Monitoramento das Produções

Title: Standardization of Plaque Reduction Neutralization Test on 96-well Plates (micro-PRNT) for Zika Virus
Authors: Simões, Marisol; Santos, Vanessa de Oliveira; Linhares, José Henrique Rezende; Setatino, Bruno Pimenta; Silva, Stephanie Almeida da; Miranda, Emily Hime; Lima, Sheila Maria Barbosa de

Title: Recombinant ZIKV envelope proteins for the arbovirosis differential diagnosis by ELISA
Authors: Freitas, Juliana Torres de; Terra, Paulo de Alvarenga C.; Gonçalves, Jorge Luís dos Santos; Cavalcanti, Marta Guimarães; Castro, Mauro Jorge Cabral; Peralta, Regina Helena Saramago; Peralta, José Mauro

by Maria T. Arévalo, Ying Huang, Cheryl A. Jones, Ted M. Ross
Introduction Chikungunya virus (CHIKV) is a re-emerging pathogen responsible for causing outbreaks of febrile disease accompanied with debilitating joint pain. Symptoms typically persist for two weeks, but more severe and chronic chikungunya illnesses have been reported, especially in the elderly. Currently, there are no licensed vaccines or antivirals against CHIKV available. In this study, we combined a CHIK virus-like particle (VLP) vaccine with different adjuvants to enhance immunogenicity and protection in both, adult and aged mice. Methods CHIK VLP-based vaccines were tested in 6-8-week-old (adult) and 18-24-month-old (aged) female C57BL/6J mice. Formulations contained CHIK VLP alone or adjuvants: QuilA, R848, or Imject Alum. Mice were vaccinated three times via intramuscular injections. CHIKV-specific antibody responses were characterized by IgG subclass using ELISA, and by microneutralization assays. In addition, CHIKV infections were characterized in vaccinated and non-vaccinated adult mice and compared to aged mice. Results In adult mice, CHIKV infection of the right hind foot induced significant swelling, which peaked by day 7 post-infection at approximately 170% of initial size. Viral titers peaked at 2.53 × 1010 CCID50/ml on day 2 post-infection. Mice vaccinated with CHIK VLP-based vaccines developed robust anti-CHIKV-specific IgG antibody responses that were capable of neutralizing CHIKV in vitro. CHIK VLP alone or CHIK plus QuilA administered by IM injections protected 100% of mice against CHIKV. In contrast, the antibody responses elicited by the VLP-based vaccines were attenuated in aged mice, with negligible neutralizing antibody titers detected. Unvaccinated, aged mice were resistant to CHIKV infection, while vaccination with CHIKV VLPs exacerbated disease. Conclusions Unadjuvanted CHIK VLP vaccination elicits immune responses that protect 100% of adult mice against CHIKV infection. However, an improved vaccine/adjuvant combination is still necessary to enhance the protective immunity against CHIKV in the aged.

by Hongyan Ren, Wei Wu, Tiegang Li, Zhicong Yang
Background Numerous urban villages (UVs) and frequent infectious disease outbreaks are major environmental and public health concerns in highly urbanized regions, especially in developing countries. However, the spatial and quantitative associations between UVs and infections remain little understood on a fine scale. Methodology and principal findings In this study, the relationships between reported dengue fever (DF) epidemics during 2012–2017, gross domestic product (GDP), the traffic system (road density, bus and/or subway stations), and UVs derived from high-resolution remotely sensed imagery in the central area of Guangzhou, were explored using geographically weighted regression (GWR) models based on a 1 km × 1 km grid scale. Accounting for 16.53%–18.07% of residential area and 16.84%–18.02% of population, UVs possessed 28.55%–38.24% of total reported DF cases in the core area of Guangzhou. The density of DF cases and the DF incidence rates in UVs were 1.81–3.13 and 1.82–3.06 times of that of normal construction land. Approximately 90% of the total cases were concentrated in the UVs and their buffering zones of radius ranged from 0 to 500 m. Significantly positive associations were observed between gridded DF incidence rates and UV area (r = 0.33, P = 0.000), the number of bus stops (r = 0.49, P = 0.000) and subway stations (r = 0.27, P = 0.000), and road density (r = 0.39, P = 0.000). About 60% of spatial variations in the gridded DF incidence rates were interpreted by the different variables of GDP, UVs, and bus stops integrated in GWR models. Conclusions UVs likely acted as special transfer stations, receiving and/or exporting DF cases during epidemics. This work increases our understanding of the influences of UVs on vector-borne diseases in highly urbanized areas, supplying valuable clues to local authorities making targeted interventions for the prevention and control of DF epidemics.

