L’interleukine 26 (IL-26) a initialement été décrite comme une molécule surexprimée par des lymphocytes humains transformés par un virus. L’IL-26 a ensuite été classée comme cytokine pro-inflammatoire en raison de sa capacité à induire la production de cytokines par les cellules myéloïdes et épithéliales. Toutefois, ses fonctions biologiques restent méconnues, notamment en raison de l’absence d’orthologue chez le rat et la souris. Le laboratoire avait précédemment rapporté que l’IL-26 est surexprimée chez les patients présentant une inflammation hépatique associée à une infection chronique par le virus de l’hépatite C (HCV) et qu’elle participe à l’immunité antivirale, notamment en conférant aux cellules NK la capacité de tuer des hépatocytes infectés par le virus. Les résultats montraient également que l’IL-26 s’accumule dans les hépatocytes infectés en l’absence d’expression du transcrit. Cette observation, suggérant un rôle non conventionnel pour une cytokine associé à une localisation inhabituelle, a constitué la base de ce projet de recherche. Utilisant un modèle original de réplication du virus HCV in vitro, nos travaux montrent que l’IL-26 protège les hépatocytes de l’infection par HCV en inhibant la réplication de l’ARN viral. Cette propriété repose notamment sur la capacité de l’IL-26 à se lier à l’ARN viral. Ces données identifient l’IL-26 comme un nouvel acteur de l’arsenal antiviral, agissant notamment en inhibant la réplication du virus HCV, et suggèrent que cette molécule peut être classée dans la famille des kinocidines. / Initially identified as a molecule over expressed in virus-transformed human T cells, IL-26 has been classified as a pro inflammatory cytokine due to its capacity to induce inflammatory cytokines production by myeloid and epithelial cells. Nevertheless, its biological functions remain largely unknown, in part because of its absence of orthologs in rat and mouse.Our team has previously reported that IL-26, overexpressed in HCV-infected patients, activates NK cells, rendering them able to kill HCV-infected hepatocytes. Results also showed that IL-26 accumulates in hepatocytes of HCV-infected patients, a localization that is not usual for a cytokine. This observation led us to evaluate whether IL-26 may exhibit non-conventional properties. In this study, we demonstrate that IL-26 protects in vitro hepatocytes from HCV infection. Confocal microscopy revealed that IL-26 colocalizes with viral dsRNA. This direct antiviral activity appears to rely on the capacity of IL-26 to bind to viral RNA and to inhibit its replication. Moreover, we investigated the capacity that IL-26 complexed to viral RNA could trigger an antiviral response by immune cells. Collectively, results show that IL-26 may have a manifold protective role during HCV infection: (i) an indirect role via its capacity to confer NK cell the capacity to kill HCV-infected cells, (ii) a direct role through its ability to inhibit viral replication, and (iii) a stimulatory role by rendering viral DNA inflammatory.

Thesis (doctoral)--Karolinska Institutet, Stockholm, 1988. / Extra t.p. with thesis statement inserted. Includes bibliographical references.

