Refolded dengue virus type 2 NS1 protein expressed in Escherichia coli preserves structural and immunological properties of the native protein

The dengue virus NS1 protein has been shown to be a protective antigen under different experimental conditions but the recombinant protein produced in bacterial expression systems is usually not soluble and loses structural and immunological features of the native viral protein In the present study, experimental conditions leading to purification and refolding of the recombinant dengue virus type 2 (DENV-2) NS1 protein expressed in Escherichia coil are described The refolded recombinant protein was recovered as heat-stable soluble dimers with preserved structural features, as demonstrated by spectroscopic methods In addition, antibodies against epitopes of the NS1 protein expressed in eukaryotic cells recognized the refolded protein expressed in E coli but not the denatured form or the same protein submitted to a different refolding condition Collectively, the results demonstrate that the recombinant NS1 protein preserved important conformation and antigenic determinants of the native virus protein and represents a valuable reagent either for the development of vaccines or for diagnostic methods. (C) 2010 Elsevier B V All rights reserved

Circulation of Different Lineages of Dengue Virus 2, Genotype American/Asian in Brazil: Dynamics and Molecular and Phylogenetic Characterization

The American/Asian genotype of Dengue virus type 2 (DENV-2) was introduced into the Americas in the 80′s. Although there is no data showing when this genotype was first introduced into Brazil, it was first detected in Brazil in 1990. After which the virus spread throughout the country and major epidemics occurred in 1998, 2007/08 and 2010. In this study we sequenced 12 DENV-2 genomes obtained from serum samples of patients with dengue fever residing in São José do Rio Preto, São Paulo (SJRP/SP), Brazil, in 2008. The whole open reading frame or envelope sequences were used to perform phylogenetic, phylogeographic and evolutionary analyses. Isolates from SJRP/SP were grouped within one lineage (BR3) close to isolates from Rio de Janeiro, Brazil. Isolates from SJRP were probably introduced there at least in 2007, prior to its detection in the 2008 outbreak. DENV-2 circulation in Brazil is characterized by the introduction, displacement and circulation of three well-defined lineages in different times, most probably from the Caribbean. Thirty-seven unique amino acid substitutions were observed among the lineages, including seven amino acid differences in domains I to III of the envelope protein. Moreover, we dated here, for the first time, the introduction of American/Asian genotype into Brazil (lineage BR1) to 1988/89, followed by the introduction of lineages BR2 (1998-2000) and BR3 (2003-05). Our results show a delay between the introduction and detection of DENV-2 lineages in Brazil, reinforcing the importance and need for surveillance programs to detect and trace the evolution of these viruses. Additionally, Brazilian DENV-2 differed in genetic diversity, date of introduction and geographic origin and distribution in Brazil, and these are important factors for the evolution, dynamics and control of dengue. © 2013 Drumond et al.

Associação entre marcadores da resposta inflamatória e a imunopatogênese de agentes infecciosos de natureza viral (Vírus da dengue, HTLV-1 e HTLV-2) e bacteriana (Chlamydia trachomatis e Chlamydia pneumoniae)

A base genética das doenças é frequentemente estudada a partir dos polimorfismos dos genes de citocinas. O presente estudo investigou marcadores da resposta inflamatória associados a infecções virais e bacterianas que possam influenciar o curso da infecção. Foram medidos os níveis séricos (por ensaio imunoenzimático) e os polimorfismos de TNF-α (-308), TNF-β (+252), IFN-γ (+874) e da proteína C reativa, por meio de PCR e RFLP ou PCR alelo específico, em grupos de pessoas infectadas pelo vírus da dengue (n=80), com doença febril, não infectados (100), um grupo de infectados pelo HTLV (30 sintomáticos e 47 assintomáticos), um grupo com doença coronariana (58 com sororreatividade para Chlamydia e 31 com sorologia negativa) e um grupo controle (99 pessoas com sorologia negativa para dengue, HTLV e Chlamydia). Nenhum grupo mostrou associação com informações demográficas. O Vírus da dengue 3 (66,2%) e o HTLV-1 (90% em sintomáticos e 76,6% em assintomáticos) foram os agentes mais frequentes dentre os grupos respectivos. A maioria com doença coronariana (65,1%) apresentou anticorpos para Chlamydia (39,6% para C. trachomatis e C. pneumoniae, 58,6% apenas para C. trachomatis e 1,7% somente para C. pneumoniae). Foram significantes as diferenças encontradas entre: (i) os níveis séricos de TNF-β, IFN-γ e PrtCR dos grupos dengue positivo e dengue negativo com o grupo controle (p< 0,01); (ii) os níveis séricos de TNF-α, TNF-β, e IFN-γ dos grupos de HTLV (incluindo os tipos) e grupo controle; (iii) os níveis séricos de TNF-α, TNF-β, IFN-γ e PrtCR entre os pacientes com doença coronariana e sorologia positiva para Chlamydia e o grupo controle; (iv) a presença de anticorpos para C. trachomatis e C. pneumoniae e o grupo controle na comparação com a TNF-β, IFN-γ e PrtCR. As distribuições de frequências genotípicas foram estatisticamente significantes para os polimorfismos: (i) dos genes TNF-α (p=0,0494) e IFN-γ (p= 0,0008), entre os grupos dengue positivo, dengue negativo e controle e para o IFN-γ (p= 0,0007) entre os grupos DEN 1, DEN 2 e DEN 3 e o controle; (ii) do gene IFN-γ (p= 0,0023) nos grupos de pacientes com doença coronariana e sorologia positiva para C. trachomatis e C. pneumoniae, assim como nos monoreativos na comparação entre a positividade para C. trachomatis e o grupo controle.

