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.

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: >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.

Genetic diversity of the E Protein of Dengue Type 3 Virus

Abstract Background Dengue is the most important arbovirus disease in tropical and subtropical countries. The viral envelope (E) protein is responsible for cell receptor binding and is the main target of neutralizing antibodies. The aim of this study was to analyze the diversity of the E protein gene of DENV-3. E protein gene sequences of 20 new viruses isolated in Ribeirao Preto, Brazil, and 427 sequences retrieved from GenBank were aligned for diversity and phylogenetic analysis. Results Comparison of the E protein gene sequences revealed the presence of 47 variable sites distributed in the protein; most of those amino acids changes are located on the viral surface. The phylogenetic analysis showed the distribution of DENV-3 in four genotypes. Genotypes I, II and III revealed internal groups that we have called lineages and sub-lineages. All amino acids that characterize a group (genotype, lineage, or sub-lineage) are located in the 47 variable sites of the E protein. Conclusion Our results provide information about the most frequent amino acid changes and diversity of the E protein of DENV-3.

Assessment of the relationship between entomologic indicators of Aedes aegypti and the epidemic occurrence of dengue virus 3 in a susceptible population, São José do Rio Preto, Sao Paulo, Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Detection of serum antibodies to parainfluenza type 3 virus, respiratory syncytial virus, bovine viral diarrhea virus, and herpes virus type 1 in sheep in the region of Botucatu, São Paulo - Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Identification of a cryptic Prokaryotic promoter within the cDNA encoding the 5′ End of Dengue Virus RNA Genome

Infectious cDNA clones of RNA viruses are important research tools, but flavivirus cDNA clones have proven difficult to assemble and propagate in bacteria. This has been attributed to genetic instability and/or host cell toxicity, however the mechanism leading to these difficulties has not been fully elucidated. Here we identify and characterize an efficient cryptic bacterial promoter in the cDNA encoding the dengue virus (DENV) 5′ UTR. Following cryptic transcription in E. coli, protein expression initiated at a conserved in-frame AUG that is downstream from the authentic DENV initiation codon, yielding a DENV polyprotein fragment that was truncated at the N-terminus. A more complete understanding of constitutive viral protein expression in E. coli might help explain the cloning and propagation difficulties generally observed with flavivirus cDNA.

Recent emergence of dengue virus serotype 4 in French Polynesia results from multiple introductions from other south pacific islands

BACKGROUND: Infection by dengue virus (DENV) is a major public health concern in hundreds of tropical and subtropical countries. French Polynesia (FP) regularly experiences epidemics that initiate, or are consecutive to, DENV circulation in other South Pacific Island Countries (SPICs). In January 2009, after a decade of serotype 1 (DENV-1) circulation, the first cases of DENV-4 infection were reported in FP. Two months later a new epidemic emerged, occurring about 20 years after the previous circulation of DENV-4 in FP. In this study, we investigated the epidemiological and molecular characteristics of the introduction, spread and genetic microevolution of DENV-4 in FP. METHODOLOGY/PRINCIPAL FINDINGS: Epidemiological data suggested that recent transmission of DENV-4 in FP started in the Leeward Islands and this serotype quickly displaced DENV-1 throughout FP. Phylogenetic analyses of the nucleotide sequences of the envelope (E) gene of 64 DENV-4 strains collected in FP in the 1980s and in 2009-2010, and some additional strains from other SPICs showed that DENV-4 strains from the SPICs were distributed into genotypes IIa and IIb. Recent FP strains were distributed into two clusters, each comprising viruses from other but distinct SPICs, suggesting that emergence of DENV-4 in FP in 2009 resulted from multiple introductions. Otherwise, we observed that almost all strains collected in the SPICs in the 1980s exhibit an amino acid (aa) substitution V287I within domain I of the E protein, and all recent South Pacific strains exhibit a T365I substitution within domain III. CONCLUSIONS/SIGNIFICANCE: This study confirmed the cyclic re-emergence and displacement of DENV serotypes in FP. Otherwise, our results showed that specific aa substitutions on the E protein were present on all DENV-4 strains circulating in SPICs. These substitutions probably acquired and subsequently conserved could reflect a founder effect to be associated with epidemiological, geographical, eco-biological and social specificities in SPICs.

Comparative susceptibility of mosquito populations in North Queensland, Australia to oral infection with dengue virus

Dengue is the most prevalent arthropod-borne virus, with at least 40% of the world’s population at risk of infection each year. In Australia, dengue is not endemic, but viremic travelers trigger outbreaks involving hundreds of cases. We compared the susceptibility of Aedes aegypti mosquitoes from two geographically isolated populations with two strains of dengue virus serotype 2. We found, interestingly, that mosquitoes from a city with no history of dengue were more susceptible to virus than mosquitoes from an outbreak-prone region, particularly with respect to one dengue strain. These findings suggest recent evolution of population-based differences in vector competence or different historical origins. Future genomic comparisons of these populations could reveal the genetic basis of vector competence and the relative role of selection and stochastic processes in shaping their differences. Lastly, we show the novel finding of a correlation between midgut dengue titer and titer in tissues colonized after dissemination.

