A Genetic and Pathologic Study of a DENV2 Clinical Isolate Capable of Inducing Encephalitis and Hematological Disturbances in Immunocompetent Mice

Dengue virus (DENV) is the causative agent of dengue fever (DF), a mosquito-borne illness endemic to tropical and subtropical regions. There is currently no effective drug or vaccine formulation for the prevention of DF and its more severe forms, i.e., dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). There are two generally available experimental models for the study of DENV pathogenicity as well as the evaluation of potential vaccine candidates. The first model consists of non-human primates, which do not develop symptoms but rather a transient viremia. Second, mouse-adapted virus strains or immunocompromised mouse lineages are utilized, which display some of the pathological features of the infection observed in humans but may not be relevant to the results with regard to the wild-type original virus strains or mouse lineages. In this study, we describe a genetic and pathological study of a DENV2 clinical isolate, named JHA1, which is naturally capable of infecting and killing Balb/c mice and reproduces some of the symptoms observed in DENV-infected subjects. Sequence analyses demonstrated that the JHA1 isolate belongs to the American genotype group and carries genetic markers previously associated with neurovirulence in mouse-adapted virus strains. The JHA1 strain was lethal to immunocompetent mice following intracranial (i.c.) inoculation with a LD50 of approximately 50 PFU. Mice infected with the JHA1 strain lost weight and exhibited general tissue damage and hematological disturbances, with similarity to those symptoms observed in infected humans. In addition, it was demonstrated that the JHA1 strain shares immunological determinants with the DENV2 NGC reference strain, as evaluated by cross-reactivity of anti-envelope glycoprotein (domain III) antibodies. The present results indicate that the JHA1 isolate may be a useful tool in the study of DENV pathogenicity and will help in the evaluation of anti-DENV vaccine formulations as well as potential therapeutic approaches.

DengueTools: innovative tools and strategies for the surveillance and control of dengue

Dengue fever is a mosquito-borne viral disease estimated to cause about 230 million infections worldwide every year, of which 25,000 are fatal. Global incidence has risen rapidly in recent decades with some 3.6 billion people, over half of the world's population, now at risk, mainly in urban centres of the tropics and subtropics. Demographic and societal changes, in particular urbanization, globalization, and increased international travel, are major contributors to the rise in incidence and geographic expansion of dengue infections. Major research gaps continue to hamper the control of dengue. The European Commission launched a call under the 7th Framework Programme with the title of 'Comprehensive control of Dengue fever under changing climatic conditions'. Fourteen partners from several countries in Europe, Asia, and South America formed a consortium named 'DengueTools' to respond to the call to achieve better diagnosis, surveillance, prevention, and predictive models and improve our understanding of the spread of dengue to previously uninfected regions (including Europe) in the context of globalization and climate change. The consortium comprises 12 work packages to address a set of research questions in three areas: Research area 1: Develop a comprehensive early warning and surveillance system that has predictive capability for epidemic dengue and benefits from novel tools for laboratory diagnosis and vector monitoring. Research area 2: Develop novel strategies to prevent dengue in children. Research area 3: Understand and predict the risk of global spread of dengue, in particular the risk of introduction and establishment in Europe, within the context of parameters of vectorial capacity, global mobility, and climate change. In this paper, we report on the rationale and specific study objectives of 'DengueTools'. DengueTools is funded under the Health theme of the Seventh Framework Programme of the European Community, Grant Agreement Number: 282589 Dengue Tools.

Phylogenetic relationship of dengue virus type 3 isolated in Brazil and Paraguay and global evolutionary divergence dynamics

