Transmission Potential of Rift Valley Fever virus over the course of the 2010 epidemic in South Africa

A Rift Valley fever (RVF) epidemic affecting animals on domestic livestock farms was reported in South Africa during January-August 2010. The first cases occurred after heavy rainfall, and the virus subsequently spread countrywide. To determine the possible effect of environmental conditions and vaccination on RVF virus transmissibility, we estimated the effective reproduction number (R) for the virus over the course of the epidemic by extending the Wallinga and Teunis algorithm with spatial information. Re reached its highest value in mid-February and fell below unity around mid-March, when vaccination coverage was 7.5%-45.7% and vector-suitable environmental conditions were maintained. The epidemic fade-out likely resulted first from the immunization of animals following natural infection or vaccination. The decline in vector-suitable environmental conditions from April onwards and further vaccination helped maintain R below unity. Increased availability of vaccine use data would enable evaluation of the effect of RVF vaccination campaigns.

Dengue Envelope Domain III-Peptide Binding Analysis via Tryptophan Fluorescence Quenching Assay

In the efforts to find an anti-viral treatment for dengue, a simple tryptophan fluorescence-screening assay aimed at identifying dengue domain III envelope (EIII) protein inhibitors was developed. Residue Trp391 of EIII was used as an intrinsic probe to monitor the change in fluorescence of the tryptophan residue upon binding to a peptide. The analysis was based on the electron excitation at 280 nm and fluorescence emission at 300–400 nm of EIII, followed by quenching of fluorescence in the presence of potential peptidic inhibitors coded DS36wt, DS36opt, DN58wt and DN58opt. The present study found that the fluorescence of the recombinant EIII was quenched following the binding of DS36opt, DN58wt and DN58opt ina concentration-dependent manner. Since the λmax for emission remained unchanged, the effect was not dueto a change in the environment of the tryptophan side chain. In contrast, a minimal fluorescence-quenching effect of DS36wt at 20 and 40 µM suggested that the DS36wt does not have any binding ability to EIII. This was supported by a simple native-page gel retardation assay that showed a band shift of EIII domain whenincubated with DS36opt, DN58wt and DN58opt but not with DS36wt. We thus developed a low-cost and convenientspectrophotometric binding assay for the analysis of EIII–peptide interactions in a drug screening application.

Dengue Virus Inhibition of Autophagic Flux and Dependency of Viral Replication on Proteasomal Degradation of the Autophagy Receptor p62

Autophagic flux involves formation of autophagosomes and their degradation by lysosomes. Autophagy can either promote or restrict viral replication. In the case of Dengue virus (DENV) several studies report that autophagy supports the viral replication cycle, and describe an increase of autophagic vesicles (AVs) following infection. However, it is unknown how autophagic flux is altered to result in increased AVs. To address this question, and gain insight into the role of autophagy during DENV infection, we established an unbiased, image-based flow cytometry approach to quantify autophagic flux under normal growth conditions and in response to activation by nutrient deprivation or the mTOR inhibitor Torin1. We found that DENV induced an initial activation of autophagic flux, followed by inhibition of general and specific autophagy. Early after infection, basal and activated autophagic flux was enhanced. However, during established replication, basal and Torin1-activated autophagic flux was blocked, while autophagic flux activated by nutrient deprivation was reduced, indicating a block to AV formation and reduced AV degradation capacity. During late infection AV levels increased as a result of inefficient fusion of autophagosomes with lysosomes. Additionally, endo-lysosomal trafficking was suppressed, while lysosomal activities were increased. We further determined that DENV infection progressively reduced levels of the autophagy receptor SQSTM1/p62 via proteasomal degradation. Importantly, stable over-expression of p62 significantly suppressed DENV replication suggesting a novel role for p62 as viral restriction factor. Overall our findings indicate that in the course of DENV infection, autophagy shifts from a supporting to an anti-viral role, which is countered by DENV.

