Molecular archaeology of Flaviviridae untranslated regions: duplicated RNA structures in the replication enhancer of flaviviruses and pestiviruses emerged via convergent evolution.

RNA secondary structures in the 3'untranslated regions (3'UTR) of the viruses of the family Flaviviridae, previously identified as essential (promoters) or beneficial (enhancers) for replication, have been analysed. Duplicated enhancer elements are revealed as a global feature in the evolution of the 3'UTR of distantly related viruses within the genera Flavivirus and Pestivirus. For the flaviviruses, duplicated structures occur in the 3'UTR of all four distantly related ecological virus subgroups (tick-borne, mosquito-borne, no known vector and insect-specific flaviviruses (ISFV). RNA structural differences distinguish tick-borne flaviviruses with discrete pathogenetic characteristics. For Aedes- and Culex-associated ISFV, secondary RNA structures with different conformations display numerous short ssRNA direct repeats, exposed as loops and bulges. Long quadruplicate regions comprise almost the entire 3'UTR of Culex-associated ISFV. Extended duplicated sequence and associated RNA structures were also discovered in the 3'UTR of pestiviruses. In both the Flavivirus and Pestivirus genera, duplicated RNA structures were localized to the enhancer regions of the 3'UTR suggesting an adaptive role predominantly in wild-type viruses. We propose sequence reiteration might act as a scaffold for dimerization of proteins involved in assembly of viral replicase complexes. Numerous nucleotide repeats exposed as loops/bulges might also interfere with host immune responses acting as a molecular sponge to sequester key host proteins or microRNAs.

Ecuador Paraiso Escondido virus, a new flavivirus isolated from New World sand flies in Ecuador, is the first representative of a novel clade in the genus flavivirus

A new flavivirus, Ecuador Paraiso Escondido virus (EPEV), named after the village where it was discovered, was isolated from sand flies (Psathyromyia abonnenci, formerly Lutzomyia abonnenci) that are unique to the New World. This represents the first sand fly-borne flavivirus identified in the New World. EPEV exhibited a typical flavivirus genome organization. Nevertheless, the maximum pairwise amino acid sequence identity with currently recognized flaviviruses was 52.8%. Phylogenetic analysis of the complete coding sequence showed that EPEV represents a distinct clade which diverged from a lineage that was ancestral to the nonvectored flaviviruses Entebbe bat virus, Yokose virus, and Sokoluk virus and also the Aedes-associated mosquito-borne flaviviruses, which include yellow fever virus, Sepik virus, Saboya virus, and others. EPEV replicated in C6/36 mosquito cells, yielding high infectious titers, but failed to reproduce either in vertebrate cell lines (Vero, BHK, SW13, and XTC cells) or in suckling mouse brains. This surprising result, which appears to eliminate an association with vertebrate hosts in the life cycle of EPEV, is discussed in the context of the evolutionary origins of EPEV in the New World.The flaviviruses are rarely (if ever) vectored by sand fly species, at least in the Old World. We have identified the first representative of a sand fly-associated flavivirus, Ecuador Paraiso Escondido virus (EPEV), in the New World. EPEV constitutes a novel clade according to current knowledge of the flaviviruses. Phylogenetic analysis of the virus genome showed that EPEV roots the Aedes-associated mosquito-borne flaviviruses, including yellow fever virus. In light of this new discovery, the New World origin of EPEV is discussed together with that of the other flaviviruses.

Esporos de Clostridium botulinum em mel comercializado no Estado de São Paulo e em outros Estados brasileiros

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Molecular and serological detection of Ehrlichia spp. in cats on São Luís Island, Maranhão, Brazil

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

Oocysts of Hepatozoon canis in Rhipicephalus (Boophilus) microplus collected from a naturally infected dog

Canine hepatozoonosis is a tick-borne disease caused by protozoans of the genus Hepatozoon. Several tick species have been implicated as potential vectors. Therefore, extensive studies are needed to determine the 'natural' endemic cycle of this parasite. This paper presents the first report of the presence of Hepatozoon canis oocysts in Rhipicephalus (Boophilus) microplus collected from an infected dog. (C) 2011 Elsevier B.V. All rights reserved.