Background: Dengue is the most important mosquito-borne viral disease worldwide. Dengue virus comprises four antigenically related viruses named dengue virus type 1 to 4 (DENV1-4). DENV-3 was re-introduced into the Americas in 1994 causing outbreaks in Nicaragua and Panama. DENV-3 was introduced in Brazil in 2000 and then spread to most of the Brazilian States, reaching the neighboring country, Paraguay in 2002. In this study, we have analyzed the phylogenetic relationship of DENV-3 isolated in Brazil and Paraguay with viruses isolated worldwide. We have also analyzed the evolutionary divergence dynamics of DENV-3 viruses. Results: The entire open reading frame (ORF) of thirteen DENV-3 isolated in Brazil (n = 9) and Paraguay (n = 4) were sequenced for phylogenetic analysis. DENV-3 grouped into three main genotypes (I, II and III). Several internal clades were found within each genotype that we called lineage and sub-lineage. Viruses included in this study belong to genotype III and grouped together with viruses isolated in the Americas within the lineage III. The Brazilian viruses were further segregated into two different sub-lineage, A and B, and the Paraguayan into the sub-lineage B. All three genotypes showed internal grouping. The nucleotide divergence was in average 6.7% for genotypes, 2.7% for lineages and 1.5% for sub-lineages. Phylogenetic trees constructed with any of the protein gene sequences showed the same segregation of the DENV-3 in three genotypes. Conclusion: Our results showed that two groups of DENV-3 genotypes III circulated in Brazil during 2002-2009, suggesting different events of introduction of the virus through different regions of the country. In Paraguay, only one group DENV-3 genotype III is circulating that is very closely related to the Brazilian viruses of sub-lineage B. Different degree of grouping can be observed for DENV-3 and each group showed a characteristic evolutionary divergence. Finally, we have observed that any protein gene sequence can be used to identify the virus genotype.

Worldwide spread of Dengue virus type 1

BACKGROUND: DENV-1 is one of the four viral serotypes that causes Dengue, the most common mosquito-borne viral disease of humans. The prevalence of these viruses has grown in recent decades and is now present in more than 100 countries. Limited studies document the spread of DENV-1 over the world despite its importance for human health. METHODOLOGY/PRINCIPAL FINDINGS: We used representative DENV-1 envelope gene sequences to unravel the dynamics of viral diffusion under a Bayesian phylogeographic approach. Data included strains from 45 distinct geographic locations isolated from 1944 to 2009. The estimated mean rate of nucleotide substitution was 6.56 × 10⁻⁴ substitutions/site/year. The larger genotypes (I, IV and V) had a distinctive phylogenetic structure and since 1990 they experienced effective population size oscillations. Thailand and Indonesia represented the main sources of strains for neighboring countries. Besides, Asia broadcast lineages into the Americas and the Pacific region that diverged in isolation. Also, a transmission network analysis revealed the pivotal role of Indochina in the global diffusion of DENV-1 and of the Caribbean in the diffusion over the Americas. CONCLUSIONS/SIGNIFICANCE: The study summarizes the spatiotemporal DENV-1 worldwide spread that may help disease control.

Mosquitoes in degraded and preserved areas of the Atlantic Forest and potential for vector-borne disease risk in the municipality of Sao Paulo, Brazil

In order to assess the epidemiological potential of the Culicidae species in remaining areas of the Brazilian Atlantic Forest, specimens of this family were collected in wild and anthropic environments. A total of 9,403 adult mosquitoes was collected from May, 2009 to June, 2010. The most prevalent among species collected in the wild environment were Anopheles (Kerteszia) cruzii, the Melanoconion section of Culex (Melanoconion), and Aedes serratus, while the most common in the anthropic site were Coquillettidia chrysonotum/albifera, Culex (Culex) Coronator group, and An. (Ker.) cruzii. Mosquito richness was similar between environments, although the abundance of individuals from different species varied. When comparing diversity patterns between environments, anthropic sites exhibited higher richness and evenness, suggesting that environmental stress increased the number of favorable niches for culicids, promoting diversity. Increased abundance of opportunistic species in the anthropic environment enhances contact with culicids that transmit vector-borne diseases.

Stochastic lattice model describing a vector-borne disease

We employ the approach of stochastic dynamics to describe the dissemination of vector-borne diseases such as dengue, and we focus our attention on the characterization of the threshold of the epidemic. The coexistence space comprises two representative spatial structures for both human and mosquito populations. The human population has its evolution described by a process that is similar to the Susceptible-Infected-Recovered (SIR) dynamics. The population of mosquitoes follows a dynamic of the type of the Susceptible Infected-Susceptible (SIS) model. The coexistence space is a bipartite lattice constituted by two structures representing the human and mosquito populations. We develop a truncation scheme to solve the evolution equations for the densities and the two-site correlations from which we get the threshold of the disease and the reproductive ratio. We present a precise deØnition of the reproductive ratio which reveals the importance of the correlations developed in the early stage of the disease. According to our deØnition, the reproductive rate is directed related to the conditional probability of the occurrence of a susceptible human (mosquito) given the presence in the neighborhood of an infected mosquito (human). The threshold of the epidemic as well as the phase transition between the epidemic and the non-epidemic states are also obtained by performing Monte Carlo simulations. References: [1] David R. de Souza, T^ania Tom∂e, , Suani R. T. Pinho, Florisneide R. Barreto and M∂ario J. de Oliveira, Phys. Rev. E 87, 012709 (2013). [2] D. R. de Souza, T. Tom∂e and R. M. ZiÆ, J. Stat. Mech. P03006 (2011).