Le virus de l'hépatite B (HBV) infecte chroniquement plus de 250 millions de personnes. Des traitements existent permettant de contrôler la production de particules infectieuses. Cependant, aucun des traitements actuels ne permet d'éradiquer complètement l'infection. Il est donc nécessaire de développer de nouvelles stratégies thérapeutiques, incluant des approches immunothérapeutiques pour permettre un meilleur contrôle immunologique des infections HBV. Dans une étude récente menée au sein du laboratoire, il a été montré que l'IL-1ß est la cytokine ayant le plus fort effet antiviral contre la réplication d'HBV dans les hépatocytes. Dans le foie, la cytokine IL-1ß est principalement produite par les macrophages résidents (les cellules de Kupffer) ou infiltrant (monocytes inflammatoires différenciés en macrophages). De nombreuses études récentes ont montrés qu'HBV était capable de bloquer partiellement l'induction des réponses immunitaires innées. Il est donc important de déterminer si HBV est capable d'empêcher la production d'IL-1ß par les différents types de macrophages. L'objectif de cette thèse était d'étudier l'effet du virus sur le phénotype des macrophages et les implications de ces modifications phénotypiques sur l'établissement de l'infection dans les hépatocytes. Des monocytes du sang ou des macrophages du foie ont été purifiés, respectivement, du sang périphérique ou de résections hépatiques, et ont été exposés au virus pendant leur différentiation et/ou leur activation pour les monocytes, ou seulement pendant leur activation pour les macrophages hépatiques déjà différenciés. Il a été démontré que le virus de l'hépatite B est capable d'inhiber la sécrétion d'IL-6 et d'IL-1ß par les macrophages pro-inflammatoires. De plus, HBV est capable d'inhiber la sécrétion d'IL-1ß par les macrophages hépatiques stimulés par différents ligands. Finalement, les surnageants de macrophages pro-inflammatoires sont capables de bloquer l'établissement de l'infection, ce qui n'est pas le cas quand les macrophages ont été exposés au virus. Il apparait donc qu'HBV est capable de modifier le phénotype des macrophages pour favoriser l'établissement et la persistance de l'infection. La compréhension des mécanismes de subversion du phénotype des macrophages par le virus de l'hépatite B serait un premier pas vers le développement de nouvelles stratégies thérapeutiques / Hepatitis B virus (HBV) chronically infects over 250 million people worldwide. Several treatments can be used to prevent the formation of de novo particles. However, they do not allow the total eradication of the infection. Therefore, it is necessary to develop new therapeutic strategies, including immune-therapeutic ones, which would be more likely to lead to an immunological control of HBV infections. We have recently shown that IL-1ß is the most effective antiviral cytokine against the replication of HBV in vitro. In the liver, IL-1ß is mainly produced by resident macrophages (also called Kupffer cells) or infiltrating cells (inflammatory monocytes differentiated into macrophages). Recent studies have shown that HBV is able to partially inhibit the induction of innate immune responses. Hence, it was necessary to determine if HBV was also able to block the production of IL-1ß by the different types of macrophages.The objective of this thesis was to study the effect of HBV on macrophage phenotypes and the impact of those modifications on the establishment of HBV infection in hepatocytes.Blood monocytes and liver macrophages were purified, respectively, from peripheral blood or hepatic resections, and were exposed to HBV during their differentiation and/or activation for monocytes, or only during activation for liver macrophages which are already in a differentiated state. HBV was able to partially inhibit the secretion of IL-6 and IL-1ß by pro-inflammatory macrophages. Moreover, HBV was able to inhibit IL-1ß secretion by liver macrophages stimulated by different ligands and, conditioned medium of pro-inflammatory macrophages could inhibit the establishment of infection in hepatocytes. This effect was reverted when macrophages were exposed to HBV, concomitantly with a lower production of IL-6 and IL-1ß.In summary, HBV is able to modify macrophage phenotypes to favour the establishment and persistence of HBV infection. The full understanding of the mechanistic basis of how HBV phenotypically modifies macrophages will be a first step towards the development of new therapeutic strategies

