A dengue é uma arbovirose transmitida ao ser humano, através da picada de fêmeas do mosquito Aedes aegypti infectadas. A doença é endêmica em 112 países da África, das Américas, do Mediterrâneo, do Sudeste Asiático e do Pacífico Oeste. Conforme Estimativas da Organização Mundial de Saúde, 40% da população mundial vive em áreas de risco para dengue. A caracterização molecular contribui para compreender e acompanhar a diversidade genética do Dengue virus, assim como, a variabilidade da virulência dos isolados virais circulantes. Em Minas Gerais, circulam os quatro sorotipos do Dengue virus, porém poucos estudos vêm sendo realizados sobre sua dinâmica populacional. O isolado BR/Alfenas/2012, analisado neste estudo, foi obtido a partir da amostra de soro de um paciente com febre hemorrágica da dengue. A região codificadora da proteína E e NS5 foram amplificadas por RT-PCR e utilizadas nos estudos filogenéticos. As análises agruparam este isolado no genótipo V, linhagem L1, próxima a isolados provenientes do Rio de Janeiro, do Norte do Brasil e outros isolados da Venezuela e Colômbia. A análise filogeográfica forneceu evidências que o isolado BR/Alfenas/2012 é descendente direto de amostras do Rio de Janeiro de 2010 e 2011, tendo como ancestral comum o isolado DENV-1/VE/BID-V2468/2008, proveniente da Venezuela. A análise da região codificadora da proteína E, mostrou que o isolado BR/Alfenas/2012 apresenta uma diferença de 14 bases em relação aos outros isolados presentes no mesmo genótipo e linhagem, gerando desta forma, mutações sinônimas e não-sinônimas. Para caracterizar o potencial virulento desta amostra, camundongos Swiss foram infectados por via intraperitoneal com 1 x 104 unidades formadoras de placas. Sete dias pós-infecção os animais foram sacrificados e o fígado retirado para estudo histopatológico, onde os animais infectados apresentaram infiltrados inflamatórios, esteatose hepática, além de edema, hemorragia e pontos de necrose focais. Este é o primeiro relato do isolamento de DENV-1, no Sul do estado de Minas Gerais, o que possibilita iniciar uma inferência sobre a distribuição genética e diversidade desse vírus, nesta região. / Dengue virus is an arbovirus transmitted to humans through the bite of infected female Aedes aegypti mosquitoes. The disease is endemic in 112 countries in Africa, Americas, Mediterranean, South East Asia and Western Pacific. The World Health Organization estimates that 40% of the world population lives in areas at risk for dengue. Molecular characterization helps to understand and monitor the genetic diversity of DENV, as well as the variability in virulence of circulating viral isolated. The four serotypes of DENV circulate in Minas Gerais state, however few studies have been conducted about the population dynamics of DENV in this state. The isolate BR/Alfenas/2012 analyzed in this study was obtained from the serum sample from a patient with dengue hemorrhagic fever. The coding region of the E and NS5 protein were amplified by RT-PCR and used in phylogenetic studies. The analysis grouped this isolate in genotype V, line L1, getting very close to isolates from Rio de Janeiro, Brazil's North and other isolated from Venezuela and Colombia. The phylogeographic analysis provided evidence that the isolated BR/Alfenas/2012 is a direct descendant of samples from Rio de Janeiro in 2010 and 2011, and the common ancestor isolated DENV-1/VE/BID-V2468/2008, from Venezuela. Analysis of the coding region of the E protein showed that the isolated BR/Alfenas/2012 differ from 14 to other isolates from the same genotype and lineage, thereby generating mutations synonymous and non-synonymous. To characterize the virulent potential of this isolate, Swiss mice were infected with 1 x 104 plaque forming units. Seven days after infection, the animals were sacrified and the liver removed for histopathological analysis. The liver of infected animals showed a presence of inflammatory cells, steatosis and also focal points of edhema, hemorrhage and necrosis. This is the first report of the isolation of DENV-1, in the south of Minas Gerais, which allowed starting an inference about the distribution and genetic diversity of the virus.