Optimal and sub-optimal control in Dengue epidemics

This work concerns the application of the optimal control theory to Dengue epidemics. The dynamics of this insect-borne disease is modelled as a set of non-linear ordinary differential equations including the effect of educational campaigns organized to motivate the population to break the reproduction cycle of the mosquitoes by avoiding the accumulation of still water in open-air recipients. The cost functional is such that it reflects a compromise between actual financial spending (in insecticides and educational campaigns) and the population health (which can be objectively measured in terms of, for instance, treatment costs and loss of productivity). The optimal control problem is solved numerically using a multiple shooting method. However, the optimal control policy is difficult to implement by the health authorities because it is not practical to adjust the investment rate continuously in time. Therefore, a suboptimal control policy is computed assuming, as the admissible set, only those controls which are piecewise constant. The performance achieved by the optimal control and the sub-optimal control policies are compared with the cases of control using only insecticides when Breteau Index is greater or equal to 5 and the case of no-control. The results show that the sub-optimal policy yields a substantial reduction in the cost, in terms of the proposed functional, and is only slightly inferior to the optimal control policy. Copyright (C) 2001 John Wiley & Sons, Ltd.

Molecular characterization of Hepatozoon canis in dogs from Colombia

Hepatozoonosis is a tick-borne disease whose transmission to dogs occurs by ingestion of oocysts infected ticks or feeding on preys infested by infected ticks. Until now, there is no previous report of molecular characterization of Hepatozoon sp. in dogs from Colombia. EDTA blood samples were collected from 91 dogs from central-western region of Colombia (Bogota, Bucaramanga, and Villavicencio cities) and submitted to 18S rRNA Hepatozoon sp. PCR and blood smears confection. Phylogenetic analysis was used to access the identity of Hepatozoon species found in sampled dogs. From 91 sampled dogs, 29 (31.8%) were positive to Hepatozoon sp. (25 dogs were only positive in PCR, 1 was positive only in blood smears, and 3 were positive in both blood smears and PCR). After sequencing, the found Hepatozoon sp. DNA showed 100% of identity with Hepatozoon canis DNA isolates. The phylogenetic tree supported the identity of the found Hepatozoon sp. DNA, showing that the isolates from Colombia were placed in the same clade than other H. canis isolates from Venezuela, Spain, and Taiwan. This is the first molecular detection of H. canis in dogs from Colombia.

Amblyomma cajennense ticks induce immediate hypersensitivity in horses and donkeys

Since host immune reaction to ticks interferes with tick-borne pathogen transmission, it is important to recognize naturally occurring tick-host immune relationships to better understand the epidemiology of such infectious diseases. Amblyomma cajennense is an important tick-borne disease vector in the Neotropical region and horses maintain it in domestic environments. In the present work intradermal testing of A. cajennense tick exposed horses and donkeys using crude tick antigens was used to evaluate the type of hypersensitivity induced by infestations. Animals sensitized by A. cajennense infestation displayed an immediate hypersensitivity reaction at the antigen inoculation site. Foals sensitized with experimental infestations and field sensitized horses presented the most intense reactions (40% of ear thickness increase). Field sensitized donkeys presented less intense reaction reaching no more than 22% of mean thickness increase. Control horses (non-sensitized) had the least intense reaction, with a peak of no more 12% of increase. The presence of a prominent immediate hypersensitivity in equids sensitized experimentally or by field infestations indicates that A. cajennense ticks induce in this host an immune response that is associated with IgE production and which is known to be inappropriate against intracellular pathogens. Differences observed between horses and donkeys are discussed.

Avaliação sorológica de patógenos transmitidos por carrapatos em cães urbanos e rurais do estado do Paraná, Brasil

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Molecular and serological prevalence of Babesia bovis and Babesia bigemina in water buffaloes in the north region of Brazil

Bovine babesiosis is a tick-borne disease caused mainly by Babesia bovis and Babesia bigemina, which are associated to considerable economic losses in cattle herds worldwide. Approximately 60% of buffalo herds in South America are located in Northern Brazil. Little is known about the impact of babesiosis on buffalo herds in Brazil. The present work aimed to verify the occurrence of B. bovis and B. bigemina in 542 water buffaloes in the state of Pará, Northern Brazil, using molecular and serological techniques. The percentage of seropositive animals for B. bovis and B. bigemina was 41.2% and 19.0%, respectively, by ELISA. B. bovis and B. bigemina DNA were detected in 15 and 16% of sampled buffaloes, respectively. A high correlation (Kappa index of 0.9) between serological and molecular tests suggests that the combination of the utilized techniques in the present study is suitable for babesiosis diagnosis in an endemic unstable area. Significantly difference of positivity for serological and molecular assays was verified to localities and reproductive status of sampled animals, but not between buffalo breeds. The immune status of sampled buffaloes associated to the circulation of babesiosis agents in sampled population suggests that the studied area is at risk to clinical babesiosis outbreaks. Furthermore, this study demonstrated that this region can be classified as endemically unstable. © 2013 Elsevier B.V.

