Origin and evolution of dengue virus type 3 in Brazil

The incidence of dengue fever and dengue hemorrhagic fever in Brazil experienced a significant increase since the emergence of dengue virus type-3 (DENV-3) at the early 2000s. Despite the major public health concerns, there have been very few studies of the molecular epidemiology and time-scale of this DENV lineage in Brazil. In this study, we investigated the origin and dispersion dynamics of DENV-3 genotype III in Brazil by examining a large number (n = 107) of E gene sequences sampled between 2001 and 2009 from diverse Brazilian regions. These Brazilian sequences were combined with 457 DENV-3 genotype III E gene sequences from 29 countries around the world. Our phylogenetic analysis reveals that there have been at least four introductions of the DENV-3 genotype III in Brazil, as signified by the presence of four phylogenetically distinct lineages. Three lineages (BR-I, BR-II, and BR-III) were probably imported from the Lesser Antilles (Caribbean), while the fourth one (BR-IV) was probably introduced from Colombia or Venezuela. While lineages BR-I and BR-II succeeded in getting established and disseminated in Brazil and other countries from the Southern Cone, lineages BR-III and BR-IV were only detected in one single individual each from the North region. The phylogeographic analysis indicates that DENV-3 lineages BR-I and BR-II were most likely introduced into Brazil through the Southeast and North regions around 1999 (95% HPD: 1998–2000) and 2001 (95% HPD: 2000–2002), respectively. These findings show that importation of DENV-3 lineages from the Caribbean islands into Brazil seems to be relatively frequent. Our study further suggests that the North and Southeast Brazilian regions were the most important hubs of introduction and spread of DENV-3 lineages and deserve an intense epidemiological surveillance.     

The dynamics of dengue virus serotype 3 introduction and dispersion in the state of Bahia, Brazil

By 2002, dengue virus serotype 1 (DENV-1) and DENV-2 had circulated for more than a decade in Brazil. In 2002, the introduction of DENV-3 in the state of Bahia produced a massive epidemic and the first cases of dengue hemorrhagic fever. Based on the standardized frequency, timing and location of viral isolations by the state's Central Laboratory, DENV-3 probably entered Bahia through its capital, Salvador, and then rapidly disseminated to other cities, following the main roads. A linear regression model that included traffic flow, distance from the capital and DENV-1 circulation (r2 = 0.24, p = 0.001) supported this hypothesis. This pattern was not seen for serotypes already in circulation and was not seen for DENV-3 in the following year. Human population density was another important factor in the intensity of viral circulation. Neither DENV-1 nor DENV-2 fit this model for 2001 or 2003. Since the vector has limited flight range and vector densities fail to correlate with intensity of viral circulation, this distribution represents the movement of infected people and to some extent mosquitoes. This pattern may mimic person-to-person spread of a new infection.     

Comparative immunological recognition of proteins from New Guinea "C" dengue virus type 2 prototype and from a dengue virus type 2 strain isolated in the State of Rio de Janeiro,Brazil

The protein profiles of the New Guinea "C" dengue virus type 2 (DENV-2)prototype and those of a Brazilian DENV-2 isolated in the State of Rio de Janeiro in 1995 were compared. SDS-PAGE analysis showed that the virus from Rio de Janeiro expresses NS5 (93.0 kDa), NS3 (66.8 kDa) E (62.4 kDa) and NS1 (41.2 kDa) proteins differently from the New Guinea "C" virus. The immunoblot revealed specificity and antigenicity for the NS3 protein from DENV-2 Rio de Janeiro mainly in primary infections, convalescent cases, and in secondary infections in both cases and only antigenicity for E and NS1 proteins for both viruses in primary and secondary infections.     