by Kristal An Agrupis, Michelle Ylade, Josephine Aldaba, Anna Lena Lopez, Jacqueline Deen

Dengue is an important public health problem in the Philippines. We sought to describe the trends in dengue research in the country. We searched four databases and identified published studies on dengue research in the Philippines during the past 60 years. We reviewed 135 eligible studies, of which 33% were descriptive epidemiologic studies or case series, 16% were entomologic or vector control studies, 12% were studies on dengue virology and serologic response, 10% were socio-behavioral and economics studies, 8% were clinical trials, 7% were on burden of disease, 7% were investigations on markers of disease severity, 5% were on dengue diagnostics, and 2% were modeling studies. During the last decade, dengue research in the Philippines has increased and evolved from simple descriptive studies to those with more complex and diverse designs. We identified several key topics where more research would be useful.

by Uma Sangumathi Kamaraj, Jun Hao Tan, Ong Xin Mei, Louise Pan, Tanu Chawla, Anna Uehara, Lin-Fa Wang, Eng Eong Ooi, Duane J. Gubler, Hasitha Tissera, Lee Ching Ng, Annelies Wilder-Smith, Paola Florez de Sessions, Timothy Barkham, Danielle E. Anderson, October Michael Sessions

The frequency of epidemics caused by Dengue viruses 1–4, Zika virus and Chikungunya viruses have been on an upward trend in recent years driven primarily by uncontrolled urbanization, mobility of human populations and geographical spread of their shared vectors, Aedes aegypti and Aedes albopictus. Infections by these viruses present with similar clinical manifestations making them challenging to diagnose; this is especially difficult in regions of the world hyperendemic for these viruses. In this study, we present a targeted-enrichment methodology to simultaneously sequence the complete viral genomes for each of these viruses directly from clinical samples. Additionally, we have also developed a customized computational tool (BaitMaker) to design these enrichment baits. This methodology is robust in its ability to capture diverse sequences and is amenable to large-scale epidemiological studies. We have applied this methodology to two large cohorts: a febrile study based in Colombo, Sri Lanka taken during the 2009–2015 dengue epidemic (n = 170) and another taken during the 2016 outbreak of Zika virus in Singapore (n = 162). Results from these studies indicate that we were able to cover an average of 97.04% ± 0.67% of the full viral genome from samples in these cohorts. We also show detection of one DENV3/ZIKV co-infected patient where we recovered full genomes for both viruses.

by Mikhail Churakov, Christian J. Villabona-Arenas, Moritz U. G. Kraemer, Henrik Salje, Simon Cauchemez

Dengue continues to be the most important vector-borne viral disease globally and in Brazil, where more than 1.4 million cases and over 500 deaths were reported in 2016. Mosquito control programmes and other interventions have not stopped the alarming trend of increasingly large epidemics in the past few years. Here, we analyzed monthly dengue cases reported in Brazil between 2001 and 2016 to better characterise the key drivers of dengue epidemics. Spatio-temporal analysis revealed recurring travelling waves of disease occurrence. Using wavelet methods, we characterised the average seasonal pattern of dengue in Brazil, which starts in the western states of Acre and Rondônia, then travels eastward to the coast before reaching the northeast of the country. Only two states in the north of Brazil (Roraima and Amapá) did not follow the countrywide pattern and had inconsistent timing of dengue epidemics throughout the study period. We also explored epidemic synchrony and timing of annual dengue cycles in Brazilian regions. Using gravity style models combined with climate factors, we showed that both human mobility and vector ecology contribute to spatial patterns of dengue occurrence. This study offers a characterization of the spatial dynamics of dengue in Brazil and its drivers, which could inform intervention strategies against dengue and other arboviruses.