Differential Display technique was used for gene profiling in trnasformed human umbilical vein endothelial cell line (ECV 304) and primary human umbilical vein endothelial cells (HUVECs) to study the cellular response to viral infection. After screening the mRNA from uninfected and infected HUVECs and ECV 304 cells with 16 different random primers we identified 8 gene targets. These genes included the human inhibitor of apoptosis-1 (h-IAP1), 2'-5' oligoadenylate synthetase (2'-5' OAS), 2'-5' oligoadenylate synthetase-like (2'-5' OAS-like), Galectin-9 (Gal-9), MxA, Mx1, Regulator of g-protein signaling (RGS2) and endothelial and smooth muscle cell-derived neuropilin-like protein (ESDN). We found that HUVECs were a better model to study gene expression dureing dengue 2 virus infection but not the transformed cell line, ECV 304. Of the 41 primer combinations utilized in ECV 304 cells detected only one upregulated gene, h-IAP1 and 8 out of the 16 primer combinations tried for HUVECs. We hypothesize the activation of two novel signaling pathways (Tumor necrosis factor- alpha (TNF-alpha), Interleukin1-beta (IL1-beta) in endothelial cells during D2V infection. ALso, our data detected genes that are activated in the Type-I IFN (IFN alpha/beta) signaling pathway during dengue 2 virus infection in HUVEC.

abstract: Antibodies are naturally occurring proteins that protect a host during infection through direct neutralization and/or recruitment of the innate immune system. Unfortunately, in some infections, antibodies present unique hurdles that must be overcome for a safer and more efficacious antibody-based therapeutic (e.g., antibody dependent viral enhancement (ADE) and inflammatory pathology). This dissertation describes the utilization of plant expression systems to produce N-glycan specific antibody-based therapeutics for Dengue Virus (DENV) and Chikungunya Virus (CHIKV). The Fc region of an antibody interacts with Fcγ Receptors (FcγRs) on immune cells and components of the innate immune system. Each class of immune cells has a distinct action of neutralization (e.g., antibody dependent cell-mediated cytotoxicity (ADCC) and antibody dependent cell-mediated phagocytosis (ADCP)). Therefore, structural alteration of the Fc region results in novel immune pathways of protection. One approach is to modulate the N-glycosylation in the Fc region of the antibody. Of scientific significance, is the plant’s capacity to express human antibodies with homogenous plant and humanized N-glycosylation (WT and GnGn, respectively). This allows to study how specific glycovariants interact with other components of the immune system to clear an infection, producing a tailor-made antibody for distinct diseases. In the first section, plant-produced glycovariants were explored for reduced interactions with specific FcγRs for the overall reduction in ADE for DENV infections. The results demonstrate a reduction in ADE of our plant-produced monoclonal antibodies in in vitro experiments, which led to a greater survival in vivo of immunodeficient mice challenged with lethal doses of DENV and a sub-lethal dose of DENV in ADE conditions. In the second section, plant-produced glycovariants were explored for increased interaction with specific FcγRs to improve ADCC in the treatment of the highly inflammatory CHIKV. The results demonstrate an increase ADCC activity in in vitro experiments and a reduction in CHIKV-associated inflammation in in vivo mouse models. Overall, the significance of this dissertation is that it can provide a treatment for DENV and CHIKV; but equally importantly, give insight to the role of N-glycosylation in antibody effector functions, which has a broader implication for therapeutic development for other viral infections. / Dissertation/Thesis / Doctoral Dissertation Molecular and Cellular Biology 2019

