Los flavivirus se encuentran distribuidos en las principales zonas tropicales del mundo, nuestra Amazonía es endémica para virus como el dengue; sin embargo, alberga también otros virus que aún no han sido estudiados. El objetivo fue determinar la presencia de anticuerpos neutralizantes contra los virus Rocío, Ilheus y Oeste del Nilo; utilizando la técnica del ELISA como tamizaje para la detección de muestras positivas y la microneutralización como prueba confirmatoria. Se analizaron un total de 400 sueros por ELISA IgG de personas en fase convaleciente de la localidad de Iquitos entre los años 2003 y 2007. 152 sueros fueron negativos y 248 dieron positivo por ELISA IgG, de las cuales se confirmó mediante microneutralización 23 sueros positivos para el virus Ilheus y 7 sueros positivos para el virus Rocío. El valor de reacción cruzada para la prueba de ELISA fue de 87%. Los resultados indican la existencia de anticuerpos neutralizantes contra el virus Ilheus en un 5.75% de los sueros analizados y también la presencia de anticuerpos neutralizantes contra el virus Rocío en un 1.75% de los sueros, afortunadamente las infecciones por estos virus en la localidad de Iquitos no es significativa con respecto a la observada para el virus dengue (34.7%). / The flavivirus are distributed in major tropical areas around the world, our Amazonia is endemic to viruses such as dengue, but also houses many others which have not yet been studied. The aim was to evaluate the presence of neutralizing antibodies against Rocio, Ilheus and West Niles viruses using the technique of ELISA as screening for the detection of positive samples and the microneutralization as confirmatory test. Were analyzed a total of 400 sera by Elisa IgG from people in convalescent phase of the city of Iquitos during the years 2003 - 2007. 152 were negative and 248 were positive by Elisa IgG, of which was confirmed by microneutralization 23 sera positive for the virus Ilheus and 7 sera positive for the virus Rocio. The value of cross-reactivity for the ELISA was 87%. The results indicate the existence of neutralizing antibodies against the Ilheus virus in 5.75% of the sera analyzed and the presence of neutralizing antibodies against the Rocio virus in 1.75% of the sera. Fortunately the presence of these viruses in the city of Iquitos is not significant compared to that observed for the dengue virus (34.7%).

The study of the evolution and phylogeny of West Nile virus (WNV) has been an important area of research since the introduction of WNV in 1999. However, genome sequencing of isolates from the Southeastern part of the United States has been somewhat limited. To determine how WNV has evolved at a more localized level, ten isolates from Florida and Georgia from 2003-2012 were completely sequenced using Illumina's next-generation technology. In addition, a phylogenetic comparison of both the complete genome and select partial genomes was completed to ensure consistency among the results. This study further demonstrated the dominance of the North American WN02 genotype within the Southeastern United States. In addition, phylogenetic analyses revealed the continued presence of genetic variance in 2012 with the finding of a new group within the North American clade. In conclusion, WNV has continued to evolve within the Southeastern US.

The flavivirus resistance gene, Flv, in mice has been identified as 2'-5' oligoadenylate synthetase 1b (Oas1b). Susceptible mice produce a protein that is truncated (Oas1btr) at the C-terminus due to a premature stop codon encoded by a C820T transition. Mice produce 8 Oas1 proteins, Oas1a-Oas1h. In the present study, Oas1a, Oas1b and Oas1btr were expressed as MBP-fusion proteins in bacteria and purified. 2-5A synthetase activity was demonstrated using MBP-Oas1a, while neither MBP-Oas1b nor MBP-Oas1btr were functionally active. The 2-5A synthetase activity of MBP-Oas1a was inhibited in a dose-dependent manner by the addition of MBP-Oas1b but not MBPOas1btr. Finally, three RNA probes were synthesized from the 3' end of the WNV Eg101 genome and used to test the ability of the expressed Oas1 proteins to bind to viral RNA. Results of the RNA binding activity assays suggest Oas1 proteins may specifically interact with regions of WNV RNA.

