) neste vetor. As linhas celulares de carraça foram mantidas, em paralelo, com células Vero E6 e SW-13. As três linhagens celulares de carraça foram infectadas com HAZV em diferentes multiplicidades de infecção e o seu sobrenadante foi recuperado em vários pontos temporais pós-infecção, sendo posteriormente processado para extração de RNA usando tri-reagente. O RNA viral foi quantificado por meio de uma nova reação em cadeia da polimerase via transcriptase reversa em tempo real (RT-QPCR), usando uma sonda TaqMan, complementar a sequencia específica do segmento S do genoma de HAZV. Os resultados obtidos, evidenciam a formação de uma infecção persistente, presente em todas as três linhagens celulares, sem prejudicar a sobrevivência das mesmas. Os títulos virais obtidos através de RT-QPCR revelam um pico na deteção de genomas virais, aproximadamente 7-8 dias após a infecção. Estes resultados demonstram igualmente que a linhagem celular HLE/LULS42, infetada com HAZV apresenta os valores de título viral mais elevado em todas as três linhagens celulares estudadas, indicando uma diferença na cinética de replicação viral. É necessário realizar estudos futuros, usando a linha celular HLE/LULS42, para uma melhor compreensão dos mecanismos moleculares implícitos na manutenção da persistência da infeção viral sem danificar o hospedeiro. Concluindo, este trabalho fornece novos dados para a compreensão das interações entre vetor e agente patogénico e permitirá realizar novo estudos para a prevenção e tratamento de ameaças virais.