Analysis of human immunodeficiency virus type 1 drug resistance in children receiving nucleoside analogue reverse-transcriptase inhibitors plus nevirapine, nelfinavir, or ritonavir (Pediatric AIDS Clinical Trials Group 377)

In Pediatric AIDS Clinical Trials Group 377, antiretroviral therapy-experienced children were randomized to 4 treatment arms that included different combinations of stavudine, lamivudine (3TC), nevirapine (Nvp), nelfinavir (Nfv), and ritonavir (Rtv). Previous treatment with zidovudine (Zdv), didanosine (ddI), or zalcitabine (ddC) was acceptable. Drug resistance ((R)) mutations were assessed before study treatment (baseline) and at virologic failure. Zdv(R), ddI(R), and ddC(R) mutations were detected frequently at baseline but were not associated with virologic failure. Children with drug resistance mutations at baseline had greater reductions in virus load over time than did children who did not. Nvp(R) and 3TC(R) mutations were detected frequently at virologic failure, and Nvp(R) mutations were more common among children receiving 3-drug versus 4-drug Nvp-containing regimens. Children who were maintained on their study regimen after virologic failure accumulated additional Nvp(R) and 3TC(R) mutations plus Rtv(R) and Nfv(R) mutations. However, Rtv(R) and Nfv(R) mutations were detected at unexpectedly low rates.

Completion of the genome sequence of Lettuce necrotic yellows virus, type species of the genus Cytorhabdovirus

We completed the genome sequence of Lettuce necrotic yellows virus (LNYV) by determining the nucleotide sequences of the 4a (putative phosphoprotein), 4b, M (matrix protein), G (glycoprotein) and L (polymerase) genes. The genome consists of 12,807 nucleotides and encodes six genes in the order 3′ leader-N-4a(P)-4b-M-G-L-5′ trailer. Sequences were derived from clones of a cDNA library from LNYV genomic RNA and from fragments amplified using reverse transcription-polymerase chain reaction. The 4a protein has a low isoelectric point characteristic for rhabdovirus phosphoproteins. The 4b protein has significant sequence similarities with the movement proteins of capillo- and trichoviruses and may be involved in cell-to-cell movement. The putative G protein sequence contains a predicted 25 amino acids signal peptide and endopeptidase cleavage site, three predicted glycosylation sites and a putative transmembrane domain. The deduced L protein sequence shows similarities with the L proteins of other plant rhabdoviruses and contains polymerase module motifs characteristic for RNA-dependent RNA polymerases of negative-strand RNA viruses. Phylogenetic analysis of this motif among rhabdoviruses placed LNYV in a group with other sequenced cytorhabdoviruses, most closely related to Strawberry crinkle virus.

Prediction of protein-protein interactions in dengue virus coat proteins guided by low resolution cryoEM structures

Background: Dengue virus along with the other members of the flaviviridae family has reemerged as deadly human pathogens. Understanding the mechanistic details of these infections can be highly rewarding in developing effective antivirals. During maturation of the virus inside the host cell, the coat proteins E and M undergo conformational changes, altering the morphology of the viral coat. However, due to low resolution nature of the available 3-D structures of viral assemblies, the atomic details of these changes are still elusive. Results: In the present analysis, starting from C alpha positions of low resolution cryo electron microscopic structures the residue level details of protein-protein interaction interfaces of dengue virus coat proteins have been predicted. By comparing the preexisting structures of virus in different phases of life cycle, the changes taking place in these predicted protein-protein interaction interfaces were followed as a function of maturation process of the virus. Besides changing the current notion about the presence of only homodimers in the mature viral coat, the present analysis indicated presence of a proline-rich motif at the protein-protein interaction interface of the coat protein. Investigating the conservation status of these seemingly functionally crucial residues across other members of flaviviridae family enabled dissecting common mechanisms used for infections by these viruses. Conclusions: Thus, using computational approach the present analysis has provided better insights into the preexisting low resolution structures of virus assemblies, the findings of which can be made use of in designing effective antivirals against these deadly human pathogens.