Our innate immunity is our first line of defence against pathogens. We require this immunity to control the numerous viral infections we are challenged with during our lives. However, little is known about the exact molecular mechanisms of our innate immunity, particularly components that have specific antiviral potential. One potent mediator of this antiviral activity is the interferon system. Activation of the interferon system leads to the production of several interferon-induced proteins, which inhibit the multiplication of viruses by distinct mechanisms. A key example of one of these mediators is the human MxA protein. Human MxA has the capacity to inhibit many different viruses from diverse families. In many cases it is proposed that MxA interferes with key viral components, such as incoming or newly formed nucleocapsids. It is speculated that MxA traps and missorts these viral components so they are no longer available for virus production and virus dissemination is inhibited. West Nile virus belongs to Flaviviridae family of viruses and was involved in the outbreak of virus-associated encephalitis in New York City in 1999. In this thesis I show that West Nile virus is insensitive to antiviral activity of MxA and describe how West Nile virus has developed a replication strategy that avoids MxA recognition and activation. I show that virus-induced changes of cytoplasmic membranes provide a protective microenvironment for viral replication and the viral components essential for viral replication. This hypothesis was proven by preventing the formation of these membrane structures with the fungal chemical Brefeldin A. Under these conditions I observed that stable expression of MxA could partially restrict West Nile virus RNA replication. Subsequently, I showed that the assembly mechanism of West Nile virus prevents interaction between the MxA protein and the viral capsid proteins. This was achieved by the use of a trans-packaging cell line whereby the West Nile virus structural proteins are expressed stably in trans instead of in cis from the polyprotein. When this cell line was transfected with a West Nile virus replicon expressing the human MxA protein distinct co-localisation and redistribution of the MxA with West Nile virus capsid proteins into large tubular structures within the cytoplasm of transfected cells was observed. Strikingly, these tubular aggregates are visually analogous to structures observed during infection of MxA expressing cells infected with members of the Bunyaviridae, particularly La Crosse virus. Moreover, retargeting MxA to specific sites of the endoplasmic reticulum in cells transfected with the West Nile virus infectious clone resulted in co-localisation between MxA and the West Nile virus capsid proteins and significantly inhibited the production of infectious particles. These results suggest that the sequestering of viral capsids within cytoplasmic inclusions maybe a conserved mechanism for antiviral activity of the MxA protein across the viruses families and highlight the innate ability of such molecules to recognise key molecular patterns within pathogens. Finally, I sought to exploit the antiviral potential of MxA as a therapeutic agent against infection with Influenza A viruses; viruses that have a very high sensitivity for the antiviral activity of MxA. By expressing MxA from the West Nile virus replicon, infection with the highly pathogenic Influenza virus H5N1 strain could be inhibited in vitro. Furthermore, in vivo studies in Mx-negative mice indicated that intranasal inoculation with MxA expressed from the West Nile virus replicon can protect these mice against an otherwise lethal infection with a low pathogenic Influenza A virus. Taken all together, in this thesis I provide evidence that strongly supports the existence of an evolutionary working mechanism of a significant mediator of our immune system, the antiviral MxA protein. Furthermore, I show how an important human pathogen, such as West Nile virus has evolved a replication strategy to evade this antiviral protein. These results will open new pathways for the development of a new type of antiviral therapies that utilize the potent antiviral activity of the MxA protein.