An assessment of the economic impact of climate change on the health sector In Jamaica

Climate change is a naturally occurring phenomenon in which the earth‘s climate goes through cycles of warming and cooling; these changes usually take place incrementally over millennia. Over the past century, there has been an anomalous increase in global temperature, giving rise to accelerated climate change. It is widely accepted that greenhouse gas emissions from human activities such as industries have contributed significantly to the increase in global temperatures. The existence and survival of all living organisms is predicated on the ability of the environment in which they live not only to provide conditions for their basic needs but also conditions suitable for growth and reproduction. Unabated climate change threatens the existence of biophysical and ecological systems on a planetary scale. The present study aims to examine the economic impact of climate change on health in Jamaica over the period 2011-2050. To this end, three disease conditions with known climate sensitivity and importance to Jamaican public health were modelled. These were: dengue fever, leptospirosis and gastroenteritis in children under age 5. Historical prevalence data on these diseases were obtained from the Ministry of Health Jamaica, the Caribbean Epidemiology Centre, the Climate Studies Group Mona, University of the West Indies Mona campus, and the Meteorological Service of Jamaica. Data obtained spanned a twelve-year period of 1995-2007. Monthly data were obtained for dengue and gastroenteritis, while for leptospirosis, the annual number of cases for 1995-2005 was utilized. The two SRES emission scenarios chosen were A2 and B2 using the European Centre Hamburg Model (ECHAM) global climate model to predict climate variables for these scenarios. A business as usual (BAU) scenario was developed using historical disease data for the period 2000-2009 (dengue fever and gastroenteritis) and 1995-2005 (leptospirosis) as the reference decades for the respective diseases. The BAU scenario examined the occurrence of the diseases in the absence of climate change. It assumed that the disease trend would remain unchanged over the projected period and the number of cases of disease for each decade would be the same as the reference decade. The model used in the present study utilized predictive empirical statistical modelling to extrapolate the climate/disease relationship in time, to estimate the number of climate change-related cases under future climate change scenarios. The study used a Poisson regression model that considered seasonality and lag effects to determine the best-fit model in relation to the diseases under consideration. Zhang and others (2008), in their review of climate change and the transmission of vector-borne diseases, found that: ―Besides climatic variables, few of them have included other factors that can affect the transmission of vector-borne disease….‖ (Zhang 2008) Water, sanitation and health expenditure are key determinants of health. In the draft of the second communication to IPCC, Jamaica noted the vulnerability of public health to climate change, including sanitation and access to water (MSJ/UNDP, 2009). Sanitation, which in its broadest context includes the removal of waste (excreta, solid, or other hazardous waste), is a predictor of vector-borne diseases (e.g. dengue fever), diarrhoeal diseases (such as gastroenteritis) and zoonoses (such as leptospirosis). In conceptualizing the model, an attempt was made to include non-climate predictors of these climate-sensitive diseases. The importance of sanitation and water access to the control of dengue, gastroenteritis and leptospirosis were included in the Poisson regression model. The Poisson regression model obtained was then used to predict the number of disease cases into the future (2011-2050) for each emission scenario. After projecting the number of cases, the cost associated with each scenario was calculated using four cost components. 1. Treatment cost morbidity estimate. The treatment cost for the number of cases was calculated using reference values found in the literature for each condition. The figures were derived from studies of the cost of treatment and represent ambulatory and non-fatal hospitalized care for dengue fever and gastroenteritis. Due to the paucity of published literature on the health care cost associated with leptospirosis, only the cost of diagnosis and antibiotic therapy were included in the calculation. 2. Mortality estimates. Mortality estimates are recorded as case fatality rates. Where local data were available, these were utilized. Where these were unavailable, appropriate reference values from the literature were used. 3. Productivity loss. Productivity loss was calculated using a human capital approach, by multiplying the expected number of productive days lost by the caregiver and/or the infected person, by GDP per capita per day (US$ 14) at 2008 GDP using 2008 US$ exchange rates. 4. No-option cost. The no-option cost refers to adaptation strategies for the control of dengue fever which are ongoing and already a part of the core functions of the Vector Control Division of the Ministry of Health, Jamaica. An estimated US$ 2.1 million is utilized each year in conducting activities to prevent the post-hurricane spread of vector borne diseases and diarrhoea. The cost includes public education, fogging, laboratory support, larvicidal activities and surveillance. This no-option cost was converted to per capita estimates, using population estimates for Jamaica up to 2050 obtained from the Statistical Institute of Jamaica (STATIN, 2006) and the assumption of one expected major hurricane per decade. During the decade 2000-2009, Jamaica had an average inflation of 10.4% (CIA Fact book, last updated May 2011). This average decadal inflation rate was applied to the no-option cost, which was inflated by 10% for each successive decade to adjust for changes in inflation over time.