Characterization and immune response of a protein produced by a cDNA clone of foot and mouth disease virus, type Asia 1 63/72

A 0.9 kb double stranded cDNA of foot and mouth disease virus (FMDV) Type Asia 1, 63/72 was cloned in an expression vector, pUR222. A protein of 38 kd was produced by the clone which reacted with the antibodies raised against the virus. A 20 kd protein which may be derived from the 38 kd protein contained the antigenic epitopes of the protein VP1 of the virus. Injection of 10-20 micrograms of the partially purified 38 and 20 kd proteins or a lysate of cells containing 240 micrograms of the proteins elicited high titers of FMDV specific antibodies in guinea pigs and cattle respectively. Also, at these concentrations, the proteins protected 5 of 8 guinea pigs and 3 of 8 cattle when challenged with a virulent virus.

Identification and characterization of a new class of human immunodeficiency virus type 1 recombinants comprised of two circulating recombinant forms, CRF07_BC and CRF08_BC, in China

We identified a new class of human immunodeficiency virus type 1 (HIV-1) recombinants (00CN-HH069 and 00CN-HH086) in which further recombination occurred between two established circulating recombinant forms (CRFs). These two isolates were found among 57 HIV-1 samples from a cohort of injecting drug users in eastern Yunnan Province of China. Informative-site analysis in conjunction with bootscanning plots and exploratory tree analysis revealed that these two strains were closely related mosaics comprised of CRF07_BC and CRF08_BC, which are found in China. The genotype screening based on gag-reverse transcriptase sequences if 57 samples from eastern Yunnan identified 47 CRF08_BC specimens (82.5%), 5 CRF07_BC specimens (8.8%), and 3 additional specimens with the novel recombinant structure. These new "second-generation" recombinants thus constitute a substantial proportion (5 of 57; 8.8%) of HIV-1 strains in this population and may belong to a new but yet-undefined class of CRF. This might be the first example of CRFs recombining with each other, leading to the evolution of second-generation inter-CRF recombinants.

Differential inhibition of human immunodeficiency virus type 1 in peripheral blood mononuclear cells and TZM-bl cells by endotoxin-mediated chemokine and gamma interferon production.

Bacterial lipopolysaccharide (endotoxin) is a frequent contaminant of biological specimens and is also known to be a potent inducer of beta-chemokines and other soluble factors that inhibit HIV-1 infection in vitro. Though lipopolysaccharide (LPS) has been shown to stimulate the production of soluble HIV-1 inhibitors in cultures of monocyte-derived macrophages, the ability of LPS to induce similar inhibitors in other cell types is poorly characterized. Here we show that LPS exhibits potent anti-HIV activity in phytohemagglutinin-stimulated peripheral blood mononuclear cells (PBMCs) but has no detectable anti-HIV-1 activity in TZM-bl cells. The anti-HIV-1 activity of LPS in PBMCs was strongly associated with the production of beta-chemokines from CD14-positive monocytes. Culture supernatants from LPS-stimulated PBMCs exhibited potent anti-HIV-1 activity when added to TZM-bl cells but, in this case, the antiviral activity appeared to be related to IFN-gamma rather than to beta-chemokines. These observations indicate that LPS stimulates PBMCs to produce a complex array of soluble HIV-1 inhibitors, including beta-chemokines and IFN-gamma, that differentially inhibit HIV-1 depending on the target cell type. The results also highlight the need to use endotoxin-free specimens to avoid artifacts when assessing HIV-1-specific neutralizing antibodies in PBMC-based assays.

Age-related immune reconstitution during highly active antiretroviral therapy in human immunodeficiency virus type 1-infected children.

OBJECTIVES: To evaluate the immune reconstitution in HIV-1-infected children in whom highly active antiretroviral therapy (HAART) controlled viral replication and to assess the existence of a relation between the magnitude of this restoration and age. METHODS: All HIV-1-infected children in whom a new HAART decreased plasma viral load below 400 copies/ml after 3 months of therapy were prospectively enrolled in a study of their immune reconstitution. Viral load, lymphocyte phenotyping, determination of CD4+ and CD8+ T cell receptor repertoires and proliferative responses to mitogens and recall antigens were assessed every 3 months during 1 year. RESULTS: Nineteen children were evaluated. Naive and memory CD4+ percentages were already significantly increased after 3 months of HAART. In contrast to memory CD4+ percentages, naive CD4+ percentages continued to rise until 12 months. Age at baseline was inversely correlated with the magnitude of the rise in naive CD4+ cells after 3, 6 and 9 months of therapy but not after 12 months. Although memory and activated CD8+ cells were already decreasing after 3 months, abnormalities of the CD8 T cell receptor repertoire and activation of CD8+ cells persisted at 1 year. HAART increased the response to mitogens as early as 3 months after starting therapy. CONCLUSIONS: In children the recovery of naive CD4+ cells occurs more rapidly if treatment is started at a younger age, but after 1 year of viral replication control, patients of all ages have achieved the same level of restoration. Markers of chronic activation in CD8+ cells persist after 1 year of HAART.