Genome 3'-end repair in dengue virus type 2

Genomes of RNA viruses encounter a continual threat from host cellular ribonucleases. Therefore, viruses have evolved mechanisms to protect the integrity of their genomes. To study the mechanism of 3′-end repair in dengue virus-2 in mammalian cells, a series of 3′-end deletions in the genome were evaluated for virus replication by detection of viral antigen NS1 and by sequence analysis. Limited deletions did not cause any delay in the detection of NS1 within 5 d. However, deletions of 7–10 nucleotides caused a delay of 9 d in the detection of NS1. Sequence analysis of RNAs from recovered viruses showed that at early times, virus progenies evolved through RNA molecules of heterogeneous lengths and nucleotide sequences at the 3′ end, suggesting a possible role for terminal nucleotidyl transferase activity of the viral polymerase (NS5). However, this diversity gradually diminished and consensus sequences emerged. Template activities of 3′-end mutants in the synthesis of negative-strand RNA in vitro by purified NS5 correlate well with the abilities of mutant RNAs to repair and produce virus progenies. Using the Mfold program for RNA structure prediction, we show that if the 3′ stem–loop (3′ SL) structure was abrogated by mutations, viruses eventually restored the 3′ SL structure. Taken together, these results favor a two-step repair process: non-template-based nucleotide addition followed by evolutionary selection of 3′-end sequences based on the best-fit RNA structure that can support viral replication.     

Complete genetic characterization of a Brazilian dengue virus type 3 strain isolated from a fatal outcome

We have determined the complete nucleotide and the deduced amino acid sequences of Brazilian dengue virus type 3 (DENV-3) from a dengue case with fatal outcome, which occurred during an epidemic in the state of Rio de Janeiro, Brazil, in 2002. This constitutes the first complete genetic characterization of a Brazilian DENV-3 strain since its introduction into the country in 2001. DENV-3 was responsible for the most severe dengue epidemic in the state, based on the highest number of reported cases and on the severity of clinical manifestations and deaths reported.     

Filamentous structures in dengue type 3 virus infected mouse neurones

Dengue type 3 (H-87) virus was inoculated into suckling mouse brain and animals were sacrificed at 24 hr intervals. Parallel filamentous structures were found in infected neurones in close association with virus particles in the distended endoplasmic cisternae. They were usually arranged in a crystalloid pattern, oriented in different directions within the cisternae. Faint helical features were sometimes observed. These filamentous structures measured 15-25 nm in width and varied in length. Their possible involvement with viral material or a viral core is postulated.     

Dengue virus type 3 in Rio de Janeiro, Brazil.

Dengue virus type 3 was isolated for the first time in the country as an indigenous case from a 40 year-old woman presenting signs and symptoms of a classical dengue fever in the municipality of Nova Igua?u, State of Rio de Janeiro. This serotype has been associated with dengue haemorrhagic epidemics and the information could be used to implement appropriate prevention and control measures. Virological surveillance was essential in order to detected this new serotype.     

分子绝对进化速率与物种分歧时间之间的定量关系

通过对美国国家生物技术信息中心数据库Gen Bank提供的一些蛋白质和核苷酸序列进行比对和分析,发现生物分子绝对进化速率k与进化时间或物种分歧时间t之间存在下列定量关系:lnk=-Ea/Rt+lnK0,式中Ea为位点突变活化能,k0为分子极限绝对进化速率,R为常数,并对其生物学意义进行了初步的探讨;数据分析还揭示出物种的分子极限绝对进化速率与进化时间或物种分歧时间之间也服从相似的定量公式,也就是说生物分子进化过程可能同时受到序列位点突变和控制物种分子极限绝对进化速率进化的两个"分子钟"作用,即存在"双重分子钟"现象。     

Circulation of different lineages of dengue virus type 2 in Central America, their evolutionary time-scale and selection pressure analysis

Dengue is caused by any of the four serotypes of dengue virus (DENV-1 to 4). Each serotype is genetically distant from the others, and each has been subdivided into different genotypes based on phylogenetic analysis. The study of dengue evolution in endemic regions is important since the diagnosis is often made by nucleic acid amplification tests, which depends upon recognition of the viral genome target, and natural occurring mutations can affect the performance of these assays. Here we report for the first time a detailed study of the phylogenetic relationships of DENV-2 from Central America, and report the first fully sequenced DENV-2 strain from Guatemala. Our analysis of the envelope (E) protein and of the open reading frame of strains from Central American countries, between 1999 and 2009, revealed that at least two lineages of the American/Asian genotype of DENV-2 have recently circulated in that region. In occasions the co-circulation of these lineages may have occurred and that has been suggested to play a role in the observed increased severity of clinical cases. Our time-scale analysis indicated that the most recent common ancestor for Central American DENV-2 of the American/Asian genotype existed about 19 years ago. Finally, we report positive selection in DENV-2 from Central America in codons of the genes encoding for C, E, NS2A, NS3, and NS5 proteins. Some of these identified codons are novel findings, described for the first time for any of the DENV-2 genotypes.     