Flaviviruses are a group of medically relevant pathogens, known to cause serious disease in animals and humans. The previously defined roles of the flavivirus non-structural protein 2A (NS2A) in RNA replication, and modulation of the host antiviral response, has recently been extended to include virus assembly and secretion. In West Nile virus subtype Kunjin (KUN), an Isoleucine (I)-to-Asparagine (N) substitution at position 59 of the NS2A protein blocked the production of secreted virus particles in cells electroporated with viral RNA carrying this mutation. In this study, prolonged incubation of mutant KUN NS2A-I59N replicon RNA, in an inducible BHK-derived packaging cell line (expressing KUN structural proteins C, prM, and E), generated escape mutants that rescued the secretion of infectious virus-like particles. Sequencing identified three groups of revertants that included (i) reversions to wild-type, hydrophobic Ile, (ii) pseudorevertants to more hydrophobic residues (Ser, Thr, and Tyr) at codon 59, and (iii) pseudorevertants retaining Asn at NS2A codon 59, but containing a compensatory mutation (Thr-to-Pro) at NS2A codon 149. Engineering hydrophobic residues at NS2A position 59, or the compensatory T149P mutation into NS2A-I59N replicon RNA, restored the assembly of secreted virus-like particles in packaging cells. T149P mutation also rescued virus production when introduced into the full-length KUN RNA containing an NS2A-I59N mutation. Immunofluorescence and electron microscopy analyses of NS2A-I59N replicon-expressing cells showed a distinct lack of virus-induced membranes normally present in cells expressing wild-type replicon RNA. The compensatory mutation NS2A-T149P restored the induction of membrane structures to a level similar to those observed during wild-type replication. These results further confirm the role of NS2A in virus assembly, demonstrate the importance of hydrophobic residues at codon 59 in this process, implicate the involvement of NS2A in the biogenesis of virus-induced membranes, and suggest a vital role for these induced membranes in virus assembly. To further our understanding of how mutations within NS2A are able to affect the induction of virus-induced membranes, leading to a block in virus assembly, the membrane topology of KUN NS2A was investigated. Using a plasmid encoding NS1 and NS2A proteins with C-terminal c-myc and FLAG epitopes, NS2A proteins containing N-linked acceptor sites and C-terminal truncations were generated. Assays were performed to identify the subcellular localization of specific sequences within NS2A by Western blot and immunofluorescence analyses. While the membrane topology could not be determined experimentally, the findings of this study support the assertion that cleavage at the NS1/NS2A junction requires the majority, if not all of the NS2A protein for proper processing to occur, and suggests that the interaction between hydrophilic loops and -helical transmembrane segments plays an important role in the formation and stability of the flavivirus NS2A protein topology. Based on the knowledge of polyprotein processing events, and utilizing a range of software packages, a topology model of NS2A was predicted. The likelihood of additional sequences within NS2A affecting the ability to induce virus-specific membranes, and facilitate virion assembly, has led to the development of an invasive bacterial screening system, as a delivery vehicle to screen libraries of mutated KUN replicon clones. Using these invasive bacteria to deliver mutated KUN replicons into BHK-derived packaging cells, mutations causing a deficiency in either RNA replication or encapsidation can be identified by performing -gal assays on cells maintained in the presence, or absence of Doxycycline (suppressing the expression of structural proteins), respectively. Furthermore, this system was adapted for use in a 96-well plate format, allowing for high-throughput screening. Thus, KUN replicon clones capable of RNA replication, but unable to assemble and secrete virus-like particles can be identified and further analyzed, in the hope of mapping amino acid residues and motifs involved in encapsidation of flavivirus RNA. Finally, a range of hypotheses are discussed, explaining the possible mechanisms by which NS2A is involved in flavivirus assembly. A number of future directions and applications are also presented.