Ocorrências comuns de infecções virais graves, aparecimento de cepas resistentes e um limitado número de quimioterápicos antivirais disponíveis mostram a necessidade da busca por novas substâncias ativas como antivirais. Muitas substâncias derivadas de plantas são candidatas ao estudo do seu potencial na terapia sistêmica e/ou profilaxia de herpes simplex virus 1 (HSV-1). Dentre as plantas com atividade antiviral, destacam-se as do gênero Baccharis. O objetivo geral do trabalho foi o estudo da composição química e avaliação da atividade antiviral das folhas de Baccharis anomala D.C. através de fracionamento bioguiado. Utilizando precipitação com etanol e fracionamento por permeação molecular foi possível separar os constituintes químicos ativos contra o vírus HSV-1 presentes no extrato aquoso de Baccharis anomala. Os testes de prospecção de constituintes químicos indicaram a presença de taninos, catequinas e saponinas no extrato aquoso da espécie. Através da análise cromatográfica foi possível detectar a presença de compostos fenólicos, utilizando-se coloração com cloreto férrico e reagente natural. Em relação à atividade antiviral, a fração ativa denominada AQ PPT FR 4-5 apresentou pronunciada atividade antiviral, inibindo a replicação viral em 100 % nas concentrações de 1,25, 0,625, 0,312 e 0,156 mg/mL, contra as cepas ATCC-VR733 e Aciclovir-resistente 29-R do HSV-1. Em relação ao mecanismo de ação, observou-se atividade virucida da fração AQ PPT FR 4-5. Estes resultados são muito importantes, pois, de acordo com a literatura, ainda não foram relatados compostos com atividade virucida úteis clinicamente para o tratamento de infecções por HSV-1. Conclui-se, portanto, que Baccharis anomala possui potente atividade antiviral contra o vírus HSV-1 e é promissora para estudos posteriores que visem ao isolamento, identificação e estudo do mecanismo de ação antiviral de compostos ativos da espécie, considerando a emergência de cepas resistentes e a necessidade de compostos com novos mecanismos de ação. / Common occurrences of severe viral infections, emergence of resistant strains and a limited number of available antiviral chemotherapeutics show the need to search for new active substances as antiviral. Many compounds derived from plants are candidates for the study of their potential in systemic therapy and/or prophylaxis of herpes simplex virus type 1 (HSV-1). Among the plants with antiviral activity, those from the genus Baccharis are remarkable. The main objective of the work was the study of the chemical composition and evaluation of the antiviral activity of extracts from Baccharis anomala D.C., using bioactivity guided fractionation. Through precipitation with ethanol and fractionation by molecular permeation it was achieved the separation of the active chemical constituents against HSV-1 virus in the aqueous extract of Baccharis anomala. Phytochemical tests indicated the presence of tannins, catechins and saponins in the aqueous extract. By thin layer chromatography it was detected the presence of phenolic compounds using ferric chloride and natural reagent. Concerning the antiviral activity, the active fraction named AQ PPT FR 4-5 showed pronounced antiviral activity, represented by 100 % inhibition of viral replication at concentrations of 1.25, 0.625, 0.312 and 0.156 mg/mL, against the strains ATCC-VR733 and Acyclovir-resistant 29-R of HSV-1 virus. Regarding the mechanism of action, virucidal activity on the fraction AQ PPT FR 4-5 was detected, which is also very important because, as far as we know, compounds with virucidal activity for clinical use in the treatment of HSV-1 infections were not reported yet. In conclusion, Baccharis anomala displayed pronounced antiviral activity against HSV-1 virus and it is promising for further studies aimed to the isolation, identification and mechanism of action of antiviral active compounds of the species, considering the emergence of resistant strains and the need for compounds with new mechanisms of action.

Ocorrências comuns de infecções virais graves, aparecimento de cepas resistentes e um limitado número de quimioterápicos antivirais disponíveis mostram a necessidade da busca por novas substâncias ativas como antivirais. Muitas substâncias derivadas de plantas são candidatas ao estudo do seu potencial na terapia sistêmica e/ou profilaxia de herpes simplex virus 1 (HSV-1). Dentre as plantas com atividade antiviral, destacam-se as do gênero Baccharis. O objetivo geral do trabalho foi o estudo da composição química e avaliação da atividade antiviral das folhas de Baccharis anomala D.C. através de fracionamento bioguiado. Utilizando precipitação com etanol e fracionamento por permeação molecular foi possível separar os constituintes químicos ativos contra o vírus HSV-1 presentes no extrato aquoso de Baccharis anomala. Os testes de prospecção de constituintes químicos indicaram a presença de taninos, catequinas e saponinas no extrato aquoso da espécie. Através da análise cromatográfica foi possível detectar a presença de compostos fenólicos, utilizando-se coloração com cloreto férrico e reagente natural. Em relação à atividade antiviral, a fração ativa denominada AQ PPT FR 4-5 apresentou pronunciada atividade antiviral, inibindo