<p>Inducible Heat shock protein (Hsp70i) is a protein chaperone that is utilized during tumorigenesis and viral infections for efficient propagation. Overexpression of Hsp70i is observed in a wide spectrum of human tumors, and this overexpression correlates with metastasis, poor outcomes, and resistance to chemotherapy in patients. Hsp70i aids in cancer cell propagation through regulation of anti-apoptotic and cell survival pathways. Furthermore, Hsp70i is induced following infection for several viruses and aids viral propagation, in part through regulation of anti-apoptotic pathways as well as promoting the folding of newly synthesized proteins. Due to the parallel role of Hsp70i in both cancer and viral pathogenesis, identification of small-molecule inhibitors selective for Hsp70i could provide tools for the development of novel therapeutics and further elucidate the role of Hsp70i in both cancer and viral infections.</p><p>To date, few Hsp70 inhibitors have been identified and characterized, and their efficacy in clinical settings is unknown. Through the fluorescence-linked enzyme chemoproteomic strategy (FLECS) screen, an allosteric inhibitor selective for Hsp70i was identified, called HS-72. We show that HS-72 is highly selective for Hsp70i, over the broader purinome and other Hsp70 family members, in particular the closely related constitutively active Hsp70 family member, Hsc70. Additionally, HS-72 acts as an allosteric inhibitor to induce a conformational change and inhibit Hsp70i activity. HS-72 displays hallmarks of Hsp70i inhibition in vitro by promoting Hsp70i substrate protein degradation, protein aggregation, and selective growth inhibition of cancer cells. In wild type mice HS-72 is well tolerated and a limited PK study shows HS-72 is bioavailable. Furthermore, in a MMTV-neu breast cancer mouse model, HS-72 shows efficacy to inhibit tumor growth and promote survival.</p><p>Due to the similar utilization of Hsp70i in cancer and viral pathogenesis, this suggests the potential for HS-72 as an antiviral agent. Dengue virus (DENV) is of great public health importance due to estimates of up to 400 million infections per year, coupled with the geographic distribution of the virus, which is now endemic in over 100 countries worldwide. There is also a pressing need for DENV interventions, owing to the lack of approved vaccines or antiviral therapies. DENV is reliant on host factors throughout the viral life cycle and Hsp70i has been implicated as a host factor in DENV pathogenesis. Additionally, the complete role of Hsp70i in DENV pathogenesis remains to be elucidated, highlighting a unique opportunity to use HS-72 as a tool to specifically probe Hsp70i function. In monocytes, Hsp70i is expressed at low levels preceding DENV infection, but Hsp70i expression is induced upon DENV infection. Furthermore, inducing Hsp70i expression prior to infection, correlates with an increase in DENV infection. Targeting Hsp70i with HS-72, results in a dose dependent reduction in DENV infected monocytes, while cell viability was maintained, through inhibiting the entry stage of the viral life cycle. Following infection, Hsp70i localizes to the cell surface and interacts with the DENV receptor complex to mediate viral entry. While, HS-72 treatment results in a disruption of the interaction of Hsp70i with the DENV receptor complex, yielding a reduction in infected cells. </p><p>Collectively this work further supports Hsp70i as an anticancer and anti-dengue virus target, and identifies HS-72, a chemical scaffold that is amenable to resynthesis and iteration, as an ideal starting point for a new generation of therapeutics targeting Hsp70i.</p> / Dissertation

Thesis (M.Sc. (Microbiology))--Mahidol University, 1984.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2010. / Title from PDF title page (viewed on July 7, 2010). Includes bibliographical references.

Submitted by Programa de Pós-graduação em Biotecnologia (mebiotec.ufba@gmail.com) on 2017-04-06T12:53:22Z No. of bitstreams: 1 Dissertação Final - Adriana Andrade.pdf: 2860120 bytes, checksum: 9c25748f97bf9e85c8b9bcc05e3f32d5 (MD5) / Approved for entry into archive by Delba Rosa (delba@ufba.br) on 2017-06-29T14:41:03Z (GMT) No. of bitstreams: 1 Dissertação Final - Adriana Andrade.pdf: 2860120 bytes, checksum: 9c25748f97bf9e85c8b9bcc05e3f32d5 (MD5) / Made available in DSpace on 2017-06-29T14:41:03Z (GMT). No. of bitstreams: 1 Dissertação Final - Adriana Andrade.pdf: 2860120 bytes, checksum: 9c25748f97bf9e85c8b9bcc05e3f32d5 (MD5) / CAPES / O vírus da dengue (DENV) é um arbovírus pertencente à família Flaviviridae, gênero Flavivirus, apresentando quatro sorotipos denominados DENV-1, DENV-2, DENV-3 e DENV-4. No Brasil, a infecção pelo DENV-4 ressurgiu em 2010, após 31 anos, expondo a população ao alto risco de desenvolvimento da dengue