Amazonian malaria: Asymptomatic human reservoirs, diagnostic challenges, environmentally driven changes in mosquito vector populations, and the mandate for sustainable control strategies

Across the Americas and the Caribbean, nearly 561,000 slide-confirmed malaria infections were reported officially in 2008. The nine Amazonian countries accounted for 89% of these infections; Brazil and Peru alone contributed 56% and 7% of them, respectively. Local populations of the relatively neglected parasite Plasmodium vivax, which currently accounts for 77% of the regional malaria burden, are extremely diverse genetically and geographically structured. At a time when malaria elimination is placed on the public health agenda of several endemic countries, it remains unclear why malaria proved so difficult to control in areas of relatively low levels of transmission such as the Amazon Basin. We hypothesize that asymptomatic parasite carriage and massive environmental changes that affect vector abundance and behavior are major contributors to malaria transmission in epidemiologically diverse areas across the Amazon Basin. Here we review available data supporting this hypothesis and discuss their implications for current and future malaria intervention policies in the region. Given that locally generated scientific evidence is urgently required to support malaria control interventions in Amazonia, we briefly describe the aims of our current field-oriented malaria research in rural villages and gold-mining enclaves in Peru and a recently opened agricultural settlement in Brazil. (C) 2011 Elsevier B.V. All rights reserved.

Spatial distribution of arboviral mosquito vectors (Diptera, Culicidae) in Vale do Ribeira in the South-eastern Brazilian Atlantic Forest

Mosquitoes are vectors of arboviruses that can cause encephalitis and hemorrhagic fevers in humans. Aedes serratus (Theobald), Aedes scapularis (Rondani) and Psorophora ferox (Von Humboldt) are potential vectors of arboviruses and are abundant in Vale do Ribeira, located in the Atlantic Forest in the southeast of the State of Sao Paulo, Brazil. The objective of this study was to predict the spatial distribution of these mosquitoes and estimate the risk of human exposure to mosquito bites. Results of the analyses show that humans are highly exposed to bites in the municipalities of Cananeia, Iguape and Ilha Comprida. In these localities the incidence of Rocio encephalitis was 2% in the 1970s. Furthermore, Ae. serratus, a recently implicated vector of yellow fever virus in the State of Rio Grande do Sul, should be a target for the entomological surveillance in the southeastern Atlantic Forest. Considering the continental dimensions of Brazil and the inherent difficulties in sampling its vast area, the habitat suitability method used in the study can be an important tool for predicting the distribution of vectors of pathogens.

Possible Host Adaptation as an Evolution Factor of Cowpea aphid-borne mosaic virus Deduced by Coat Protein Gene Analysis

Cowpea aphid-borne mosaic virus (CABMV) causes major diseases in cowpea and passion flower plants in Brazil and also in other countries. CABMV has also been isolated from leguminous species including, Cassia hoffmannseggii, Canavalia rosea, Crotalaria juncea and Arachis hypogaea in Brazil. The virus seems to be adapted to two distinct families, the Passifloraceae and Fabaceae. Aiming to identify CABMV and elucidate a possible host adaptation of this virus species, isolates from cowpea, passion flower and C.hoffmannseggii collected in the states of Pernambuco and Rio Grande do Norte were analysed by sequencing the complete coat protein genes. A phylogenetic tree was constructed based on the obtained sequences and those available in public databases. Major Brazilian isolates from passion flower, independently of the geographical distances among them, were grouped in three different clusters. The possible host adaptation was also observed in fabaceous-infecting CABMV Brazilian isolates. These host adaptations possibly occurred independently within Brazil, so all these clusters belong to a bigger Brazilian cluster. Nevertheless, African passion flower or cowpea-infecting isolates formed totally different clusters. These results showed that host adaptation could be one factor for CABMV evolution, although geographical isolation is a stronger factor.