a replicação viral em 100 % nas concentrações de 1,25, 0,625, 0,312 e 0,156 mg/mL, contra as cepas ATCC-VR733 e Aciclovir-resistente 29-R do HSV-1. Em relação ao mecanismo de ação, observou-se atividade virucida da fração AQ PPT FR 4-5. Estes resultados são muito importantes, pois, de acordo com a literatura, ainda não foram relatados compostos com atividade virucida úteis clinicamente para o tratamento de infecções por HSV-1. Conclui-se, portanto, que Baccharis anomala possui potente atividade antiviral contra o vírus HSV-1 e é promissora para estudos posteriores que visem ao isolamento, identificação e estudo do mecanismo de ação antiviral de compostos ativos da espécie, considerando a emergência de cepas resistentes e a necessidade de compostos com novos mecanismos de ação. / Common occurrences of severe viral infections, emergence of resistant strains and a limited number of available antiviral chemotherapeutics show the need to search for new active substances as antiviral. Many compounds derived from plants are candidates for the study of their potential in systemic therapy and/or prophylaxis of herpes simplex virus type 1 (HSV-1). Among the plants with antiviral activity, those from the genus Baccharis are remarkable. The main objective of the work was the study of the chemical composition and evaluation of the antiviral activity of extracts from Baccharis anomala D.C., using bioactivity guided fractionation. Through precipitation with ethanol and fractionation by molecular permeation it was achieved the separation of the active chemical constituents against HSV-1 virus in the aqueous extract of Baccharis anomala. Phytochemical tests indicated the presence of tannins, catechins and saponins in the aqueous extract. By thin layer chromatography it was detected the presence of phenolic compounds using ferric chloride and natural reagent. Concerning the antiviral activity, the active fraction named AQ PPT FR 4-5 showed pronounced antiviral activity, represented by 100 % inhibition of viral replication at concentrations of 1.25, 0.625, 0.312 and 0.156 mg/mL, against the strains ATCC-VR733 and Acyclovir-resistant 29-R of HSV-1 virus. Regarding the mechanism of action, virucidal activity on the fraction AQ PPT FR 4-5 was detected, which is also very important because, as far as we know, compounds with virucidal activity for clinical use in the treatment of HSV-1 infections were not reported yet. In conclusion, Baccharis anomala displayed pronounced antiviral activity against HSV-1 virus and it is promising for further studies aimed to the isolation, identification and mechanism of action of antiviral active compounds of the species, considering the emergence of resistant strains and the need for compounds with new mechanisms of action.

Em 2009, foi demonstrado um potente antiviral na hemolinfa de L. obliqua. Esta proteína purificada reduziu o título viral (TCID50 ml-1) do sarampo, pólio e H1N1 em 157, 61 e 64 vezes. Recentemente, expressamos a proteína antiviral em sistema baculovírus, que reduziu o título de vírus do herpes em 106 vezes, da rubéola e EMC em 105 vezes. No entanto, este sistema de expressão é muito caro e trabalhoso. Com isso, clonamos e expressamos esta proteína com atividade antiviral em sistema bacteriano. A sequência antiviral foi clonada em vetor de expressão pET28a. As construções resultantes foram expressas em E. coli Bl21 DE3 pLysS induzidas com IPTG 1,0 mM. Após expressão, o pellet bacteriano foi sonicado e o material foi purificado por afinidade e gel filtração. Testou-se contra o vírus EMC mostrando uma proteção de 104 vezes em relação ao controle. E utilizando qPCR para determinar os níveis de transcrição de RNA do vírus do herpes e da rubéola em células infectadas mostrou uma inibição respectivamente de 106 e 105 vezes, em relação ao controle. / In 2009, we demonstrated a potent antiviral in the hemolymph of L. obliqua. This purified protein has reduced the viral titer (TCID50 ml-1), measles and polio H1N1 157, 61 and 64 times. Recently, the antiviral protein expressed in baculovirus system, which reduced the title of the herpes virus in 106 times, rubella and EMC in 105 times. However, this expression system is very expensive and laborious. Thus, cloned and expressed this protein with antiviral activity in bacterial system. The antiviral sequence was cloned into pET28a expression vector. The resulting constructs were expressed in E. coli BL21 DE3 pLysS cells induced with 1.0 mM IPTG. Following expression, the bacterial pellet was sonicated and the material was purified by affinity and gel filtration. Was tested against EMC virus showing a protection of 104 times compared to the control. And using qPCR to determine the levels of viral RNA transcription of the herpes virus and rubella-infected cells showed an inhibition of respectively 106 and 105 times compared to the control.