IMPORTANCE: Autophagic flux is a dynamic process starting with the formation of autophagosomes and ending with their degradation after fusion with lysosomes. Autophagy impacts the replication cycle of many viruses. However, thus far the dynamics of autophagy in case of Dengue virus (DENV) infections has not been systematically quantified. Therefore, we employed high-content, imaging-based flow cytometry to quantify autophagic flux and endo-lysosomal trafficking in response to DENV infection. We report that DENV induced an initial activation of autophagic flux, followed by inhibition of general and specific autophagy. Further, lysosomal activity was increased, but endo-lysosomal trafficking was suppressed confirming the block of autophagic flux. Importantly, we provide evidence that p62, an autophagy receptor, restrict DENV replication and was specifically depleted in DENV-infected cells via increased proteasomal degradation. These results suggest that during DENV infection autophagy shifts from a pro- to an antiviral cellular process, which is counteracted by the virus.

Relapsing Fever Borreliae: A Global Review

Relapsing fever borreliae were notorious and feared infectious agents that earned their place in history through their devastating impact as causes of both epidemic and endemic infection. More recently they are considered more as an oddity and their burden of infection is largely overshadowed by other infections such as malaria, which presents in a similar clinical way. Despite this, they remain the most common bacterial infection in some developing countries. Transmitted by soft ticks or lice, these fascinating spirochaetes have evolved a myriad of mechanisms to survive within their diverse environments.

Quimioterapia da dengue: fármacos disponíveis e perspectivas de novas terapêuticas

Dissertação de mestrado, Ciências Farmacêuticas, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2015

Structural proteins of enveloped virus : from biochemistry to molecular medicine : a focus on dengue virus

Tese de doutoramento, Ciências Biomédicas (Bioquímica Médica), Universidade de Lisboa, Faculdade de Medicina, 2016

Relação entre as variáveis sociais, económicas e ambientais com o padrão da distribuição espaço-temporal dos casos de Dengue por munícipio no Brasil : de 2008 até 2012, utilizando o SOM

Atualmente, um dos principais desafios que afeta a saúde pública no Brasil é a crescente evolução no número de casos e epidemias provocados pelo vírus da dengue. Não existem estudos suficientes que consigam elucidar quais fatores contribuem para a evolução das epidemias de Dengue. Fatores como condições sanitárias, localização geográfica, investimentos financeiros em infraestrutura e qualidade de vida podem estar relacionados com a incidência de Dengue. Além disso, outra questão que merece um maior destaque é o estudo para se identificar o grau de impacto das variáveis determinantes da dengue e se existe um padrão que está correlacionado com a taxa de incidência. Desta forma, este trabalho tem como objetivo principal a correlação da taxa de incidência da dengue na população de cada município brasileiro, utilizando dados relativos aos aspectos sociais, econômicos, demográficos e ambientais. Outra contribuição relevante do trabalho, foi a análise dos padrões de distribuição espacial da taxa de incidência de Dengue e sua relação com os padrões encontrados utilizando as variáveis socioeconômicas e ambientais, sobretudo analisando a evolução temporal no período de 2008 até 2012. Para essa análises, utilizou-se o Sistema de Informação Geográfica (SIG) aliado com a mineração de dados, através da metodologia de rede neural mais especificamente o mapa auto organizável de Kohonen ou self-organizing maps (SOM). Tal metodologia foi empregada para a identificação de padrão de agrupamentos dessas variáveis e sua relação com as classes de incidência de dengue no Brasil (Alta, Média e Baixa). Assim, este projeto contribui de forma significativa para uma melhor compreensão dos fatores que estão associados à ocorrência de Dengue, e como essa doença está correlacionada com fatores como: meio ambiente, infraestrutura e localização no espaço geográfico.

Feasibility and clinical outcomes when using practice guidelines for evaluation of fever in returning travelers and migrants : a validation study.