Prevalência de auto-anticorpos contra antígenos celulares em pacientes com infecção pelos Vírus da dengue e Vírus linfotrópico de células T humanas, HTLV - 1 e 2

A pesquisa de anticorpos contra antígenos celulares requer permanente revisão das informações sobre a interpretação dos resultados, visto que a positividade é observada em parte da população normal e desencadeada transitoriamente por processos infecciosos. O objetivo deste trabalho foi determinar, através da técnica da pesquisa de auto-anticorpos anti-nucleares (ANA) em células HEp-2, a prevalência de auto-anticorpos contra antígenos celulares em três grupos de pessoas: Grupo 1- pacientes com infecção pelo Virus da dengue (VD) (n= 30); Grupo 2 - pacientes com infecção pelos HTLV 1 e 2 (n= 30), Grupo 3 - indivíduos doadores de sangue (n= 100) não infectados e sem manifestações clínicas aparentes. A prevalência de ANA nos Grupos 1 (40%) e 2 (40%) foi altamente significativa em relação ao Grupo 3 (2%) (p<0,0001), com predomínio do padrão citoplasmático em relação ao padrão nuclear. Os indivíduos do Grupo 1 estavam infectados por três espécies do VD, com predominância (p= 0,002) para o DEN 3 (66,7%), entretanto a distribuição da freqüência de ANA de acordo com a espécie, mostrou uma diferença significante (p= 0,0260) entre as infecções pelo VD1 (p= 0,0644) e VD2 (p= 0,0249), em relação ao VD3, mas sem diferença entre os padrões (p= 0,2479). No Grupo 2 a prevalência e o padrão de ANA não mostraram correlação com o tipo de HTLV, embora tenha predominado indivíduos infectados pelo HTLV 1 (p= 0,0035) (76,7%); a maioria não apresentava sintomas clínicos (p= 0,0136), 36,7% mostrava doença compatível com PET/MAH, e a presença de ANA não mostrou diferença significativa entre sintomáticos e assintomáticos (p> 0,05). Não houve correlação de soropositividade com sexo entre os grupos. Concluiu-se que o quadro infeccioso é um importante desencadeador de respostas auto-imunes detectadas laboratorialmente, não se observando influencia nas manifestaçõe clínícas dos agravos. Estudos prospectivos, com controles destes casos, poderão trazer as respostas quanto a importância e significado dos resultados obtidos.

Immune transcript variations among Aedes aegypti populations with distinct susceptibility to dengue virus serotype 2

The innate immune response of insects is one of the factors that may dictate their susceptibility to viral infection. Two immune signaling pathways, Toll and JAK-STAT, and the RNA interference (RNAi) pathway are involved in Aedes aegypti responses against dengue virus (DENV), however natural differences in these antiviral defenses among mosquito populations have not been studied. Here, two field Ae. aegypti populations from distinct ecological environments, one from Recife and the other from Petrolina (Brazil), and a laboratory strain were studied for their ability to replicate a primary isolate of dengue virus serotype 2 (DENV-2). Virus infectivity and replication were determined in insect tissues collected after viral exposure through reverse-transcription real time PCR (RT-PCR). The expression of a transcript representing these defense mechanisms (Toll, JAK-STAT and RNAi) in the midgut and fat body was studied with RTPCR to evaluate variations in innate immune mechanisms possibly employed against DENV. Analyses of infection rates indicated that the field populations were more susceptible to DENV-2 infection than the lab strain. There were distinct expression patterns among mosquito populations, in both control and infected insects. Moreover, lower expression of immune molecules in DENV-2-infected insects compared to controls was observed in the two field populations. These results suggest that natural variations in vector competence against DENV may be partly due to differences in mosquito defense mechanisms, and that the down-regulation of immune transcripts after viral infection depends on the insect strain. (C) 2012 Elsevier B.V. All rights reserved.