A dengue é a mais importante arbovirose no mundo; aproximadamente 50 milhões de infecções ocorrem anualmente acarretando 500.000 casos de dengue hemorrágica e 22.000 mortes. A febre amarela é uma doença hemorrágica viral com elevada mortalidade que é transmitida por mosquitos. Vacinas eficazes contra a febre amarela já estão disponíveis há quase 70 anos e são responsáveis por uma redução significativa de ocorrências da doença no mundo, no entanto, cerca de 200.000 casos de febre amarela ainda ocorrem anualmente, principalmente na África. Dessa forma, o desenvolvimento de fármacos antivirais contra essas viroses é uma prioridade de saúde pública. Os produtos naturais sejam de origem vegetal ou animal, possuem uma extensa diversidade química, sendo uma fonte inesgotável de compostos com promissoras atividades biológicas. No Brasil, é grande a incidência de animais venenosos ou peçonhentos, tais como serpentes, sapos e escorpiões. Os venenos desses animais são fontes de diversas substâncias químicas que ainda não possuem a sua atividade biológica e farmacológica completamente estudada. Neste trabalho avaliamos a potencial ação antiviral de peçonhas de serpentes (Crotalus durissus terrificus, Bothrops jararacussu, Bothrops jararaca, Bothrops pirajai, Bothrops moojeni, Bothrops brasili e Bothrops fonseca) e escorpião (Tityus serrulatus) contra os virus da febre amarela e dengue usando diferentes estratégias metodológicas (pré-tratamento, pós-tratamento, virucida, adsorção e internalização). Primeiramente realizamos um screening com as peçonhas brutas, observando que a peçonha de Crotalus durissus terrificus inibiu a replicação viral apresentando os maiores índices de seletividade (IS). Crotoxina, crotamina, crotapotina, convulxina, giroxina, PLA2-CB e PLA2-IC, isoladas de Crotalus durissus terrificus, foram então testadas nas diferentes estratégias metodológicas contra os vírus dengue e febre amarela. Foi possível verificar que crotoxina, PLA2-CB e PLA2-IC inibiram a replicação viral com altos índices de seletividade (IS). A ação verificada ocorreu na fase inicial do ciclo de replicação viral (pré-tratamento, virucida, adsorção). A ação antiviral verificada neste estudo foi atribuida a ação da PLA2, visto que a crotoxina é um complexo protéico composto pela crotapotina e pela PLA2-CB. Posteriormente avaliamos uma fosfolipase sem atividade catalítica isolada de Bothrops jararacussu, a BthTX-I. Essa fosfolipase apresentou baixa inibição da replicação viral, sugerindo que a atividade catalítica da fosfolipase é importante, mas possivelmente não a única responsável pela ação antiviral. Os resultados obtidos permitem sugerir também que as fosfolipases apresentam ação tanto sobre a partícula viral quanto sobre receptores celulares, o que justifica os altos índices de seletividade observados. / Dengue is the most important arbovirus disease in the world; nearly 50 million infections occur annually resulting in 500,000 cases of DHF and 22,000 deaths. Yellow fever is a viral haemorrhagic fever with high mortality that is transmitted by mosquitoes. Effective vaccines against yellow fever have been available for almost 70 years and are responsible for a significant reduction of the disease worldwide. However, about 200,000 cases of yellow fever still occur annually, mainly in Africa. Thus, the development of antiviral drugs against these viruses is a public health priority. Natural products of plant or animal origin have an extensive chemical diversity, and an inexhaustible source of compounds with promising biological activities. In Brazil, there is a high incidence of poisonous or venomous animals such as snakes, frogs and scorpions occur. The venoms of these animals are a source of several chemicals that does not possess biological and pharmacological activity completely studied. In this