by Perran A. Ross, Scott A. Ritchie, Jason K. Axford, Ary A. Hoffmann

Wolbachia bacteria are now being introduced into Aedes aegypti mosquito populations for dengue control. When Wolbachia infections are at a high frequency, they influence the local transmission of dengue by direct virus blocking as well as deleterious effects on vector mosquito populations. However, the effectiveness of this strategy could be influenced by environmental temperatures that decrease Wolbachia density, thereby reducing the ability of Wolbachia to invade and persist in the population and block viruses. We reared wMel-infected Ae. aegypti larvae in the field during the wet season in Cairns, North Queensland. Containers placed in the shade produced mosquitoes with a high Wolbachia density and little impact on cytoplasmic incompatibility. However, in 50% shade where temperatures reached 39°C during the day, wMel-infected males partially lost their ability to induce cytoplasmic incompatibility and females had greatly reduced egg hatch when crossed to infected males. In a second experiment under somewhat hotter conditions (>40°C in 50% shade), field-reared wMel-infected females had their egg hatch reduced to 25% when crossed to field-reared wMel-infected males. Wolbachia density was reduced in 50% shade for both sexes in both experiments, with some mosquitoes cleared of their Wolbachia infections entirely. To investigate the critical temperature range for the loss of Wolbachia infections, we held Ae. aegypti eggs in thermocyclers for one week at a range of cyclical temperatures. Adult wMel density declined when eggs were held at 26–36°C or above with complete loss at 30–40°C, while the density of wAlbB remained high until temperatures were lethal. These findings suggest that high temperature effects on Wolbachia are potentially substantial when breeding containers are exposed to partial sunlight but not shade. Heat stress could reduce the ability of Wolbachia infections to invade mosquito populations in some locations and may compromise the ability of Wolbachia to block virus transmission in the field. Temperature effects may also have an ecological impact on mosquito populations given that a proportion of the population becomes self-incompatible.

Title: Duration of immunogenicity after 17-DD Yellow Fever vaccine in adults and children
Authors: Noronha, Tatiana Guimarães de; Maia, Maria de Lourdes de Sousa; Camacho, Luiz Antônio Bastos

by Nazish Badar, Muhammad Salman, Jamil Ansari, Aamer Ikram, Javaria Qazi, Muhammad Masroor Alam

Title: Reduction of the cycle of lyophilization of the vaccine atenuated Yellow Fever presentation 02 and 05 doses of Bio-Manguinhos
Authors: Agra, Patrícia Alvarenga; Assumpção, Sergio Luiz de Lima; Crespo, Celso de Farias; Calado, Verônica

by Anna S. Jaeger, Reyes A. Murrieta, Lea R. Goren, Chelsea M. Crooks, Ryan V. Moriarty, Andrea M. Weiler, Sierra Rybarczyk, Matthew R. Semler, Christopher Huffman, Andres Mejia, Heather A. Simmons, Michael Fritsch, Jorge E. Osorio, Jens C. Eickhoff, Shelby L. O’Connor, Gregory D. Ebel, Thomas C. Friedrich, Matthew T. Aliota

Congenital Zika virus (ZIKV) infection was first linked to birth defects during the American outbreak in 2015/2016. It has been proposed that mutations unique to the Asian/American-genotype explain, at least in part, the ability of Asian/American ZIKV to cause congenital Zika syndrome (CZS). Recent studies identified mutations in ZIKV infecting humans that arose coincident with the outbreak in French Polynesia and were stably maintained during subsequent spread to the Americas. Here we show that African ZIKV can infect and harm fetuses and that the S139N substitution that has been associated with the American outbreak is not essential for fetal harm. Our findings, in a vertical transmission mouse model, suggest that ZIKV will remain a threat to pregnant women for the foreseeable future, including in Africa, Southeast Asia, and the Americas. Additional research is needed to better understand the risks associated with ZIKV infection during pregnancy, both in areas where the virus is newly endemic and where it has been circulating for decades.

by Catherine A. Lippi, Anna M. Stewart-Ibarra, M. E. Franklin Bajaña Loor, Jose E. Dueñas Zambrano, Nelson A. Espinoza Lopez, Jason K. Blackburn, Sadie J. Ryan