Our innate immunity is our first line of defence against pathogens. We require this immunity to control the numerous viral infections we are challenged with during our lives. However, little is known about the exact molecular mechanisms of our innate immunity, particularly components that have specific antiviral potential. One potent mediator of this antiviral activity is the interferon system. Activation of the interferon system leads to the production of several interferon-induced proteins, which inhibit the multiplication of viruses by distinct mechanisms. A key example of one of these mediators is the human MxA protein. Human MxA has the capacity to inhibit many different viruses from diverse families. In many cases it is proposed that MxA interferes with key viral components, such as incoming or newly formed nucleocapsids. It is speculated that MxA traps and missorts these viral components so they are no longer available for virus production and virus dissemination is inhibited. West Nile virus belongs to Flaviviridae family of viruses and was involved in the outbreak of virus-associated encephalitis in New York City in 1999. In this thesis I show that West Nile virus is insensitive to antiviral activity of MxA and describe how West Nile virus has developed a replication strategy that avoids MxA recognition and activation. I show that virus-induced changes of cytoplasmic membranes provide a protective microenvironment for viral replication and the viral components essential for viral replication. This hypothesis was proven by preventing the formation of these membrane structures with the fungal chemical Brefeldin A. Under these conditions I observed that stable expression of MxA could partially restrict West Nile virus RNA replication. Subsequently, I showed that the assembly mechanism of West Nile virus prevents interaction between the MxA protein and the viral capsid proteins. This was achieved by the use of a trans-packaging cell line whereby the West Nile virus structural proteins are expressed stably in trans instead of in cis from the polyprotein. When this cell line was transfected with a West Nile virus replicon expressing the human MxA protein distinct co-localisation and redistribution of the MxA with West Nile virus capsid proteins into large tubular structures within the cytoplasm of transfected cells was observed. Strikingly, these tubular aggregates are visually analogous to structures observed during infection of MxA expressing cells infected with members of the Bunyaviridae, particularly La Crosse virus. Moreover, retargeting MxA to specific sites of the endoplasmic reticulum in cells transfected with the West Nile virus infectious clone resulted in co-localisation between MxA and the West Nile virus capsid proteins and significantly inhibited the production of infectious particles. These results suggest that the sequestering of viral capsids within cytoplasmic inclusions maybe a conserved mechanism for antiviral activity of the MxA protein across the viruses families and highlight the innate ability of such molecules to recognise key molecular patterns within pathogens. Finally, I sought to exploit the antiviral potential of MxA as a therapeutic agent against infection with Influenza A viruses; viruses that have a very high sensitivity for the antiviral activity of MxA. By expressing MxA from the West Nile virus replicon, infection with the highly pathogenic Influenza virus H5N1 strain could be inhibited in vitro. Furthermore, in vivo studies in Mx-negative mice indicated that intranasal inoculation with MxA expressed from the West Nile virus replicon can protect these mice against an otherwise lethal infection with a low pathogenic Influenza A virus. Taken all together, in this thesis I provide evidence that strongly supports the existence of an evolutionary working mechanism of a significant mediator of our immune system, the antiviral MxA protein. Furthermore, I show how an important human pathogen, such as West Nile virus has evolved a replication strategy to evade this antiviral protein. These results will open new pathways for the development of a new type of antiviral therapies that utilize the potent antiviral activity of the MxA protein.

Submitted by Tatiana Silva (tsilva@icict.fiocruz.br) on 2013-01-25T16:35:56Z No. of bitstreams: 1 raquel_t_nogueira_ioc_bcm_0036_2011.pdf: 26030733 bytes, checksum: f5f3242630bf6c38b49320fb704ac2e4 (MD5) / Made available in DSpace on 2013-01-25T16:35:56Z (GMT). No. of bitstreams: 1 raquel_t_nogueira_ioc_bcm_0036_2011.pdf: 26030733 bytes, checksum: f5f3242630bf6c38b49320fb704ac2e4 (MD5) Previous issue date: 2011 / Fundação Oswaldo Cruz.Instituto Oswaldo Cruz. Rio de janeiro, RJ, Brasil / O vírus vivo atenuado de Febre Amarela (FA) YF 17D é uma das vacinas virais mais seguras e eficazes já administradas a humanos, a qual induz uma resposta imune polivalente. Estas características tornam este vírus vacinal umaplataforma tecnológica para o desenvolvimento de novas vacinas. Através da tecnologia do clone infeccioso, nós utilizamos o arcabouço