Οι macro τομείς αποτελούν μία οικογένεια δομών, συντηρημένη εξελικτικά σε πολλά είδη οργανισμών. Ομόλογά τους βρίσκονται σε βακτήρια, αρχαία, ασπόνδυλα, φυτά καθώς και σε θηλαστικά. Η βιολογική τους λειτουργία δεν έχει αποσαφηνιστεί πλήρως αλλά το βασικό βιοχημικό τους χαρακτηριστικό είναι η ύπαρξη μιας θετικά φορτισμένης κοιλότητας η οποία χρησιμεύει ως σημείο πρόσδεσης της διφωσφορικής αδενικής ριβόζης (ADPr). Ο macro τομέας αποτελεί επίσης μέρος των μη δομικών πρωτεϊνών ιών, οι οποίοι φέρουν ως γενετικό υλικό νοηματικό μονόκλωνο RNA. Τέτοιου είδους ιοί είναι και οι άλφα ιοί στους οποίους ανήκει και ο ιός Mayaro. Στην παρούσα εργασία, πραγματοποιείται δομική μελέτη του macro τομέα του ιού Mayaro. Με χρήση της τεχνολογίας του ανασυνδυασμένου DNA, ο πρωτεϊνικός τομέας εκφράστηκε σε μεγάλες ποσότητες και κατέστη δυνατή η μελέτη του με τη χρήση πολυπυρηνικής/πολυδιάστατης φασματοσκοπίας πυρηνικού μαγνητικού συντονισμού. Η NMR δομή του macro τομέα του ιού Mayaro επιλύθηκε σε υψηλή διακριτικότητα (tf=1.03+/-9.7*10e-2, RMSD=0.90+/-0.15 και RMSD=1.44+/-0.14 για τα άτομα της πολυπεπτιδικής αλυσίδας και τα βαρέα άτομα αντίστοιχα) και αποκάλυψε μια αβα σάντουιτς δομή στο κέντρο της οποίας σχηματίζεται μια εκτεταμένη β-πτυχωτή επιφάνεια. Στη συνέχεια πραγματοποιήθηκαν πειράματα αλληλεπίδρασης, τα οποία αποκάλυψαν ότι το ADPr αποτελεί προσδέτη στο macro τομέα του ιού Mayaro. / Macro domains are a family of structures, evolutionarily conserved in many kinds of organisms such as bacteria, archaea, invertebrates, plants and mammals. Their biological function is not fully elucidated but their key biochemical feature seems to be the binding of the Adenosine Diphosphate ribose(ADPr). Macro domain is also found in non-structural proteins of several positive strand RNA viruses like Mayaro virus, a member of the genus of Alphavirus. In the present study, a structural analysis of the macro domain of virus Mayaro is being performed. Using the technology of recombinant DNA, the macro domain was expressed in high yield, making the analysis by multinuclear/multidimensional Nuclear Magnetic Resonance possible. The NMR solution structure of the macro domain od virus Mayaro was determined in high resolution(tf=1.03+/-9.7*10e-2, RMSD=0.90+/-0.15 and RMSD=1.44+/-0.14 for backbone and heavy atoms respectively) and revealed a well folded 3-Layer(aba)Sandwich structure in the center of which, an extended beta sheet is formed. Furthermore, interaction experiments were performed which revealed that the ADPr is a ligand for the macro domain of virus Mayaro.