BRAZILIAN MOSQUITO (DIPTERA: CULICIDAE) FAUNA. I. Anopheles SPECIES FROM PORTO VELHO, RONDONIA STATE, WESTERN AMAZON, BRAZIL

This study contributes to knowledge of Anopheles species, including vectors of Plasmodium from the western Brazilian Amazon in Porto Velho, Rondonia State. The sampling area has undergone substantial environmental changes as a consequence of agricultural and hydroelectric projects, which have caused intensive deforestation and favored habitats for some mosquito species. The purpose of this study was to diagnose the occurrence of anopheline species from collections in three locations along an electric-power transmission line. Each locality was sampled three times from 2010 to 2011. The principal adult mosquitoes captured in Shannon trap were Anopheles darlingi, An. triannulatus, An. nuneztovari l.s., An. gilesi and An. costai. In addition, larvae were collected in ground breeding sites for Anopheles braziliensis, An. triannulatus, An. darlingi, An. deaneorum, An. marajoara, An. peryassui, An. nuneztovari l.s. and An. oswaldoi-konderi. Anopheles darlingi was the most common mosquito in the region. We discuss Culicidae systematics, fauna distribution, and aspects of malaria in altered habitats of the western Amazon.

A Third Amblyomma Species and the First Tick-Borne Rickettsia in Chile

During November 2010, three ticks were collected from three dogs living in the rural area of Arica, northern Chile. Morphological analyses of the ticks in the laboratory revealed that they were most similar to Amblyomma maculatum Koch and Amblyomma triste Koch. However, because of unique metatarsal spurs, neither of the Chilean specimens could be assigned with certainty to A. maculatum or A. triste, based on external morphology. The mitochondrial 16S rRNA gene partial sequences obtained from two Chilean specimens were 99.5% identical to A. triste from Uruguay, and 99.0% identical to A. maculatum from the United States. Through phylogenetic analysis inferred from partial 16S rRNA sequences, the Chilean specimens were classified as A. triste. Molecular analyses also showed that one of the three Chilean ticks was infected by Candidatus 'Rickettsia andeanae'. These findings extend the geographical distribution of A. triste to Chile, where no tick-associated rickettsia had been reported previously.

Lactobacillus sakei 2a and its Concentrated Acid Extract on Inhibition of Food-Borne Salmonella strains

Lactic acid bacteria are used in food production to provide desirable organoleptic characteristics, and can also act as biopreservatives, controlling the growth of undesirable microorganisms. In this study, we examined the antimicrobial action of Lactobacillus sakei 2a and its concentrated acid extract against food-borne Salmonella spp. The extract was obtained by acid extraction from culture broth of L. sakei 2a and was designated extract 2a. We determined that extract 2a had significant activity (approximately 500 AU ml(-1)). We used different antimicrobial substances alone or in combination with extract 2a to evaluate the inhibitory activity of the various treatments on a pool of five Salmonella strains. The pathogen Listeria monocytogenes Scott A Cm-r Em(r) was used as an indicator strain of inhibitory activity. In summary, all antimicrobials substances that were tested showed an inhibitory effect against the growth of Salmonella, andthis action was enhanced in the presence of extract 2a. Moreover, among the treatments applied, the combination of extract 2a and 0.1% lactic acid exhibited the most potent inhibitory effect towards the pool of Salmonella strains. Our findings indicate that L. sakei 2a and extract 2a, especially in combination with other antimicrobials, present potential technological application in the control of salmonellae in foods.