Estudo quantitativo da infecção por Babesia bovis em bovinos de corte de diferentes grupos genéticos

Pós-graduação em Genética e Melhoramento Animal - FCAV

Identification of Novel Compounds Inhibiting Chikungunya Virus-Induced Cell Death by High Throughput Screening of a Kinase Inhibitor Library

Chikungunya virus (CHIKV) is a mosquito-borne arthrogenic alphavirus that causes acute febrile illness in humans accompanied by joint pains and in many cases, persistent arthralgia lasting weeks to years. The re-emergence of CHIKV has resulted in numerous outbreaks in the eastern hemisphere, and threatens to expand in the foreseeable future. Unfortunately, no effective treatment is currently available. The present study reports the use of resazurin in a cell-based high-throughput assay, and an image-based high-content assay to identify and characterize inhibitors of CHIKV-infection in vitro. CHIKV is a highly cytopathic virus that rapidly kills infected cells. Thus, cell viability of HuH-7 cells infected with CHIKV in the presence of compounds was determined by measuring metabolic reduction of resazurin to identify inhibitors of CHIKV-associated cell death. A kinase inhibitor library of 4,000 compounds was screened against CHIKV infection of HuH-7 cells using the resazurin reduction assay, and the cell toxicity was also measured in non-infected cells. Seventy-two compounds showing >= 50% inhibition property against CHIKV at 10 mu M were selected as primary hits. Four compounds having a benzofuran core scaffold (CND0335, CND0364, CND0366 and CND0415), one pyrrolopyridine (CND0545) and one thiazol-carboxamide (CND3514) inhibited CHIKV-associated cell death in a dose-dependent manner, with EC50 values between 2.2 mu M and 7.1 mu M. Based on image analysis, these 6 hit compounds did not inhibit CHIKV replication in the host cell. However, CHIKV-infected cells manifested less prominent apoptotic blebs typical of CHIKV cytopathic effect compared with the control infection. Moreover, treatment with these compounds reduced viral titers in the medium of CHIKV-infected cells by up to 100-fold. In conclusion, this cell-based high-throughput screening assay using resazurin, combined with the image-based high content assay approach identified compounds against CHIKV having a novel antiviral activity -inhibition of virus-induced CPE - likely by targeting kinases involved in apoptosis.

Low genetic diversity associated with low prevalence of Anaplasma marginale in water buffaloes in Marajo Island, Brazil

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Metodologia Microbiológica Convencional e Reação em Cadeia pela Polimerase (PCR) na detecção de Salmonella spp em carcaças de frango

Salmonella is the etiological agent responsible for one of the most important Food Borne Disease (FBD), Salmonellosis, which generates significant economic consequences in several countries, including Brazil. Poultry meat is one of the most important disseminators of the pathogen. Accordingly, several countries have developed programs trying to reduce the prevalence of Salmonella in poultry meat. Such programs are based on the research of the pathogen in the carcasses, establishing a maximum limit of positive samples at each set of analysis. The Salmonella scans are usually made using the conventional microbiological methods, which tend to be expensive and time consuming. In recent years were developed rapid methods such as polymerase chain reaction (PCR), which can greatly shorten the results time, showing greater sensitivity and specificity than conventional methodology