Arboviral disease transmission by Aedes mosquitoes poses a major challenge to public health systems in Ecuador, where constraints on health services and resource allocation call for spatially informed management decisions. Employing a unique dataset of larval occurrence records provided by the Ecuadorian Ministry of Health, we used ecological niche models (ENMs) to estimate the current geographic distribution of Aedes aegypti in Ecuador, using mosquito presence as a proxy for risk of disease transmission. ENMs built with the Genetic Algorithm for Rule-Set Production (GARP) algorithm and a suite of environmental variables were assessed for agreement and accuracy. The top model of larval mosquito presence was projected to the year 2050 under various combinations of greenhouse gas emissions scenarios and models of climate change. Under current climatic conditions, larval mosquitoes were not predicted in areas of high elevation in Ecuador, such as the Andes mountain range, as well as the eastern portion of the Amazon basin. However, all models projected to scenarios of future climate change demonstrated potential shifts in mosquito distribution, wherein range contractions were seen throughout most of eastern Ecuador, and areas of transitional elevation became suitable for mosquito presence. Encroachment of Ae. aegypti into mountainous terrain was estimated to affect up to 4,215 km2 under the most extreme scenario of climate change, an area which would put over 12,000 people currently living in transitional areas at risk. This distributional shift into communities at higher elevations indicates an area of concern for public health agencies, as targeted interventions may be needed to protect vulnerable populations with limited prior exposure to mosquito-borne diseases. Ultimately, the results of this study serve as a tool for informing public health policy and mosquito abatement strategies in Ecuador.

by Aditya Lia Ramadona, Yesim Tozan, Lutfan Lazuardi, Joacim Rocklöv

Only a few studies have investigated the potential of using geotagged social media data for predicting the patterns of spatio-temporal spread of vector-borne diseases. We herein demonstrated the role of human mobility in the intra-urban spread of dengue by weighting local incidence data with geo-tagged Twitter data as a proxy for human mobility across 45 neighborhoods in Yogyakarta city, Indonesia. To estimate the dengue virus importation pressure in each study neighborhood monthly, we developed an algorithm to estimate a dynamic mobility-weighted incidence index (MI), which quantifies the level of exposure to virus importation in any given neighborhood. Using a Bayesian spatio-temporal regression model, we estimated the coefficients and predictiveness of the MI index for lags up to 6 months. Specifically, we used a Poisson regression model with an unstructured spatial covariance matrix. We compared the predictability of the MI index to that of the dengue incidence rate over the preceding months in the same neighborhood (autocorrelation) and that of the mobility information alone. We based our estimates on a volume of 1·302·405 geotagged tweets (from 118·114 unique users) and monthly dengue incidence data for the 45 study neighborhoods in Yogyakarta city over the period from August 2016 to June 2018. The MI index, as a standalone variable, had the highest explanatory power for predicting dengue transmission risk in the study neighborhoods, with the greatest predictive ability at a 3-months lead time. The MI index was a better predictor of the dengue risk in a neighborhood than the recent transmission patterns in the same neighborhood, or just the mobility patterns between neighborhoods. Our results suggest that human mobility is an important driver of the spread of dengue within cities when combined with information on local circulation of the dengue virus. The geotagged Twitter data can provide important information on human mobility patterns to improve our understanding of the direction and the risk of spread of diseases, such as dengue. The proposed MI index together with traditional data sources can provide useful information for the development of more accurate and efficient early warning and response systems.

by Ju-Mei Wang, Yang Cheng, Zuo-Kun Shi, Xiao-Feng Li, Long-Sheng Xing, Hong Jiang, Dan Wen, Yong-Qiang Deng, Ai-Hua Zheng, Cheng-Feng Qin, Zhen Zou

Mosquitoes act as vectors of numerous pathogens that cause human diseases. Dengue virus (DENV) transmitted by mosquito, Aedes aegypti, is responsible for dengue fever epidemics worldwide with a serious impact on human health. Currently, disease control mainly relies on vector targeted intervention strategies. Therefore, it is imperative to understand the molecular mechanisms underlying the innate immune response of mosquitoes against pathogens. In the present study, the expression profiles of immunity-related genes in the midgut responding to DENV infection by feeding were analyzed by transcriptome and quantitative real-time PCR. The level of Antimicrobial peptides (AMPs) increased seven days post-infection (d.p.i.), which could be induced by the Toll immune pathway. The expression of reactive oxygen species (ROS) genes, including antioxidant genes, such as HPX7, HPX8A, HPX8B, HPX8C were induced at one d.p.i. and peaked again at ten d.p.i. in the midgut. Interestingly, down-regulation of the antioxidant gene HPX8C by RNA interference led to reduction in the virus titer in the mosquito, probably due to the elevated levels of ROS. Application of a ROS inhibitor and scavenger molecules further established the role of oxygen free radicals in the modulation of the immune response to DENV infection. Overall, our comparative transcriptome analyses provide valuable information about the regulation of immunity related genes in the transmission vector in response to DENV infection. It further allows us to identify novel molecular mechanisms underlying the host-virus interaction, which might aid in the development of novel strategies to control mosquito-borne diseases.