Submitted by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2018-04-30T14:39:02Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) DISSERTAÇÃO - MARINEIDE SOUZA DA SILVA.pdf: 2429345 bytes, checksum: e0dd6988a96097cdd1e4eb856b6b2faf (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2018-04-30T14:39:25Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) DISSERTAÇÃO - MARINEIDE SOUZA DA SILVA.pdf: 2429345 bytes, checksum: e0dd6988a96097cdd1e4eb856b6b2faf (MD5) / Made available in DSpace on 2018-04-30T14:39:25Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) DISSERTAÇÃO - MARINEIDE SOUZA DA SILVA.pdf: 2429345 bytes, checksum: e0dd6988a96097cdd1e4eb856b6b2faf (MD5) Previous issue date: 2017-09-04 / FAPEAM - Fundação de Amparo à Pesquisa do Estado do Amazonas / Dengue is currently considered the most important arbovirose. Its main vector is the mosquito Aedes aegypti, present in several regions around the world. The serological diagnosis by means of the NS1 antigen search is the one that has greater applicability, because of easy of execution and the window for detection between the 1st and the 9th day of the onset of symptoms, with a higher frequency until the 5th day. This study aimed to investigate the presence of dengue virus in samples with non-reactive results for the NS1 antigen, assessing if there was a failure in laboratorial diagnosis, or the existence of other arboviruses circulating in the state of Amazonas. Using the RT-qPCR technique, the presence of dengue virus was investigated in 306 serum samples from patients with clinical suspicion of infection. The samples that remained negative were investigated for the presence of the Zika (ZIKV), Chikungunya (CHIKV), Mayaro (MAYV) and Oropouche (OROV) viruses with the same methodology, as well as the presence of IgG and IgM for dengue virus by ELISA. Of the 306 analyzed samples, 17 (5.5%) were positive for DENV, with three sequenced for serotype 4. Thirty-four (10.8%) were positive for ZIKV, one (0.3%) for CHIKV, thirteen 4.2%) for MAYV and nine (2.9%) for OROV. In relation to the NS1 test, all kits evaluated presented 100% agreement in negativity. For the screening of anti-DENV antibodies of the IgG class, of the 306 samples tested 134 (43.8%) had positive results. Regarding the detection of the IgM antibody, different positivities were observed for commercial kits: VIRION (n = 250) 35.6% positive; FOCUS (n = 105) 10.5% positive and PANBIO (N = 80) 20% positive. Our results confirm cases of false negative results for the NS1 tests of three commercial kits, in addition to the circulation of other arboviruses among patients from different municipalities in the state of Amazonas. / A dengue é considerada atualmente a mais importante arbovirose. O seu principal vetor é o mosquito da espécie Aedes aegypti, presente em diversas regiões do mundo. O diagnóstico sorológico por meio da pesquisa do antígeno NS1 é o que tem maior aplicabilidade, pela baixa complexidade na execução e por ser detectado entre o 1º e 9º dia de início dos sintomas, porém com frequência maior até o 5º dia. Esse estudo teve por objetivo principal investigar a presença do vírus dengue em amostras com resultados “Não Reagentes” para o antígeno NS1, avaliando se houve falha no diagnóstico laboratorial, ou a existência de outros arbovírus circulando no estado do Amazonas. Utilizando a técnica de RT-qPCR, pesquisou-se a presença do vírus dengue em 306 amostras de soros de pacientes com suspeita clínica de infecção. As amostras que continuaram negativas foram pesquisadas quanto a presença dos vírus Zika (ZIKV), Chikungunya (CHIKV), Mayaro (MAYV) e Oropouche (OROV), pela mesma metodologia, bem como foi pesquisada a presença de anticorpos das classes IgG e IgM para o vírus dengue por ELISA. Das 306 amostras analisadas 17 (5,5%) foram positivas para DENV, com três sequenciadas para o sorotipo 4. Trinta e quatro (10,8%) foram positivas para o ZIKV, uma (0,3%) para CHIKV, treze (4,2%) para MAYV e nove (2,9%) para OROV. Em relação ao teste NS1 todos os kits avaliados apresentaram 100% de concordância em negatividade. Para a pesquisa de anticorpos anti-dengue da classe IgG, das 306 amostras testadas 134 (43,8%) tiveram resultados positivos. Em relação à detecção do anticorpo IgM foram observadas diferentes positividades para os kits comerciais: VIRION (n=250) 35,6% positivas; FOCUS (n=105) 10,5% positivas e PANBIO (N=80) 20% positivas. Nossos resultados confirmam casos de resultados falso-negativos, para os testes NS1 de três kits comerciais, além da circulação de outros arbovírus entre os pacientes de diferentes municípios do estado do Amazonas.