grave, devido à co-circulação dos quatro sorotipos. Nesse contexto, os anticorpos monoclonais (AcM) apresentam-se como uma ferramenta importante devido à sua potencial aplicação como ferramenta biotecnológica. O objetivo deste trabalho foi a produção e caracterização de AcM contra DENV-4. A metodologia para a produção de AcM foi desenvolvida por Köhler e Milstein (1975). Resumidamente, os animais de experimentação foram hiperimunizados com antígeno viral DENV-4, obtidos a partir da multiplicação do vírus em células de cultura C6/36, e subsequente concentração e precipitação com polietilenoglicol 8000. A triagem dos sobrenadantes dos hibridomas para reatividade ao DENV-4 foi realizada pelo Ensaio Imunoenzimático (ELISA), e a caracterização dos AcM foi realizada pelas técnicas de ELISA, Dot-Blot, Imunofluorescência Indireta (IFI), Western-Blot. Um total de dez hibridomas foram obtidos e os AcM produzidos foram denominados A2, B1, B4, C11, D2, E4, F10, F12, H1, H12. Os AcM apresentaram diferentes perfis de reatividade frente aos diversos testes de detecção antigênica, demonstrando um reconhecimento direcionado principalmente para prM. Em conclusão, este estudo mostra o potencial de utilização dos AcM produzidos como insumo biotecnológico; sejam em estudos a respeito do vírus e sua patogênese, sejam em uma possível aplicação como ferramenta imunodiagnóstica. / Dengue virus (DV) is an arbovirus belonging to family Flaviviridae, genus Flavivirus, with four serotypes named DENV-1, DENV-2, DENV-3 and DENV-4. In Brazil, the infection by DENV-4 reemerged in 2010 after 31 years, exposing the population at high risk for developing severe diseases because of the co-circulation of the four serotypes. The monoclonal antibodies (AcM) are important tools due to its potential application in biotechnology. The aim of this work was to produce and to characterize of AcM against DENV-4. The methodology to produce AcM was developed by Köhler and Milstein (1975). Briefly, experimental animals were hyperimmunized with DENV-4 viral antigen obtained to virus multiplication in C6/36 culture cells and subsequent concentration and precipitation with polyethylene glycol 8000. ELISA test was performed to screen hybridoma supernatants for reactivity to DENV-4, and the characterization of AcM was performed by ELISA, Indirect Immunofluorescence (IFI), Dot-Blot and Western-Blot techniques. A total of ten hybridomas were obtained producing AcM named A2, B1, B4, C11, D2, E4, F10, F12, H1 and H12. AcM showed different reactivity profiles in the tests, showing a dominant recognition to prM. In conclusion, this study shows the potential use of AcM produced as biotechnology tool; to study the virus and its pathogenesis, or a possible application in immunodiagnostic assays.

abstract: Despite the approval of a Dengue virus (DV) vaccine in five endemic countries, dengue prevention would benefit from an immunization strategy highly immunogenic in young infants and not curtailed by viral interference. Problematically, infants younger than 9 year of age, whom are particularly prone to Dengue severe infection and death, cannot be immunized using current approved DV vaccine. The most important issues documented so far are the lack of efficiency and enhancement of the disease in young seronegative recipients, as well as uneven protection against the four DV serotypes. Based on data from clinical trials that showed enhanced performance of dengue vaccines when the host has previous anti-flaviviral immunity, I proposed here an attractive solution to complement the current vaccine: a recombinant measles vaccine vectoring dengue protective antigens to be administered to young infants. I hypothesized that recombinant measles virus expressing Dengue 2 and 4 antigens would successfully induce neutralizing responses against DV2 and 4 and the vaccine cocktail of this recombinant measles can prime anti-flaviviral neutralizing immunity. For this dissertation, I generated and performed preclinical immune assessment for four novel Measles-Dengue (MV-DV) vaccine candidates. I generated four MVs expressing the pre membrane (prM) and full length or truncated (90%) forms of the major envelope (E) from DV2 and DV4. Two virus, MVvac2-DV2(prME)N and MVvac2-DV4(prME), expressed high levels of membrane associated full-length E, while the other two