Variable surface epitopes in the crystal structure of dengue virus type 3 envelope glycoprotein

Dengue virus is an emerging global health threat. The major envelope glycoprotein, E, mediates viral attachment and entry by membrane fusion. Antibodies that bind but fail to neutralize noncognate serotypes enhance infection. We have determined the crystal structure of a soluble fragment of the envelope glycoprotein E from dengue virus type 3. The structure closely resembles those of E proteins from dengue type 2 and tick-borne encephalitis viruses. Serotype-specific neutralization escape mutants in dengue virus E proteins are all located on a surface of domain III, which has been implicated in receptor binding. While antibodies against epitopes in domain I are nonneutralizing in dengue virus, there are neutralizing antibodies that recognize serotype-conserved epitopes in domain II. The mechanism of neutralization for these antibodies is probably inhibition of membrane fusion. Our structure shows that neighboring glycans on the viral surface are spaced widely enough (at least 32 A) that they can interact with multiple carbohydrate recognition domains on oligomeric lectins such as DC-SIGN, ensuring maximum affinity for these putative receptors.     

Evidence for the co-circulation of dengue virus type 3 genotypes III and V in the Northern region of Brazil during the 2002-2004 epidemics

The reintroduction of dengue virus type 3 (DENV-3) in Brazil in 2000 and its subsequent spread throughout the country was associated with genotype III viruses, the only DENV-3 genotype isolated in Brazil prior to 2002. We report here the co-circulation of two different DENV-3 genotypes in patients living in the Northern region of Brazil during the 2002-2004 epidemics. Complete genomic sequences of viral RNA were determined from these epidemics, and viruses belonging to genotypes V (Southeast Asia/South Pacific) and III were identified. This recent co-circulation of different DENV-3 genotypes in South America may have implications for pathological and epidemiological dynamics.     

Recombinant dengue virus type 3 envelope domain III protein from Escherichia coli

Dengue is a public health problem of global significance for which there is neither an effective antiviral therapy nor a preventive vaccine. The envelope protein of dengue virus is the major antigen to elicit neutralizing antibody response and protective immunity in hosts. Optimization of culture media was carried out for enhanced production of recombinant dengue virus type 3 envelope domain III (rDen 3 EDIII) protein in E. coli. Further, batch and fed-batch cultivation process were also developed in optimized medium. After fed-batch cultivation, the dry cell weight was about 22.80 g/L of culture. The rDen 3 EDIII protein was purified using immobilized metal affinity chromatography. This process produced ～649 mg of purified rDen 3 EDIII protein per liter of culture. The purity of the protein was determined by SDS-PAGE analysis and the reactivity was checked by Western blotting as well as ELISA. These results show that the purified protein may be used for the dengue diagnosis or further prophylactic studies for dengue infection.     

Invasion and maintenance of dengue virus type 2 and type 4 in the Americas

Dengue virus type 4 (DENV-4) was first reported in the Americas in 1981, where it caused epidemics of dengue fever throughout the region. In the same year, the region's first epidemic of dengue hemorrhagic fever was reported, caused by an Asian strain of dengue virus type 2 (DENV-2) that was distinct from the American subtype circulating previously. Despite the importance of these epidemics, little is known about the rates or determinants of viral spread among island and mainland populations or their directions of movement. We employed a Bayesian coalescent approach to investigate the transmission histories of DENV-2 and DENV-4 since their introduction in 1981 and a parsimony method to assess patterns of strain migration. For both viruses there was an initial invasion phase characterized by an exponential increase in the number of DENV lineages, after which levels of genetic diversity remained constant despite reported fluctuations in DENV-2 and DENV-4 activity. Strikingly, viral lineage numbers increased far more rapidly for DENV-4 than DENV-2, indicative of a more rapid rate of exponential population growth in DENV-4 or a higher rate of geographic dispersal, allowing this virus to move more effectively among localities. We propose that these contrasting dynamics may reflect underlying differences in patterns of host immunity. Despite continued gene flow along particular transmission routes, the overall extent of viral traffic was less than expected under panmixis. Hence, DENV in the Americas has a clear geographic structure that maintains viral diversity between outbreaks.     