Antecedentes:La infección por arbovirus es una de las causas más comunes de síndrome febril agudo y un problema de salud emergente en América del Sur. En el Perú, el número de casos de dengue se ha duplicado en el último año; sin embargo, menos del 50% de los síndromes febriles agudos fueron confirmados por laboratorio, lo que conduce a un diagnóstico limitado de otros arbovirus importantes. Objetivo:Evaluar la frecuencia de Dengue (DENV), Oropuche (OROV), Chikungunya (CHIKV), Mayaro (MAYV) and Zika ZIKV) en pacientes con síndrome febril agudo de Puerto Maldonado, Perú. Metodología:Se obtuvieron muestras de pacientes con síndrome febril agudo entre enero de 2016 y marzo de 2016. Se recolectaron un total de 139 especímenes y se evaluó la presencia de DENV, OROV, CHIKV, MAYV y ZIKV con la técnica de RT-PCR. Resultados: Los arbovirus de mayor frecuencia fueron CHIKV 9.35% (13/139) y OROV 8.63% (12/139), seguidos de DENV (6.47%) y ZIKV (5.04%). Entre todos los pacientes, los síntomas más comunes que acompañaron la fiebre fueron: Cefalea 79.86% (111/139), mialgias 65.47% (91/139) y artralgias 63.31% (88/139). Conclusiones:CHIKV y OROV fueron los arbovirus más frecuentes en nuestro estudio. Es crucial mejorar la vigilancia de los arbovirus para poder entender el papel de estos patógenos en el Perú. La PCR representa una prueba confiable para la vigilancia de arbovirus y debe ser considerada como el método más adecuado para la confirmación por laboratorio en el Perú. / Background:Arboviruses are one of the most common causes of acute febrile illness and an emerging health problem in South America. In Peru, the number of Dengue cases have double in the last year; however, less than 50% of acute febrile illnessare laboratory confirmed leading to an underdiagnoses of other important arboviruses. Aim:To assess the frequency of Dengue (DENV), Oropuche (OROV), Chikungunya (CHIKV), Mayaro (MAYV) and Zika ZIKV) in patients with acute febrile illness from Puerto Maldonado, Peru. Methodology: Samples were obtained from patients with acute febrile illness during January 2016 to March 2016.A total of 139 specimens were collected and assessed for the presence of DENV, OROV, CHIKV, MAYV and ZIKV via RT-PCR. Results: CHIKV in 9.35% (13/139) and OROV in 8.63% (12/139) were the most prevalent arboviruses, followed by DENV (6.47%) and ZIKV (5.04%). Among all patients, the most common symptoms accompanying fever were: Headache 79.86% (111/139), Muscle pain 65.47% (91/139) and Joint pain 63.31% (88/139). Conclusions: CHIKV and OROV were the most common arboviruses in our study. To enhance arbovirus surveillance is crucial to understand the role of these pathogens in Peru. PCR represents a reliable test for arboviral surveillance and should be considered as the preferred method for laboratory confirmation in Peru. / Tesis

Alphaviruses are a group of medically relevant arthropod-borne viruses (arboviruses) belonging to the Togaviridae family that are maintained by mosquito vectors. These zoonotic viruses are clustered into two groups: New World and Old World, depending on their geographical origin/distribution and clinical manifestations. Both of these groups cause disease symptoms of an acute febrile illness; however, each group has a distinct, hallmark disease symptom; New World alphaviruses, such as Eastern, Western, and Venezuelan equine encephalitis viruses (EEEV, WEEV, and VEEV, respectively), present with severe