de YF 17D para expressar epítopo indutor de linfócitos T CD8 + , TEWETGQI e um fragmento imunogênico, ambos provenientes da proteína de superfície de amastigota 2 (ASP-2) de Trypanosoma cruzi, parasita causador da Doença de Chagas. Este estudo objetivou evidenciar o potencial deste vírus em expressar antígenos heterólogos. O epítopo TEWETGQI foi clonado e expresso baseando-se em doissítios distintos do genoma: na alça fgda proteína de Envelope (E) (YF17D/E200/Tc) e no sítio de clivagem proteolítico entre NS2B e NS3 (YF17D/NS2B3/Tc). Uma terceira estratégia envolveu a montagem de um cassete heterólogo expressando um fragmento imunogênico de 120 aminoácidos de ASP-2 entre as proteínas E e NS1 (YF17D/ENS1/Tc). Nós investigamos se o sítio de expressão poderia influenciar a imunogenicidade do antígeno heterólogo. Assim, foram gerados vírus que se replicaram em cultura de células similar ao YF 17DD e permaneceram estáveis geneticamente após algumas passagens seriadas em célula Vero. A expressão dos antígenos heterólogos pelos vírus recombinantes revelou distintos padrões de detecção em diferentes regiões da célula. Outros estudos de caracterizaçãomostraram que os vírus YF17D/E200/Tc e YF17D/NS2B3/Tc são mais atenuados do que YF 17DD, quando inoculados via intracerebral em camundongos, sendo YF17D/E200/Tc o mais atenuado. Estudos de imunogenicidade revelaram que todos os vírus foram capazes de induzir anticorpos neutralizantes para Febre Amarela e o vírus YF17D/ENS1/Tc induziu anticorpos que reagem especificamente com amastigotas. Além disso, os vírus recombinantes induziram em camundongos imunizados uma resposta celular T produtora de interferon-gama(IFN-γ) antígeno-específica, além de uma resposta balanceada T CD4 + e T CD8 + para Febre Amarela. A vacinação de uma linhagem murina altamente suscetível à infecção por T. cruzicom um regime de dose-reforço homólogo que utilizou uma formulação de vírus YF 17D recombinantes induziu células T CD8 + TEWETGQI específicas após uma única dose, a qual poderia explicar o maior grau de proteção após desafio com T. cruzi. Assim, concluímos que a plataforma de YF 17D é útil para expressar antígenos de protozoários (T. cruzi) em regiões funcionais distintas do genoma com um impacto mínimo na viabilidade viral. Além disso, o uso de novas formulações contendo diferentes vírus YF 17D recombinantes parece ser uma estratégia promissora, a qual será explorada para outros patógenos. / The attenuated Yellow Fever (YF) 17D vaccine virus is one of the safest and most effective viral vaccines administered to humans, inwhich it elicits a polyvalent immune response. These characteristics make this vaccine virus a technological platform to the development of new vaccines. Herein, through the infectious clone technology, we used the YF 17D backbone to express a CD8 + T cell epitope, TEWETGQI and an immunogenic fragment, both originated from the Trypanosoma cruzi (the causative agent of Chagas disease) Amastigote Surface Protein 2 (ASP-2). This study aimed to provide further evidence for the potential of this virus to express foreign epitopes. The TEWETGQI epitope was cloned and expressed in two different genomic insertion sites:the fg loop of the viral Envelope protein (E) (YF17D/E200/Tc) and the protease cleavage site between the NS2B and NS3 (YF17D/NS2B3/Tc). A third strategy involved the construction of a heterologous cassette expressing an immunogenic ASP-2 fragment between E and NS1 (YF17D/ENS1/Tc). We investigated whether the site of expression had any influence on foreign antigen immunogenicity. In this sense, we generated virus that replicated similarly to vaccine virus YF 17DD in cell culture and remained genetically stable after some serial passages in Vero cells. The expression of the heterologous antigens by the recombinant viruses revealed distinct patterns of detection regarding different regions of the cell. Other characterization studies showed that YF17D/E200/Tc and YF17D/NS2B3/Tc were more attenuated than YF 17DD, when inoculated intracerebrally in mice, YF17D/E200/Tc being the most attenuated. Immunogenicity studies revealed that all viruses elicited neutralizing antibodies to YF virus and YF17D/ENS1/Tc virus induced antibodies that react specifically to amastigotes. Moreover, recombinant viruses generated an antigen specificgamma interferon (IFN-γ) mediated T-cell response in immunized mice as well as a balanced YF T CD4 + and T CD8 + response. Vaccination of a mouse lineage highly susceptible to infection by T. cruziwith a homologous prime-boost regimen of a YF 17D recombinant formulation elicited TEWETGQI specific CD8 + T cells after only one dose which could explain the higher degree of protection after T. cruzichallenge. We conclude that the YF 17D platform is useful to express Protozoan (T. cruzi) antigens at different functional regions of its genome with minimal reduction in vector viability. Besides, the use of new viral formulations composed of different YF 17D recombinant viruses seem to be a promising strategythat will be explored to other pathogens.