Crotoxin and phospholipases A(2) from Crotalus durissus terrificus showed antiviral activity against dengue and yellow fever viruses

Dengue is the most important arbovirus in the world with an estimated of 50 million dengue infections occurring annually and approximately 2.5 billion people living in dengue endemic countries. Yellow fever is a viral hemorrhagic 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 occurrences of the disease worldwide; however, approximately 200,000 cases of yellow fever still occur annually, principally in Africa. Therefore, it is a public health priority to develop antiviral agents for treatment of these virus infections. Crotalus durissus terrificus snake, a South American rattlesnake, presents venom with several biologically actives molecules. In this study, we evaluated the antiviral activity of crude venom and isolated toxins from Crotalus durissus terrificus and found that phospholipases A(2) showed a high inhibition of Yellow fever and dengue viruses in VERO E6 cells. (C) 2011 Elsevier Ltd. All rights reserved.

Synthese, Charakterisierung und Testung von Phenylacrylsäure-Derivaten, Benzothiazolen und Diarylthioethern als potentielle Inhibitoren der Dengue-Virus-Typ-2-NS2B-NS3-Protease

Dengue-Fieber ist eine durch Stechmücken der Gattungen Aedes aegypti und Aedes albopticus übertragene, virale Infektionskrankheit des Menschen, welche eine zunehmende Bedrohung für die Weltbevölkerung darstellt; das Infektionsrisiko betrifft vorwiegend Menschen, die in tropischen und subtropischen Gebieten der Erde (Asien, Afrika, Amerika) leben. Bei dem Erreger handelt es sich um ein Flavivirus, bestehend aus einer positiv polarisierten Einzelstrang-RNA, welches in vier verschiedenen Serotypen existiert. Eine Infektion mit Dengue-Viren zeigt sich durch drei mögliche Krankheitsbilder: Klassisches Dengue-Fieber (DF), hämorrhagisches Dengue-Fieber (DHF) oder Dengue-Schock-Syndrom (DSS). Das Dengue-Virus-Genom codiert eine Serin-Protease mit einer klassischen katalytischen Triade, bestehend aus den Aminosäuren His51, Asp75 und Ser135. Die Funktion der Dengue-Virus-Protease besteht in der post-translationalen, proteolytischen Prozessierung des viralen Polyprotein-Vorläufers, womit sie essentiell für die Virus-Replikation ist und damit einen wichtigen therapeutischen Ansatz für die Entwicklung neuer Wirkstoffe gegen Dengue-Fieber darstellt. Die Ziele der vorliegenden Arbeit bestanden darin, neue potentielle Inhibitoren der Dengue-Virus Typ 2 NS2B-NS3 Protease (DEN-2 NS2B-NS3pro) zu synthetisieren, deren Hemmwirkung sowie den Inhibitionstyp mithilfe fluorimetrischer Enzym-Assays zu bestimmen, Struktur-Wirkungs-Beziehungen (u.a. mithilfe von Molecular Docking-Rechnungen) zu analysieren und die erhaltenen Leitstrukturen zu optimieren. In der vorliegenden Arbeit wurden zwei Substanzklassen und damit zwei Teilprojekte behandelt: Phenylacrylsäureamide im ersten Teilprojekt, Benzothiazole und Diarylthioether zusammen im zweiten Teilprojekt. Im ersten Teilprojekt zeigten einige Phenylacrylsäureamide eine schwache Hemmung der DEN-2 NS2B-NS3pro zwischen ca. 50 und 61 % bei einer Inhibitorkonzentration von 50 µM sowie eine nicht-kompetitive Hemmung, welche jedoch durch vielfältige Derivatisierung kaum verändert oder verbessert werden konnte. Darüber hinaus wurden die endogenen Serin-Proteasen alpha-Chymotrypsin und Trypsin durch einige Phenylacrylsäureamide erheblich stärker gehemmt als die DEN-2 NS2B-NS3pro. Das zweite Teilprojekt befasste sich mit der Synthese und Testung von Diarylthioethern mit hydroxy-substituierten Benzothiazol-Bausteinen sowie der Testung einiger methoxy-substituierter Synthese-Vorstufen der Endverbindungen, um die Relevanz und den Einfluss der einzelnen Bausteine auf die Hemmung der DEN-2 NS2B-NS3pro zu untersuchen. Der in der vorliegenden Arbeit synthetisierte, potenteste Inhibitor der DEN-2 NS2B-NS3pro (Hemmung: 90 % [50 µM]; IC50 = 3.6 +/- 0.11 µM) und der DEN-3 NS2B-NS3pro (Hemmung: &amp;gt;99 % [100 µM]; IC50 = 9.1 +/- 1.02 µM), SH65, ein Diarylthioether-Benzothiazol-Derivat, entstand aufgrund der Vorhersage zweier möglicher Bindungsmodi (kompetitiv und nicht-kompetitiv) mithilfe von Molecular Docking-Experimenten an der Röntgen-Kristall-struktur der DEN-3 NS2B-NS3pro (PDB-Code: 3U1I). Nach experimenteller Bestimmung der IC50-Werte bei unterschiedlichen Substratkonzentrationen erwies sich SH65 jedoch als nicht-kompetitiver Inhibitor der DEN-2 NS2B-NS3pro. Trypsin wurde von SH65 vergleichbar stark gehemmt (96% [50 µM]; IC50 = 6.27 +/- 0.68 µM) wie die beiden getesteten Dengue-Virus-Proteasen, nicht jedoch alpha-Chymotrypsin (nur 21% Hemmung bei 50 µM), wodurch diesem Inhibitor zumindest eine relative Selektivität gegenüber Serin-Proteasen zugeschrieben werden kann. SH65 zeigte lediglich Protease-Hemmung in den Enzym-Assays, jedoch keine antivirale Aktivität bei der Testung an Dengue-Virus-infizierten Zellen, was aber wiederum bei der synthetisierten Vorstufe von SH65, welche anstelle der beiden Hydroxy-Gruppen über Methoxy-Gruppen verfügt, der Fall war. Diarylthioether mit mehrfach hydroxy-substituiertem Benzothiazol-Baustein stellen hiermit eine neue, vielversprechende Wirkstoffgruppe zur Hemmung sowohl der Dengue-Virus Typ 2- als auch der Dengue-Virus Typ 3 NS2B-NS3 Protease dar.