by Pabitra Saha, Moytrey Chatterjee, Sudeep Ballav, Akash Chowdhury, Nandita Basu, Ardhendu Kumar Maji
Background Aedes albopictus and Aedes aegypti are the major vectors of arboviral diseases. As effective vaccines are not available for most of the arboviral diseases, vector control by using insecticides play the key role to reduce the disease transmission. The emergence and spread of resistance to different classes of insecticides by the vectors is a major obstacle to control the disease transmission. Information about vector susceptibility to different insecticides and their mechanisms are very important for formulating proper vector control measures. The present study was designed to assess the susceptibility of Ae. aegypti against three different classes of adulticides, one larvicidal agent available and polymorphisms in the voltage-gated sodium channel (VGSC) gene related to insecticide resistance. Methods Immature stages of Ae. aegypti were collected from three dengue endemic municipal areas of West Bengal and reared in the laboratory. Larvae and adults (F1 progeny) were used for insecticide bioassay as per WHO protocols. Knock down resistance gene (kdr) mutations were assessed by direct sequencing of PCR products. Results The Ae. aegypti population was found to be susceptible to type II pyrethroids and malathion but highly resistant to DDT. A high rate of polymorphisms in the VGSC gene was observed among the collected mosquitoes. A double mutant V1016G + F1534C was found to be associated with DDT resistance but neither V1016G nor F1534C alone showed the same association. Association between the kdr mutations and the susceptibility status of pyrethroids could not be established due to very small sample size. A low to moderate level of resistance was noticed against temephos among the larval population based on WHO criteria. Conclusion The replacement of DDT by type II pyrethroids for the management of dengue vectors is an appropriate decision taken by the national program which is supported by the findings of a higher level of resistance to DDT. Persistence of polymorphisms in the VGSC gene might be an indication of emergence of resistance against pyrethroid insecticides that should be monitored at a regular interval. Attempts should be made to determine the effectiveness of other larvicides for replacement of temephos if needed in future. Along with the chemical insecticides different biological vector control methods as well as biopesticides should also be used in vector control programmes.

Title: Incidence rates of neurotropic-like and viscerotropic-like disease in three dengue-endemic countries: Mexico, Brazil, and Malaysia
Authors: Cohen, Carine; Moreira Junior, Edson Duarte; Nañez, Homero; Nachiappan, Jeyaseelan P; Arvinder-Singh, H S; Huoi, Catherine; Nealon, Joshua; Sarti, Elsa; Rosas, Esteban Puentes; Moureau, Annick; Khromava, Alena