Mosquito (Diptera: Culicidae) assemblages associated with Nidularium and Vriesea bromeliads in Serra do Mar, Atlantic Forest, Brazil

Background: The most substantial and best preserved area of Atlantic Forest is within the biogeographical sub-region of Serra do Mar. The topographic complexity of the region creates a diverse array of microclimates, which can affect species distribution and diversity inside the forest. Given that Atlantic Forest includes highly heterogeneous environments, a diverse and medically important Culicidae assemblage, and possible species co-occurrence, we evaluated mosquito assemblages from bromeliad phytotelmata in Serra do Mar (southeastern Brazil). Methods: Larvae and pupae were collected monthly from Nidularium and Vriesea bromeliads between July 2008 and June 2009. Collection sites were divided into landscape categories (lowland, hillslope and hilltop) based on elevation and slope. Correlations between bromeliad mosquito assemblage and environmental variables were assessed using multivariate redundancy analysis. Differences in species diversity between bromeliads within each category of elevation were explored using the Renyi diversity index. Univariate binary logistic regression analyses were used to assess species co-occurrence. Results: A total of 2,024 mosquitoes belonging to 22 species were collected. Landscape categories (pseudo-F value = 1.89, p = 0.04), bromeliad water volume (pseudo-F = 2.99, p = 0.03) and bromeliad fullness (Pseudo-F = 4.47, p < 0.01) influenced mosquito assemblage structure. Renyi diversity index show that lowland possesses the highest diversity indices. The presence of An. homunculus was associated with Cx. ocellatus and the presence of An. cruzii was associated with Cx. neglectus, Cx. inimitabilis fuscatus and Cx. worontzowi. Anopheles cruzii and An. homunculus were taken from the same bromeliad, however, the co-occurrence between those two species was not statistically significant. Conclusions: One of the main findings of our study was that differences in species among mosquito assemblages were influenced by landscape characteristics. The bromeliad factor that influenced mosquito abundance and assemblage structure was fullness. The findings of the current study raise important questions about the role of An. homunculus in the transmission of Plasmodium in Serra do Mar, southeastern Atlantic Forest.

Intestinal Lymph-Borne Factors Induce Lung Release of Inflammatory Mediators and Expression of Adhesion Molecules After an Intestinal Ischemic Insult

Background. Intestinal ischemia and reperfusion (I/R) is a documented cause of acute lung injury (ALI) and systemic inflammation. We previously reported that obstruction of thoracic lymphatic flow during intestinal I/R blunts pulmonary neutrophil recruitment and microvascular injury and decreases the systemic levels of tumor necrosis factor. Here, we consider the existence of a gut-lung axis promoting the induction of systemic inflammation, whereby drained intestinal lymph stimulates lung expression of adhesion molecules and matrix components and generation of inflammatory mediators. Material and Methods. Upon administration of anesthesia, male Wistar rats were subjected to occlusion of the superior mesenteric artery for 45 min, followed by 2 h of intestinal reperfusion (I/R); groups of rats were subjected to I/R with or without thoracic lymphatic duct ligation immediately before the procedure. The non-manipulated rats were used to investigate basal parameters. Results. Obstruction of thoracic lymphatic flow before intestinal I/R decreased the ability of cultured lung tissue explants to release IL-1 beta, IL-10, and VEGF. In contrast, lymphatic obstruction normalized the elevated lung expression of PECAM-1 caused by intestinal I/R. On the other hand, lung E-selectin expression was significantly reduced, whereas fibronectin expression and collagen synthesis were not affected. Lymph levels of LTB4 and TXB2 were found to be significantly increased. Conclusions. These data suggest that lymph factors drained from the intestine during ischemic trauma stimulate the lung to generate inflammatory mediators and alter the expression of adhesion molecules. Disturbances in lung homeostasis mediated by lymph might contribute to the spread of inflammatory processes, thereby accounting for the systemic inflammation induced by intestinal I/R. (C) 2012 Elsevier Inc. All rights reserved.

Early skin immunological disturbance after Plasmodium-infected mosquito bites

Although the role of regulatory T cells (Tregs) during malaria infection has been studied extensively, such studies have focused exclusively on the role of Treg during the blood stage of infection; little is known about the detailed mechanisms of Tregs and sporozoite deposition in the dermis by mosquito bites. In this paper we show that sporozoites introduced into the skin by mosquito bites increase the mobility of skin Tregs and dendritic cells (DCs). We also show differences in MHC class II and/or C086 expression on skin-resident dendritic cell subtypes and macrophages. From the observed decrease of the number of APCs into draining lymph nodes, suppression of CD28 expression in conventional CD4 T cells, and a low homeostatic proliferation of skin-migrated CD4 T found in nude mice indicate that Tregs may play a fundamental role during the initial phase of malaria parasite inoculation into the mammalian host. (C) 2012 Elsevier Inc. All rights reserved.