encephalitis while Old World alphaviruses, such as Sindbis, chikungunya, and Mayaro viruses (SINV, CHIKV, and MAYV, respectively) present with an incapacitating polyarthralgia that can persist for years following initial infection. To date, the most effective means of controlling these arboviral infections is through mosquito control programs. However, these programs have crucial limitations in their effectiveness; therefore, novel approaches are necessary to control the spread of these crippling pathogens and lessen their disease burden. Given the close phylogenetic and antigenic relationship between MAYV and CHIKV, we hypothesized that prior CHIKV immunity may affect the outcome of MAYV disease and/or limit its emergence in humans. Our work has shown that anti-CHIKV neutralizing antibodies can provide cross-protective immunity against MAYV disease. Alongside these studies, we have characterized the potency of a camelid-derived single-domain antibody (sdAb) that neutralizes a breadth of alphaviruses, including CHIKV and MAYV. With these data, we have designed and generated transgenic Aedes aegypti mosquitoes that express two anti-CHIKV sdAbs to target infection, dissemination, and transmission of MAYV and CHIKV within this deadly vector. These findings are particularly significant because they highlight the ability to co-target two emerging alphaviruses that are crippling public health and obliterating quality of life around the globe within a single defense strategy. / Doctor of Philosophy / Alphaviruses are arthropod-borne viruses (arboviruses) belonging to the Togaviridae family that infect millions of people annually via the bite of female mosquitoes. These viruses are major public health threats due to their ability to infect humans and animals and infections resulting in a range of debilitating diseases. Viruses within this genus are clustered into two groups: Old World and New World, based on geographical origin and distribution. While New World alphaviruses are known for inducing severe encephalitis (i.e., swelling in the brain), a hallmark symptom of the Old World alphaviruses is the development of incapacitating polyarthralgia (i.e., widespread joint pain) that can persist for years following initial infection. To date, the most effective means of combatting these viruses is through mosquito control programs. However, these programs have crucial limitations in their effectiveness; therefore, novel approaches are necessary to control the spread of these crippling pathogens. Given the close genetic relationship between chikungunya virus (CHIKV) and Mayaro virus (MAYV), our research has focused on harnessing cross-reactive immunity between these emerging alphaviruses. We discovered this cross-reactivity provides protective immunity to both viruses (i.e., CHIKV and MAYV) after exposure to only one (i.e., CHIKV) of the viruses. Next, we characterized the potency of a small, single-domain antibody (sdAb) to neutralize a breadth of alphaviruses, including CHIKV and MAYV. With these data, we have designed and generated transgenic Aedes aegypti mosquitoes that express this sdAb to target both CHIKV and MAYV within this deadly mosquito vector. These findings are particularly significant because they provide the foundation for a novel approach to controlling and preventing outbreaks of these emerging alphavirus pathogens that obliterate quality of life in public health settings around the globe.