study, we assess the potential antiviral action of snake venom (Crotalus durissus terrificus, Bothrops jararacussu, Bothrops jararaca, Bothrops pirajai, Bothrops moojeni, Bothrops brasili and Bothrops fonseca) and Scorpion (Tityus serrulatus) against yellow fever and dengue viruses using different methodological strategies (pre-treatment, post-treatment, virucidal, adsorption and internalization). First, we performed a screening with the crude venoms, founding that the venom of Crotalus durissus terrificus inhibited viral replication showing the highest selectivity index (SI). Crotoxin crotamin, crotapotin, convulxin, gyroxin, PLA2-CB and PLA2-IC isolated from Crotalus durissus terrificus, were then tested in the different methodological strategies against dengue and yellow fever viruses. We found that crotoxin, PLA2-CB and PLA2-IC inhibited viral replication with high SI. The action of these compounds against the virus was at the first steps of the replication cycle (pre-treatment, virucidal, adsorption). The antiviral action observed in this study was attributed to the action of PLA2, since crotoxin is a protein complex composed of crotapotin and PLA2-CB. Afterwards, we evaluated a phospholipase without catalytic activity isolated from Bothrops jararacussu, the BthTX-I. This phospholipase showed low inhibition of viral replication, showing that the catalytic activity of phospholipase is important, but perhaps not the only one responsible for the antiviral action. Our results also suggest that phospholipases have action on the viral particle and on cell receptors, which explains the high levels of selectivity observed.

A raiva é uma zoonose que afeta todos os mamíferos e causa cerca de 55.000 mortes humanas por ano, causada pelo vírus da raiva. O vírus da raiva pertence à Ordem Mononegavirales, Família Rhabdoviridae e o Gênero Lissavirus. Atualmente, o tratamento humano se baseia no uso do Protocolo de Milwaukee composto de indução do paciente ao coma e uso de massiva terapia antiviral. O protocolo, apesar de ter sido utilizado duas vezes com sucesso, inclusive em um caso brasileiro, ainda requer aprimoramentos. Neste sentido, a interferência por RNA (RNAi) é uma nova abordagem para terapia de doenças virais. O objetivo deste trabalho foi avaliar a inibição da replicação do vírus da raiva in vitro e in vivo utilizando RNAi. Para tanto, foram utilizados três siRNAs (siRNA 124, siRNA 750, siRNA B) com a fita antisenso complementar ao mRNA da nucleoproteína (N) do vírus da raiva. Para o ensaio in vitro foram utilizadas a cepa PV do vírus da raiva e as células de BHK-21 (Baby hamster kidney). As monocamadas celulares foram infectadas com a cepa PV e depois de 2 horas de incubação transfectadas com cada um dos siRNAs em combinação com Lipofectamine 2000®. Depois de 22 horas as placas teste e controle foram submetidas à imunofluorescência direta (IFD) com conjugado globulina de coelho anti-nucleocapsídeo do vírus da raiva/isotiocianato de fluoresceína (BIO-RADTM). Os resultados revelaram títulos de 5,71logTCID50/ml, 5,56logTCID50/ml e 5,65logTCID50/ml para os siRNAs 124, B e 750, respectivamente, enquanto que, para a placa controle, o título foi 6,43logTCID50/ml. Para o ensaio in vivo, foram usados camundongos albino suíços de 21 dias com peso entre 11 e 14g, infectados com a cepa PV via intracerebral. Duas horas depois da infecção foi inoculada por via intracerebral uma solução do \"pool\" dos três siRNAs com Lipofectamine 2000® . Os animais com paralisia foram sacrificados e aqueles sobreviventes foram observados até completar 30 dias de observação quando foram, então, sacrificados. O sistema nervoso central de todos os animas foi recolhido e submetido a IFD. O título viral do grupo teste foi 7.03logLD50%/ml e do grupo controle 7.13logLD50%/ml. O resultado do ensaio in vitro