BACKGROUND: Practice guidelines for examining febrile patients presenting upon returning from the tropics were developed to assist primary care physicians in decision making. Because of the low level of evidence available in this field, there was a need to validate them and assess their feasibility in the context they have been designed for. OBJECTIVES: The objectives of the study were to (1) evaluate physicians' adherence to recommendations; (2) investigate reasons for non-adherence; and (3) ensure good clinical outcome of patients, the ultimate goal being to improve the quality of the guidelines, in particular to tailor them for the needs of the target audience and population. METHODS: Physicians consulting the guidelines on the Internet (www.fevertravel.ch) were invited to participate in the study. Navigation through the decision chart was automatically recorded, including diagnostic tests performed, initial and final diagnoses, and clinical outcomes. The reasons for non-adherence were investigated and qualitative feedback was collected. RESULTS: A total of 539 physician/patient pairs were included in this study. Full adherence to guidelines was observed in 29% of the cases. Figure-specific adherence rate was 54.8%. The main reasons for non-adherence were as follows: no repetition of malaria tests (111/352) and no presumptive antibiotic treatment for febrile diarrhea (64/153) or abdominal pain without leukocytosis (46/101). Overall, 20% of diversions from guidelines were considered reasonable because there was an alternative presumptive diagnosis or the symptoms were mild, which means that the corrected adherence rate per case was 40.6% and corrected adherence per figure was 61.7%. No death was recorded and all complications could be attributed to the underlying illness rather than to adherence to guidelines. CONCLUSIONS: These guidelines proved to be feasible, useful, and leading to good clinical outcomes. Almost one third of physicians strictly adhered to the guidelines. Other physicians used the guidelines not to forget specific diagnoses but finally diverged from the proposed attitudes. These diversions should be scrutinized for further refinement of the guidelines to better fit to physician and patient needs.

Predicting adverse events in children with fever and chemotherapy-induced neutropenia: the prospective multicenter SPOG 2003 FN study.

PURPOSE To develop a score predicting the risk of adverse events (AEs) in pediatric patients with cancer who experience fever and neutropenia (FN) and to evaluate its performance. PATIENTS AND METHODS Pediatric patients with cancer presenting with FN induced by nonmyeloablative chemotherapy were observed in a prospective multicenter study. A score predicting the risk of future AEs (ie, serious medical complication, microbiologically defined infection, radiologically confirmed pneumonia) was developed from a multivariate mixed logistic regression model. Its cross-validated predictive performance was compared with that of published risk prediction rules. Results An AE was reported in 122 (29%) of 423 FN episodes. In 57 episodes (13%), the first AE was known only after reassessment after 8 to 24 hours of inpatient management. Predicting AE at reassessment was better than prediction at presentation with FN. A differential leukocyte count did not increase the predictive performance. The score predicting future AE in 358 episodes without known AE at reassessment used the following four variables: preceding chemotherapy more intensive than acute lymphoblastic leukemia maintenance (weight = 4), hemoglobin > or = 90 g/L (weight = 5), leukocyte count less than 0.3 G/L (weight = 3), and platelet count less than 50 G/L (weight = 3). A score (sum of weights) > or = 9 predicted future AEs. The cross-validated performance of this score exceeded the performance of published risk prediction rules. At an overall sensitivity of 92%, 35% of the episodes were classified as low risk, with a specificity of 45% and a negative predictive value of 93%. CONCLUSION This score, based on four routinely accessible characteristics, accurately identifies pediatric patients with cancer with FN at risk for AEs after reassessment.

Transmission of West Nile virus in the Niagara Region among a population at risk for exposure