Made available in DSpace on 2015-04-22T22:06:36Z (GMT). No. of bitstreams: 1 Dissertacao Final Gustavo Henrique.pdf: 11557113 bytes, checksum: 738eeceeb5f826798bc9ce2b09633f72 (MD5) Previous issue date: 2009-03-06 / FAPEAM - Fundação de Amparo à Pesquisa do Estado do Amazonas / Arbovirosis are currently recognized by the World Health Organization as a global problem. Most arbovirosis are summarized in diseases with acute and nonspecific symptoms such as fever, headache and muscle pain. Although self limited, these symptoms create relevant social and economic impacts. In Brazil, the arbovirosis caused by viruses belonging to the genus Alphavirus, Flavivirus and Orthobunyavirus and are the main cause of outbreaks or epidemics. This study aimed to study the circulation of arbovirus mayaro, venezuelan equine encephalitis virus, yellow fever virus, Saint Louis encephalitis virus and oropouche virus in a rural and an urban area of the State of Amazonas, Brazil. Therefore, the serology for detection of immunoglobulin G was used to assess the prevalence of antibodies against these viruses in 335 residents of a rural community in the state of Amazonas and PCR was used to assess the incidence of these viruses in 250 samples collected in urban area of Manaus. The results for serology suggest the movement of mayaro virus in the rural community. The seroprevalence detected in the samples was 41.5%. There was no significant relationship to risk for mayaro infection between genders (p value = 0.7760) or between age groups (p value = 0.9422). The positive serology detected among 39 children younger than 10 years indicates a recent infection. The factors of protection against mayaro infection detected were the use of mosquito net (p value = 0.0119) and the presence of animals in surrounding (p value = 0.0407). The risk factors identified for mayaro infection were the location of residence in towns near the forest (p value <0.0001) and presence of toilet in or near the home (p value = 0.0415). The serological results suggest that infection with mayaro occurred less than 10 years in the vicinity of residences analyzed. Molecular analysis of the samples collected in the urban area of Manaus not detected genomic fragments of arboviruses. Factors such as low viremia at the time of blood collection and storage of serum samples may have contributed to the non-detection of genomic fragments. However, the protocol for the detection of genomic fragments of arboviruses based on the PCR technique is already used in research centers and surveillance of Fundação de Medicina Tropical do Amazonas FMTAM and Instituto Leônidas e Maria Deane ILMD/FIOCRUZ. / As arboviroses são atualmente reconhecidas pela Organização Mundial da Saúde como um problema global. A maioria das arboviroses resume-se em afecções com sintomatologias agudas e inespecíficas, como febre, dores de cabeça e dores musculares. Embora sejam auto limitados, tais sintomas geram impactos sociais e econômicos relevantes. No Brasil, as arboviroses provocadas pelos por vírus pertencentes aos gêneros Alphavirus, Orthobunyavirus e Flavivirus são as principais causadoras de surtos ou epidemias. O presente estudo teve como objetivo estudar a circulação dos arbovírus mayaro, vírus da encefalite eqüina venezuelana, vírus da febre amarela, vírus da encefalite de Saint Louis e vírus oropouche em uma área rural e uma área urbana do Estado do Amazonas, Brasil. Assim sendo, a sorologia para detecção de imunoglobulinas G foi utilizada para avaliar a prevalência de anticorpos contra tais vírus em 335 moradores de uma comunidade rural do Estado do Amazonas e a PCR foi utilizada para avaliar a incidência de tais vírus em 250 amostras coletadas na área urbana de Manaus. Os resultados encontrados para a sorologia sugerem a circulação do vírus mayaro na comunidade rural. A soroprevalência detectada nas amostras foi de 41,5%. Não houve relação estatisticamente significativa de risco para a infecção por mayaro entre os gêneros (p valor=0,7760) ou entre as faixas etárias (p valor=0,9422). A sorologia positiva detectada entre 39 crianças menores de 10 anos indica uma infecção recente. Os fatores de proteção contra a infecção por mayaro detectados foram o uso de mosquiteiro (p valor=0,0119) e a presença de animais no peridomicílio (p valor=0,0407). Os fatores de risco detectados para a infecção por mayaro foram a localização do domicílio em vilas próximas à floresta (p valor<0,0001) e a presença de toalete dentro ou próximo ao domicílio (p valor=0,0415). Os resultados sorológicos sugerem que a infecção por mayaro ocorreu há menos de 10 anos nas proximidades das residências analisadas. A análise molecular das amostras coletadas na zona urbana de Manaus não detectou fragmentos genômicos de arbovírus. Fatores como baixa viremia no momento da coleta de sangue e estocagem das amostras de soro podem ter contribuído para a não detecção dos fragmentos genômicos. Contudo, o protocolo de detecção de fragmentos genômicos de arbovírus baseado na técnica de PCR está em uso nos centros de pesquisa e vigilância epidemiológica da Fundação de Medicina Tropical do Amazonas FMTAM e do Instituto Leônidas e Maria Deane ILMD/FIOCRUZ.