viruses, MVvac2-DV2(prMEsol)N and MVvac2-DV4(prMEsol)N, expressed and secreted truncated, soluble E protein to its extracellular environment. The last two vectored vaccines proved superior anti-dengue neutralizing responses comparing to its corresponding full length vectors. Remarkably, when MVvac2-DV2/4(prMEsol)N recombinant vaccines were combined, the vaccine cocktail was able to prime cross-neutralizing responses against DV 1 and the relatively distant 17D yellow fever virus attenuated strain. Thus, I identify a promising DV vaccination strategy, MVvac2-DV2/4(prMEsol)N, which can prime broad neutralizing immune responses by using only two of the four available DV serotypes. The current MV immunization scheme can be advantageus to prime broad anti-flaviviral neutralizing immunity status, which will be majorly boosted by subsequent chimeric Dengue vaccine approaches. / Dissertation/Thesis / Doctoral Dissertation Microbiology 2016

Introdução: Arbovírus são vírus transmitidos por artrópodos, pertencendo, principalmente, aos gêneros Flavivirus (Flaviviridae), Alphavirus (Togaviridae) e Orthobunyavirus (Bunyavirus). Os Flavivirus, em sua maioria, são associados a zoonoses, causando doenças humanas febrís, febres hemorrágicas e encefalites. Inclusive, causam epidemias que são sério problema de saúde pública. Este estudo mostra uma pesquisa de Flavivirus em culicídeos, de diferentes regiões do país, utilizando uma técnica para identificação viral por RT-PCR com primers Flavivirusespecíficos e uma Multiplex-nested-PCR com primers espécie-específicos. Métodos: Culicídeos foram capturados, quantificados, identificados, agrupados em lotes por espécie e congelados. No laboratório, os animais foram macerados e tiveram o RNA extraído. Estes extratos foram submetidos a RT-PCR gênero-específica e à Multiplex-nested-PCR, para detecção e identificação dos vírus a nível de espécie. Resultado: De 3317 culicídios adultos e 571 larvas coletados em 4 diferentes regiões do Brasil, Sul, Sudeste, Norte e Nordeste, fez-se 246 lotes de mosquitos e desses foi possível obter amplicon sugestivo de Flavivirus em 16 (6,5%). Em 3 lotes contendo larvas de Aedes albopictus obteve-se amplicon sugestivo de vírus do dengue tipo 3. Também, em 13 lotes contendo Haemagogus leucocelaenus, Aedes aegypti e Aedes albopictus foi possível obter amplicons sugestivos de vírus do dengue tipos 1 e 2. Dos amplicons obtidos, 4 tiveram nucleotídios seqüenciados o que permitiu confirmar a presença dos vírus do dengue tipo 3 e Cacipacoré. Conclusão: O trabalho permitiu concluir que: a metodologia de RT-PCR para Flavivirus seguida de Multiplex-nested-PCR espécie-específica foi adequada para detecção e identificação destes vírus em culicídios; amplificaram-se genomas de Flavivirus em 6,5% dos lotes de culicídios estudados; vírus do dengue tipo 1 e tipo 2 foram encontrados infectando Aedes aegypti de Santos em 1999, Manaus em 2005-2006 e Foz do Iguaçu; vírus do dengue tipos 2 e 3 foram encontrados em Aedes albopictus de Santos em 1999 e Manaus em 2005-2006, sugerindo que este mosquito participe na transmissão de dengue; vírus do dengue tipo 3 foi encontrado em larvas de Aedes albopictus mostrando transmissão vertical do vírus; vírus do dengue tipo 1 foi encontrado infectando Haemagogus sp. sugerindo existência de ciclo silvático deste vírus; Aedes aegypti do Amazonas estavam infectados com o vírus Cacipacoré. / Introduction: Arbovirus are rodent-borne viruses mostly from Flavivirus (Flaviviridae), Alphavirus (Togaviridae) e Orthobunyavirus (Bunyavirus) genus. Flavivirus, are commonly zoonotic and can cause febrile illness, haemorrhagic fever and encephalitis. Flavivirus outbreaks occur in Brazil and are a major public health problem. We show here a research looking for Flavivirus infections in Culicidae by a RT-PCR using Flavivirus-especific primers and a Multiplex-nested-PCR using specie-specific primers for virus identification. Methods: Culicidae were captured, quantified, identified, pooled based on the specie and frozzen. In the laboratory, the animals were crushed and had the RNA extracted. These extracts were tested by a Flavivirus genus-specific RT-PCR followed by a specie-specific Multiplex-nested-PCR. Results: From 3317 captured adult Culicidae and 571 collected larvae in 4 different regions of Brazil, 246 pools were obtained