Background. West Nile Virus (WNV), a mosquito-borne flavivirus, is one of an increasing number of infectious diseases that have been emerging or re-emerging in the last two decades. Since the arrival ofWNV to Canada to present date, the Niagara Region has only reported 30 clinical cases, a small number compared to the hundreds reported in other regions of similar conditions. Moreover, the last reported human case in Niagara was in 2006. As it has been demonstrated that the majority of WNV infections are asymptomatic, the question remains whether the lack of clinical cases in Niagara truly reflects the lack of transmission to humans or if infections are still occurring but are mostly asymptomatic. Objectives. The general objective of this study was to establish whether or not active WNV transmission could be detected in a human population residing in Niagara for the 2007 transmission season. To fullfil this objective, a cross-sectional seroprevalence study was designed to investigate for the presence of anti-WNV antibodies in a sample of Mexican migrant agricultural workers employed in farms registered with the Seasonal Agricultural Workers Program (SAWP). Due to the Mexican origin of the study participants, three specific research objectives were proposed: a) determine the seroprevalence ofanti-WNV antibodies as well as anti-Dengue virus antibodies (a closely related virus prevalent in Mexico and likely to confound WNV serology); b) analyze risk factors associated with WNV and Dengue virus seropositivity; and c) assess the awareness of study participants about WNV infection as well as their understanding of the mode of transmission and clinical importance of the infection. Methodology: After obtaining ethics clearance from Brock University, farms were visited and workers invited to participate. Due to time constraints, only a small number of farms were enrolled with a resulting convenience and non-randomized study sample. Workers' demographic and epidemiological data were collected using a standardized questionnaire and blood samples were drawn to determine serum anti-WNV and anti- Dengue antibodies with a commercial ELISA. All positive samples were sent to the National Microbiology Laboratory in Winnipeg, Manitoba for confirmation with the Plaque Reduction Neutralization Test (PRNT). Data was analyzed with Stata 10.0. Antibody determinations were reported as seroprevalence proportions for both WNV and Dengue. Logistic regression was used to analyze risk factors that may be associated with seropositivity and awareness was reported as a proportion of the number of individuals possessing awareness over the total number of participants. Results and Discussion. In total 92 participants working in 5 farms completed the study. Using the commercial ELISA, seropositivity was as follows: 2.2% for WNV IgM, 20.7% for WNV IgG, and 17.1 % for Dengue IgG. Possible cross-reactivity was demonstrated in 15/20 (75.0%) samples that were positive for both WNV IgG and Dengue IgG. Confirmatory testing with the PRNT demonstrated that none of the WNV ELISA positive samples had antibodies to WNV but 13 samples tested positive for anti-Dengue antibodies (14.1 % Dengue sereoprevalence). The findings showed that the ELISA performance was very poor for assessing anti-WNV antibodies in individuals previously exposed to Dengue virus. However, the ELISA had better sensitivity and specificity for assessing anti-Dengue antibodies. Whereas statistical analysis could not be done for WNV seropositivity, as all samples were PRNT negative, logistic regression demonstrated several risk factors for Dengue exposure_ The first year coming to Canada appeared to be significantly associated with increased exposure to Dengue while lower socio-economic housing and the presence of a water basin in the yard in Mexico appeared to be significantly associated with a decreased exposure to Dengue_ These seemingly contradictory results illustrate that in mobile populations such as migrant workers, risk factors for exposure to Dengue are not easily identified and more research is needed. Assessing the awareness of WNV and its clinical importance showed that only 23% of participants had some knowledge of WNV, of which 76% knew that the infection was mosquito-borne and 47% recognized fever as a symptom. The identified lack of understanding and awareness was not surprising since WNV is not a visible disease in Mexico. Since WNV persists in an enzootic cycle in Niagara and the occurrence of future outbreaks is unpredictable, the agricultural workers remain at risk for transmission. Therefore it important they receive sufficient health education regarding WNV before leaving Mexico and during their stay in Canada. Conclusions. Human transmission of WNV could not be proven among the study participants even when due to their occupation they are at high risk for mosquito bites. The limitations of the study sample do not permit generalizable conclusions, however, the study findings are consistent with the absence of clinical cases in the Niagara Region, so it is likely that human transmission is indeed neglible or absent. As evidenced by our WNV serology results, PRNT must be utilized as a confirmatory test since false positivity occurs frequently. This is especially true when previous exposure to Dengue virus is likely.

Respuesta funcional de Toxorhynchites theobaldi (DYAR y KNAB) sobre larvas de Aedes aegypti (LINN.) vector del dengue

Tesis (Maestría en Ciencias con Especialidad en Entomología Médica) UANL

Control del vector del dengue Aedes aegypti (l) con la participación de estudiantes de primaria como educadores de salud en la cuidad de Guadalupe Nuevo Léon

Tesis (Maestria en Ciencias con Especialidad en Entomología Médica) UANL

Comparación de la eficiencia de rociados domiciliares en ultra bajo volumen (ULV) y termonebulización para el control de poblaciones adultas del vector del dengue Aedes aegypti (L.) en una colonia de Guadalupe, Nuevo León, México

Tesis (Maestría en Ciencias con Especialidad en Entomología Médica) UANL

Análisis espacial y temporal de los casos de dengue en el área metropolitana de Monterrey, N.L., México, durante 1998

Tesis (Maestría en Ciencias con Especialidad en Entomología Médica) UANL