demonstra que os siRNAs utilizados são efetivos em inibir a replicação do vírus da raiva, com eficiências equivalentes. A utilização do \"pool\" dos três siRNA em camundongos resultou em 30% de animais sobreviventes frente a 100 DL50% do vírus PV, enquanto que a mesma dose levou a 100% de mortalidade nos animais não tratados. Pode-se atribuir a menor eficiência em inibir a replicação do vírus da raiva in vivo quando se compara com os resultados in vitro possivelmente às elevadas doses virais utilizadas. Estes resultados, ainda que indiquem a necessidade de mais estudos, permitem concluir que a RNAi é uma tecnologia promissora como antiviral contra a raiva. / Rabies is a zoonotic disease that affects all mammals and causes more than 55.000 human deaths every year, caused by rabies virus (RABV) a virus of the Mononegavirales order, Family Rhabdoviridae and the Lissavirus genus. After the onset of the symptoms, the illness has a fast progression and the patients feel intense physical suffering. Currently, human rabies treatment has been based on the Milwaukee Protocol which consists on the induction of coma and massive antiviral therapy. Despite this protocol has been successful in two cases, including a Brazilian case (Recife State), more studies on antiviral for human rabies treatment are required. RNA interference is a new antiviral approach, which gives hope to the possibility of rabies antiviral treatment. The aim of this study was to assess the decrease in the titer of rabies virus in vitro and in vivo using short-interfering RNAs. For this purpose, three siRNAs (siRNA 124, siRNA B, siRNA 750) were used with antisense strands complementary to rabies virus nucleoprotein (N) mRNA. Pasteur virus strain (PV) of rabies virus and BHK-21 cells were used, and the monolayers were transfected with each of tree RNAs with Lipofectamine-2000. After 22 hours, the siRNA-treated and the control plates were tested by direct fluorescent antibody test (DFAT) with anti-rabies virus nucleocapsid antibody conjugate with fluorescein isothiocianate. Virus titers were calculated by the Spearman-Karber method. The results revealed that all three siRNAs reduced the titer of PV strain and a more intense effect was obtained with siRNA B. The titer of the PV strain in the control plate was 6.43lg TCID50%/ml and 5.56lg TCID50%/ml in the plate treated with siRNA B, respectively. The similar result was obtained in plates treated with siRNAs 124 and 750. The title of PV strain of these plates was 5.71lg TCID50%/ml of siRNA 124 and 5.65lg TCID50%/ml of siRNA750, respectively. No cytopathic or cytotoxic effect was observed in the monolayers treated with Lipofectamine-2000®. Swiss albino mice with 21 days weighing between 11 and 14g infected with strain PV by the intracerebral route used in this in vivo essay. After two hours of infection, a pool solution of 3 siRNAs with lipofectamine was also inoculated by the intracerebral route. The animals with paralysis were euthanized and those which survived were observed until the 30th day when they were also euthanized. The central nervous system of all animals were collected and induced to IFD. The title viral test group was 7.03logLD50%/ml and the control group was 7.13logLD50%/ml. The in vitro test results indicate that siRNAS are effective in inhibiting the replication of rabies virus with similar efficiencies. The use of the pool of three siRNA in mice resulted in 30% of survivors for 100 LD50% of PV virus, while the same dose led to 100% mortality in untreated animals. A lower efficiency in inhibiting the replication of rabies virus in vivo when compared with results in vitro could be possibly due to the high viral doses used. These results, although indicative of the need for further studies, show that RNAi is a promising technology as antiviral against rabies.