and from these, Flavivirus indicative amplicons were obtained in 16 (6.5%). Amplicons of dengue type 3 were obtained from 3 pools of Aedes albopictus larvae. It was also possible to obtain indicative amplicons of dengue types 1 and 2 in 13 pools of Haemagogus leucocelaenus, Aedes aegypti and Aedes albopictus. Besides, 4 amplicons had the nucleotides sequenced, confirming the mosquito infection by dengue type 3 and Cacipacoré viruses. Conclusion: The technique combining a Flavivirus genus-specific RT-PCR followed by a specie-specific Multiplex-nested-PCR was suitable for detection of these viruses in the mosquitoes; Flavivirus infecting Culicidae were detected in 6.5% of the analyzed mosquito pools; dengue virus type 1 and type 2 were found infecting Aedes aegypti from Santos (1999), Manaus (2005-2006) and Foz do Iguaçu cities; dengue type 2 virus was found in Aedes albopictus from Santos city (1999) and Manaus city (2005-2006), suggesting that this mosquito could be participating on dengue transmition; dengue type 3 virus was found in Aedes albopictus larvae showing the vertical transmission of this virus; dengue type 1 virus was found infecting Haemagogus sp. what suggests on the existence of a sylvatic maintenance cycle of this virus; Aedes aegypti from Amazonas state were found infeted by Cacipacoré virus.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2016-01-29T10:59:58Z No. of bitstreams: 2 Dissertação - Marielton dos Passos Cunha - 2015.pdf: 3794305 bytes, checksum: 63dd946c15b0a01f4a247e2bd60fa1e2 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-01-29T11:01:41Z (GMT) No. of bitstreams: 2 Dissertação - Marielton dos Passos Cunha - 2015.pdf: 3794305 bytes, checksum: 63dd946c15b0a01f4a247e2bd60fa1e2 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2016-01-29T11:01:41Z (GMT). No. of bitstreams: 2 Dissertação - Marielton dos Passos Cunha - 2015.pdf: 3794305 bytes, checksum: 63dd946c15b0a01f4a247e2bd60fa1e2 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2015-04-17 / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / Dengue viruses (DENV) serotypes 1, 2, 3, and 4 have been causing yearly outbreaks in Brazil. Nevertheless, the population structure of the viruses transmitted in Goiás state is not well understood. In this study, we investigated the phylogenetic pattern of DENV samples identified in Goiânia, Goiás, Brazil during the 2012/2013 epidemic. Therefore, the entire region of the gene of the envelope (E) protein (1485bp) of 16 DENV-1 samples as well as partial region of this gene (363bp) of seven DENV-4 samples were sequenced. Phylogenetic analysis showed that DENV-1 belongs to the genotype V and presents itself divided into two clades, suggesting co-circulation of two distinct lineages in this region. Still, the molecular analyzes indicated a significant change in the amino acid level E348 position. For DENV-4, the sequences were segregate in a monophyletic group and are classified as genotype II American subclade. The molecular and phylogenetic analysis showed that the region suffered multiple introductions by dengue virus. This is the first report of co-circulation of two lineages of DENV-1, and the circulation of DENV-4 in Goiás state. / Os vírus dengue (DENV) sorotipos 1, 2, 3 e 4 tem causado surtos anuais no Brasil. No entanto, a estrutura populacional dos vírus transmitidos em Goiás não é bem compreendida. Neste estudo, investigamos o padrão filogenético de amostras do DENV identificadas em Goiânia, Goiás, Brasil, durante a epidemia de 2012/2013. Para isso, a região completa do gene codificante da proteína do envelope (E) (1485pb) de 16 amostras DENV-1 assim como a região parcial deste gene (363pb) de sete amostras DENV-4, foram sequenciadas. A análise filogenética mostrou que o DENV-1 pertence ao genótipo V e apresenta-se dividido em dois clados, sugerindo a cocirculação de duas linhagens distintas na região. Ainda, as análises moleculares indicaram uma alteração significativa na posição do aminoácido E348. Para DENV-4, as sequências segregaram em um grupo monofilético, sendo classificadas como genótipo II subclado americano. As análises moleculares e filogenéticas indicaram que a região sofreu múltiplas introduções pelo DENV. Este é o primeiro relato de cocirculação de duas linhagens de DENV-1, assim como circulação do DENV-4 no estado de Goiás.