Made available in DSpace on 2016-04-12T12:39:35Z (GMT). No. of bitstreams: 2 noemia_lima_ioc_dout_2015.pdf: 29567185 bytes, checksum: 3de9ba1c4aa0b0fd32cd0580439ffbde (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2015 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / A vacina contra febre amarela, constituída pelo vírus febre amarela (FA) vivo atenuado da cepa 17D, é uma das mais seguras e eficazes vacinas desenvolvidas até hoje. Seu correlato de proteção é a indução de anticorpos neutralizantes, que é acompanhada de intensa resposta imune celular, com ativação de um perfil balanceado de células TH1 e TH2, devido à ampla estimulação da resposta imune inata. Estas boas características tornam atraente o uso do vírus FA 17D como vetor para o desenvolvimento de novas vacinas contra outras doenças. O nosso laboratório utiliza uma estratégia única para expressão de proteínas heterólogas ao vírus FA 17D, através da inserção de cassetes de expressão na região intergênica E/NS1. Neste trabalho, determinamos a importância de alguns motivos funcionais introduzidos nas extremidades da GFP recombinante expressa por este vetor, que causaram impacto sobre a sua expressão, através do correto enovelamento proteico e transporte para a via secretora. Além disto, apresentamos diferentes plataformas, através de modificações no vetor previamente desenvolvido, que exibiram diferenças quanto à estabilidade genética do inserto, menor interferência com a replicação viral, menor indução de morte celular e capacidade de secreção da proteína recombinante. Adicionalmente, expressamos proteínas de envelope de SIV através de alguns dos novos vetores FA 17D desenvolvidos, que foram capazes de estimular anticorpos específicos em camundongos, demonstrando o potencial do vírus FA 17D como vetor para o desenvolvimento de vacina recombinante contra AIDS baseada na indução de resposta imune humoral / The yellow fever vaccine, composed of the live attenuated Yellow fever (YF) virus strain 17D, is one of the safest and most effective vaccines ever developed. Its correlate of protection is the induction of neutralizing antibodies, which is accompanied by intense cellular immune response with activation of a balanced profile of TH1 and TH2 cells due to the broad stimulation of the innate immune response. These characteristics make this virus attractive to be used as a vector for development of new vaccines against other diseases. Our laboratory uses a unique strategy for expression of heterologous proteins in the YF 17D virus by insertion of expression cassettes in the intergenic region E/NS1. Here, we determine the importance of some functional motifs introduced at the ends of the recombinant GFP expressed by this vector, which were able to improve its expression through the proper protein folding and its transport to the secretory pathway. Moreover, we present different platforms, which show differences in genetic stability of the insert, less interference with viral replication, less induction of cell death and enhanced capacity of secretion of the recombinant protein. Additionally, we express SIV envelope proteins by some of the new vectors developed that were able to elicit specific antibodies in mice, demonstrating the potential of the YF 17D virus as a vector for the development of recombinant vaccine against AIDS based on the induction of humoral immune response / 2100-Fev-02

Submitted by Geyciane Santos (geyciane_thamires@hotmail.com) on 2015-08-24T14:40:04Z No. of bitstreams: 1 Dissertação - Samuel Franco Lopes.pdf: 9746386 bytes, checksum: 9949bc4c8abaac65b768f7a3f4108131 (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2015-08-26T20:25:24Z (GMT) No. of bitstreams: 1 Dissertação - Samuel Franco Lopes.pdf: 9746386 bytes, checksum: 9949bc4c8abaac65b768f7a3f4108131 (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2015-08-26T20:28:31Z (GMT) No. of bitstreams: 1 Dissertação - Samuel Franco Lopes.pdf: 9746386 bytes, checksum: 9949bc4c8abaac65b768f7a3f4108131 (MD5) / Made available in DSpace on 2015-08-26T20:28:31Z (GMT). No. of bitstreams: 1 Dissertação - Samuel Franco Lopes.pdf: 9746386 bytes, checksum: 9949bc4c8abaac65b768f7a3f4108131 (MD5) Previous issue date: 2011-11-24 