Co-circulação dos vírus dengue tipo 1, 2 e 4 nos Estados do Rio Grande do Norte e Paraíba, 2013

Dengue is a viral disease transmitted by female mosquitoes from genus Aedes, the principal urban vector is Aedes aegypti. Actually dengue has caused, in global scale, substantial morbidity and mortality. Four serotypes (antigenically distinct) are known: DENV-1, DENV-2, DENV-3 and DENV-4. The objective of this study was described the epidemiological profile dengue in the states of Rio Grande do Norte (RN) and Paraíba (PB), 2013. For that, suspected cases of dengue were studied, received for Laboratory of Molecular Biology of infectious disease and cancer (LADIC-UFRN) from different Health Units from RN and PB between January and December of 2013. The viral RNA was obtained from serum samples of patient from health units from RN and PB. It were studied 478 suspected cases of dengue , 252 (52,7%) from Rio Grande do Norte and 226 (47,3%) from Paraíba, showeds a global rate of infection global prevalence of 29,7% (142/478). The co-circulation of three serotypes was observed: DENV-1 (9,8% [14/142]), DENV-2 (3,5% [5/142]) and DENV-4 (86,7% [123/142]). People between 21-30 years old were the most affected by the disease during all the period of the study, representing 63,7% of the cases in both states. The genus most affected was female, representing 63,3% of cases in both states. Pau dos Ferros, Rio Grande do Norte, had the highest circulation of disease, with 8,2% (8/97) of cases. In Paraíba, the city most affected was João Pessoa, with (80% (36/45) of cases. The months with the biggest viral circulation in RN and PB were March and August, respectively. These results are very important to understanding the dengue viral activity in RN and PB, providing data that can guide control actions of this disease in support to local control programs

Elimination of dengue by community programs using Mesocyclops(Copepoda) against Aedes aegypti in central Vietnam