Climate change influences on global distributions of dengue and chikungunya virus vectors

Numerous recent studies have illuminated global distributions of human cases of dengue and other mosquito-transmitted diseases, yet the potential distributions of key vector species have not been incorporated integrally into those mapping efforts. Projections onto future conditions to illuminate potential distributional shifts in coming decades are similarly lacking, at least outside Europe. This study examined the global potential distributions of Aedes aegypti and Aedes albopictus in relation to climatic variation worldwide to develop ecological niche models that, in turn, allowed anticipation of possible changes in distributional patterns into the future. Results indicated complex global rearrangements of potential distributional areas, which—given the impressive dispersal abilities of these two species—are likely to translate into actual distributional shifts. This exercise also signalled a crucial priority: digitization and sharing of existing distributional data so that models of this sort can be developed more rigorously, as present availability of such data is fragmentary and woefully incomplete.     

Characterization of antibody responses to combinations of a dengue virus type 2 DNA vaccine and two dengue virus type 2 protein vaccines in rhesus macaques

We evaluated three nonreplicating dengue virus type 2 (DENV-2) vaccines: (i) a DNA vaccine containing the prM-E gene region (D), (ii) a recombinant subunit protein vaccine containing the B domain (i.e., domain III) of the E protein as a fusion with the Escherichia coli maltose-binding protein (R), and (iii) a purified inactivated virus vaccine (P). Groups of four rhesus macaques each were primed once and boosted twice using seven different vaccination regimens. After primary vaccination, enzyme-linked immunosorbent assay (ELISA) antibody levels increased most rapidly for groups inoculated with the P and DP combination, and by 1 month after the second boost, ELISA titers were similar for all groups. The highest plaque reduction neutralization test (PRNT) titers were seen in those groups that received the DR/DR/DR combination (geometric mean titer [GMT], 510), the P/P/P vaccine (GMT, 345), the DP/DP/DP combination (GMT, 287), and the R/R/R vaccine (GMT, 200). The next highest titers were seen in animals that received the D/R/R vaccine (GMT, 186) and the D/P/P vaccine (GMT, 163). Animals that received the D/D/D vaccine had the lowest neutralizing antibody titer (GMT, 49). Both ELISA and PRNT titers declined at variable rates. The only significant protection from viremia was observed in the P-vaccinated animals (mean of 0.5 days), which also showed the highest antibody concentration, including antibodies to NS1, and highest antibody avidity at the time of challenge.     

Local phylogenetic divergence and global evolutionary convergence of skull function in reef fishes of the family Labridae

The Labridae is one of the most structurally and functionally diversified fish families on coral and rocky reefs around the world, providing a compelling system for examination of evolutionary patterns of functional change. Labrid fishes have evolved a diverse array of skull forms for feeding on prey ranging from molluscs, crustaceans, plankton, detritus, algae, coral and other fishes. The species richness and diversity of feeding ecology in the Labridae make this group a marine analogue to the cichlid fishes. Despite the importance of labrids to coastal reef ecology, we lack evolutionary analysis of feeding biomechanics among labrids. Here, we combine a molecular phylogeny of the Labridae with the biomechanics of skull function to reveal a broad pattern of repeated convergence in labrid feeding systems. Mechanically fast jaw systems have evolved independently at least 14 times from ancestors with forceful jaws. A repeated phylogenetic pattern of functional divergence in local regions of the labrid tree produces an emergent family-wide pattern of global convergence in jaw function. Divergence of close relatives, convergence among higher clades and several unusual 'breakthroughs' in skull function characterize the evolution of functional complexity in one of the most diverse groups of reef fishes.     

Genetic exchange and evolutionary divergence in prokaryotes

Recent work shows that genetic exchange in prokaryotes is less frequent but more promiscuous than that in eukaryotes. As a result, genetic exchange plays very different roles in determining the patterns of evolutionary divergence in these major groups. Because sexual isolation is not a prerequisite for divergence in the prokaryotic world, the biological species concept is not appropriate for bacteria. However, there is a species concept that may apply universally.