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The Amazon region has the highest biodiversity on the planet, as well as the largest number of arboviruses isolated, mainly due to the great diversity of species of wild vertebrates and hematophagous arthropods. Among the arboviruses, Flavivirus stand out both for producing the highest number of infections and human diseases, such as the severity of these diseases. Moreover, it is known that birds act as reservoir of some poorly studied Flavivirus, such as: Ilheus, Saint Louis Encephalits, Rocio, Cacipacoré and Bussuquara. Despite intensive studies in the brazilian amazon, especially in certain areas of the state of Para, few epidemiological information about most of these viruses were obtained. In this study, a specie of Flavivirus has been identified in whole blood samples from wild birds, captured in-situ (Alter do Chão/PA) and ex-situ (Manaus) by polymerase chain reaction preceded by reverse transcription (RT- PCR) followed by Multiplex-Nested PCR (MN-PCR) tests for species-specific identification. Among the 189 samples, 7(4,23%) were suggestive of Ilheus virus. The diagnostic technique used was effective in identifying the genus Flavivirus in samples of wild birds presenting itself as practical, fast and secure for the identification of brazilian arboviruses. The circulation of enzootic viruses both in captive and free-living birds, increase the role of birds as host in the cycles of transmission of zoonoses. / A Amazônia apresenta os maiores índices de biodiversidade do planeta, assim como o maior número de arbovírus isolados, principalmente em função da grande diversidade de espécies de artrópodes hematófagos e vertebrados silvestres. Entre os arbovírus, destacam-se os Flavivírus tanto por produzirem o maior número de infecções e doenças humanas, como pela gravidade destas doenças. Além disso, sabe-se que as aves atuam como reservatório de alguns Flavivirus pouco estudados como: Ilhéus, Saint Louis, Rocio, Cacipacoré e Bussuquara. Apesar de estudos intensivos realizados na Amazônia brasileira, sobretudo em certas áreas do Estado do Pará, poucas informações epidemiológicas a respeito da maioria desses vírus foram obtidas. Nesse estudo, foi identificada uma espécie de Flavivirus em amostras de sangue total de aves silvestres, capturadas in-situ (Alter do chão/PA) e ex-situ (Manaus/AM) através da reação em cadeia de polimerase conjugada a transcrição reversa (RT-PCR) seguida por multiplex nested-PCR (MN-PCR) para testes de identificação espécie-específicos. Entre as 189 amostras analisadas, foram encontrados 7 (4,23%) amplicons sugestivos do vírus Ilhéus entre as amostras positivas. A técnica de diagnóstico utilizada mostrou-se eficaz para a identificação do gênero Flavivírus em amostras de aves silvestres apresentando-se como uma alternativa prática, rápida e segura para a identificação de arboviroses brasileiras. A circulação enzoótica dos vírus tanto em aves de cativeiro como de vida livre reforçam o papel das aves como hospedeiro nos ciclos de transmissão destas zoonoses.

Objectives. Acute flaccid paralysis (AFP) has recently emerged as a major central nervous system complication associated with West Nile virus (WNV) infection. The spectrum of clinical presentations of AFP in WNV infection and its sequelae have not been well-studied. Methods. We describe three patients with AFP due to WNV infection and review the clinical presentations of 56 patients with this complication derived from published studies. Results. Patients with AFP and WNV presented with a spectrum of illness ranging from single extremity paralysis to quadriparalysis with cranial nerve involvement. Patients commonly developed respiratory failure (54%) and bladder dysfunction (22%). While fever was nearly universal (92%), signs of meningismus were less common (17%). Cerebrospinal fluid (CSF) analysis generally revealed a modest pleocytosis, and imaging studies were not diagnositic. Persistent neurologic impairment occurred in all survivors; overall mortality rate was high (22%) and was associated with both the extent of paralysis and advanced age. Conclusion. AFP in the setting of WNV is associated with significant mortality and long-term morbidity.