From September 2000 to June 2003, a community-based program for dengue control using local predacious copepods of the genus Mesocyclops was conducted in three rural communes in the central Vietnam provinces of Quang Nam, Quang Ngai, and Khanh Hoa. Post-project, three subsequent entomologic surveys were conducted until March 2004. The number of households and residents in the communes were 5,913 and 27,167, respectively, and dengue notification rates for these communes from 1996 were as high as 2,418.5 per 100,000 persons. Following knowledge, attitude, and practice evaluations, surveys of water storage containers indicated that Mesocyclops spp. already occurred in 3-17% and that large tanks up to 2,000 liters, 130-300-liter jars, wells, and some 220-liter metal drums were the most productive habitats for Aedes aegypti. With technical support, the programs were driven by communal management committees, health collaborators, schoolteachers, and pupils. From quantitative estimates of the standing crop of third and fourth instars from 100 households, Ae. aegypti were reduced by approximately 90% by year 1, 92.3-98.6% by year 2, and Ae. aegypti immature forms had been eliminated from two of three communes by June 2003. Similarly, from resting adult collections from 100 households, densities were reduced to 0-1 per commune. By March 2004, two communes with no larvae had small numbers but the third was negative; one adult was collected in each of two communes while one became negative. Absolute estimates of third and fourth instars at the three intervention communes and one left untreated had significant correlations (P = 0.009-< 0.001) with numbers of adults aspirated from inside houses on each of 15 survey periods. By year 1, the incidence of dengue disease in the treated communes was reduced by 76.7% compared with non-intervention communes within the same districts, and no dengue was evident in 2002 and 2003, compared with 112.8 and 14.4 cases per 100,000 at district level. Since we had similar success in northern Vietnam from 1998 to 2000, this study demonstrates that this control model is broadly acceptable and achievable at community level but vigilance is required post-project to prevent reinfestation.

The wMel Wolbachia strain blocks dengue and invades caged Aedes aegypti populations

Dengue fever is the most important mosquito-borne viral disease of humans with more than 50 million cases estimated annually in more than 100 countries. Disturbingly, the geographic range of dengue is currently expanding and the severity of outbreaks is increasing. Control options for dengue are very limited and currently focus on reducing population abundance of the major mosquito vector, Aedes aegypti. These strategies are failing to reduce dengue incidence in tropical communities and there is an urgent need for effective alternatives. It has been proposed that endosymbiotic bacterial Wolbachia infections of insects might be used in novel strategies for dengue control. For example, the wMelPop-CLA Wolbachia strain reduces the lifespan of adult A. aegypti mosquitoes in stably transinfected lines. This life-shortening phenotype was predicted to reduce the potential for dengue transmission. The recent discovery that several Wolbachia infections, including wMelPop-CLA, can also directly influence the susceptibility of insects to infection with a range of insect and human pathogens has markedly changed the potential for Wolbachia infections to control human diseases. Here we describe the successful transinfection of A. aegypti with the avirulent wMel strain of Wolbachia, which induces the reproductive phenotype cytoplasmic incompatibility with minimal apparent fitness costs and high maternal transmission, providing optimal phenotypic effects for invasion. Under semi-field conditions, the wMel strain increased from an initial starting frequency of 0.65 to near fixation within a few generations, invading A. aegypti populations at an accelerated rate relative to trials with the wMelPop-CLA strain. We also show that wMel and wMelPop-CLA strains block transmission of dengue serotype 2 (DENV-2) in A. aegypti, forming the basis of a practical approach to dengue suppression.

Surveillance of dengue fever virus: a review of epidemiological models and early warning systems

Dengue fever is one of the world’s most important vector-borne diseases. The transmission area of this disease continues to expand due to many factors including urban sprawl, increased travel and global warming. Current preventative techniques are primarily based on controlling mosquito vectors as other prophylactic measures, such as a tetravalent vaccine are unlikely to be available in the foreseeable future. However, the continually increasing dengue incidence suggests that this strategy alone is not sufficient. Epidemiological models attempt to predict future outbreaks using information on the risk factors of the disease. Through a systematic literature review, this paper aims at analyzing the different modeling methods and their outputs in terms of accurately predicting disease outbreaks. We found that many previous studies have not sufficiently accounted for the spatio-temporal features of the disease in the modeling process. Yet with advances in technology, the ability to incorporate such information as well as the socio-environmental aspect allowed for its use as an early warning system, albeit limited geographically to a local scale.