Ocorrências comuns de infecções virais graves, aparecimento de cepas resistentes e um limitado número de quimioterápicos antivirais disponíveis mostram a necessidade da busca por novas substâncias ativas como antivirais. Muitas substâncias derivadas de plantas são candidatas ao estudo do seu potencial na terapia sistêmica e/ou profilaxia de herpes simplex virus 1 (HSV-1). Dentre as plantas com atividade antiviral, destacam-se as do gênero Baccharis. O objetivo geral do trabalho foi o estudo da composição química e avaliação da atividade antiviral das folhas de Baccharis anomala D.C. através de fracionamento bioguiado. Utilizando precipitação com etanol e fracionamento por permeação molecular foi possível separar os constituintes químicos ativos contra o vírus HSV-1 presentes no extrato aquoso de Baccharis anomala. Os testes de prospecção de constituintes químicos indicaram a presença de taninos, catequinas e saponinas no extrato aquoso da espécie. Através da análise cromatográfica foi possível detectar a presença de compostos fenólicos, utilizando-se coloração com cloreto férrico e reagente natural. Em relação à atividade antiviral, a fração ativa denominada AQ PPT FR 4-5 apresentou pronunciada atividade antiviral, inibindo a replicação viral em 100 % nas concentrações de 1,25, 0,625, 0,312 e 0,156 mg/mL, contra as cepas ATCC-VR733 e Aciclovir-resistente 29-R do HSV-1. Em relação ao mecanismo de ação, observou-se atividade virucida da fração AQ PPT FR 4-5. Estes resultados são muito importantes, pois, de acordo com a literatura, ainda não foram relatados compostos com atividade virucida úteis clinicamente para o tratamento de infecções por HSV-1. Conclui-se, portanto, que Baccharis anomala possui potente atividade antiviral contra o vírus HSV-1 e é promissora para estudos posteriores que visem ao isolamento, identificação e estudo do mecanismo de ação antiviral de compostos ativos da espécie, considerando a emergência de cepas resistentes e a necessidade de compostos com novos mecanismos de ação. / Common occurrences of severe viral infections, emergence of resistant strains and a limited number of available antiviral chemotherapeutics show the need to search for new active substances as antiviral. Many compounds derived from plants are candidates for the study of their potential in systemic therapy and/or prophylaxis of herpes simplex virus type 1 (HSV-1). Among the plants with antiviral activity, those from the genus Baccharis are remarkable. The main objective of the work was the study of the chemical composition and evaluation of the antiviral activity of extracts from Baccharis anomala D.C., using bioactivity guided fractionation. Through precipitation with ethanol and fractionation by molecular permeation it was achieved the separation of the active chemical constituents against HSV-1 virus in the aqueous extract of Baccharis anomala. Phytochemical tests indicated the presence of tannins, catechins and saponins in the aqueous extract. By thin layer chromatography it was detected the presence of phenolic compounds using ferric chloride and natural reagent. Concerning the antiviral activity, the active fraction named AQ PPT FR 4-5 showed pronounced antiviral activity, represented by 100 % inhibition of viral replication at concentrations of 1.25, 0.625, 0.312 and 0.156 mg/mL, against the strains ATCC-VR733 and Acyclovir-resistant 29-R of HSV-1 virus. Regarding the mechanism of action, virucidal activity on the fraction AQ PPT FR 4-5 was detected, which is also very important because, as far as we know, compounds with virucidal activity for clinical use in the treatment of HSV-1 infections were not reported yet. In conclusion, Baccharis anomala displayed pronounced antiviral activity against HSV-1 virus and it is promising for further studies aimed to the isolation, identification and mechanism of action of antiviral active compounds of the species, considering the emergence of resistant strains and the need for compounds with new mechanisms of action.

Influenza viruses are respiratory pathogens that are responsible for annual influenza epidemics and sporadic influenza pandemics. Oseltamivir (Tamiflu((R))) is currently the only FDA-approved oral drug that is available for the prevention and treatment of influenza virus infection. However, its narrow therapeutic window, coupled with the increasing incidence of drug resistance, calls for the next generation of influenza antivirals. In this study, we discovered hesperadin, an aurora B kinase inhibitor, as a broad-spectrum influenza antiviral through forward chemical genomics screening. Hesperadin inhibits multiple human clinical isolates of influenza A and B viruses with single to submicromolar efficacy, including oseltamivir-resistant strains. Mechanistic studies revealed that hesperadin inhibits the early stage of viral replication by delaying the nuclear entry of viral ribonucleoprotein complex, thereby inhibiting viral RNA transcription and translation as well as viral protein synthesis. Moreover, a combination of hesperadin with oseltamivir shows synergistic antiviral activity, therefore hesperadin can be used either alone to treat infections by oseltamivir-resistant influenza viruses or used in combination with oseltamivir to delay resistance evolution among oseltamivir-sensitive strains. In summary, the discovery of hesperadin as a broad-spectrum influenza antiviral offers an alternative to combat future influenza epidemics and pandemics.