Title: Estudo da resposta imune inata celular in vitro contra Dengue virus e Zika virus
Authors: Pessoa, Natália Lima
Abstract: Dengue virus (DENV) e Zika virus (ZIKV) pertencem ao gênero Flavivirus da família Flaviviridae. São arbovirus transmitidos por mosquitos do gênero Aedes, porém, outros modos de transmissão vêm sendo relatados para ZIKV, como a transmissão vertical e sexual. DENV possui quatro sorotipos distintos (DENV 1 - 4), cada um com vários genótipos e todos são capazes de causar a dengue não grave, com e sem sinais de alarme, e a dengue grave. O ZIKV possui duas linhagens, a asiática e a africana. Já foi comprovado que a infecção tanto por DENV como por ZIKV podem causar complicações neurológicas graves. E, recentemente, ZIKV vem sendo relacionado com microcefalia e Síndrome de Guillain-Barre. Como somos um país endêmico para Flavivirus, o primeiro passo do trabalho foi comprovar que as amostras de ZIKV recebidas por nossa instituição eram realmente este vírus, e não havia coinfecção com DENV. Para isto, foi feita PCR em tempo real com iniciadores específicos para ZIKV e DENV e também nested PCR com iniciadores para DENV, demonstrando que o ZIKV foi positivo na PCR em tempo real e foi negativo para DENV pelo nested PCR. O objetivo principal desta dissertação foi comparar a resposta imune celular desencadeada por dois isolados distintos de DENV 3, um neurotrópico (genótipo I) e outro não neurotrópico (genótipo III). E também comparar a resposta imune desencadeada por dois isolados brasileiros de ZIKV (PE243 e SPH). O estudo a resposta imune desencadeada pelos vírus foi feito através da quantificação da produção de óxido nítrico e de diferentes citocinas, avaliando a susceptibilidade à infecção e multiplicação viral das diferentes células utilizadas neste trabalho e avaliando a ativação de TLR2 e/ou TLR4. Assim, macrófagos intraperitoneais de camundongos C57BL/6, células KG-1 e THP-1 foram infectadas com DENV e ZIKV, e os sobrenadantes destas células foram coletados em diferentes dias após a infecção. Os sobrenadantes foram analisados por reação de Griess, para a quantificação do óxido nítrico produzido pelas células e por CBA para a quantificação das citocinas. Também foi avaliado se estas células foram susceptíveis à infecção dos vírus, por titulação. Nas células KG-1, foi detectado DENV 3 genótipo I até o 10° d.p.i. e ZIKV até o 5º d.p.i., com títulos mais altos em comparação com os macrófagos murinos. Não foi detectado DENV 3 genótipo III nas células KG-1. Como só foram detectados virus nos 1º e 2º d.p.i. nos macrófagos intraperitoneais de camundongo, é possível sugerir que os vírus foram inibidos pela capacidade destas células de produzir uma resposta imune inata eficaz contra a infecção por DENV e por ZIKV. Foi demonstrado que o NO e o IFNγ são importantes no controle da infecção por DENV 3. E que as citocinas TNF, IL-6 e a quimiocina MCP-1 estão relacionadas à infecção pelo sorotipo neurovirulento. E ainda, a ativação de TLR2 também é maior quando as células são infectadas pelo genótipo neurovirulento. Portanto, a resposta imune desencadeada pelo genótipo neurovirulento foi diferente em comparação com a resposta imune desencadeada pelo genótipo não neurovirulento. Também foi demonstrado que os dois isolados de ZIKV utilizados desencadeiam respostas imunes completamente diferentes, sendo que a amostra ZIKV PE243 desencadeou uma resposta imune mais forte, com produção de NO, maior produção das citocinas e ativação de TLR2 enquanto que a resposta imune desencadeada por ZIKV SPH foi mais branda, sem produção de NO, com menor produção de citocinas e sem a ativação de TLR2.

by Leon E. Hugo, Liesel Stassen, Jessica La, Edward Gosden, O’mezie Ekwudu, Clay Winterford, Elvina Viennet, Helen M. Faddy, Gregor J. Devine, Francesca D. Frentiu
Background Recent epidemics of Zika virus (ZIKV) in the Pacific and the Americas have highlighted its potential as an emerging pathogen of global importance. Both Aedes (Ae.) aegypti and Ae. albopictus are known to transmit ZIKV but variable vector competence has been observed between mosquito populations from different geographical regions and different virus strains. Since Australia remains at risk of ZIKV introduction, we evaluated the vector competence of local Ae. aegypti and Ae. albopictus for a Brazilian epidemic ZIKV strain. In addition, we evaluated the impact of daily temperature fluctuations around a mean of 28°C on ZIKV transmission and extrinsic incubation period. Methodology/Principal findings Mosquitoes were orally challenged with a Brazilian ZIKV strain (8.8 log CCID50/ml) and maintained at either 28°C constant or fluctuating temperature conditions. At 3, 7 and 14 days post-infection (dpi), ZIKV RNA copies were quantified in mosquito bodies, as well as wings and legs, using qRT-PCR, while virus antigen in saliva (a proxy for transmission) was detected using a cell culture ELISA. Despite high body and disseminated infection rates in both vectors, the transmission rates of ZIKV in saliva of Ae. aegypti (50–60%) were significantly higher than in Ae. albopictus (10%) at 14 dpi. Both species supported a high viral load in bodies, with no significant differences between constant and fluctuating temperature conditions. However, a significant difference in viral load in wings and legs between species was observed, with higher titres in Ae. aegypti maintained at constant temperature conditions. For ZIKV transmission to occur in Ae. aegypti, a disseminated virus load threshold of 7.59 log10 copies had to be reached. Conclusions/Significance Australian Ae. aegypti are better able to transmit a Brazilian ZIKV strain than Ae. albopictus. The results are in agreement with the global consensus that Ae. aegypti is the major vector of ZIKV.

Title: TLR expression and NK cell activation after human yellow fever vaccination
Authors: Neves, Patrícia Cristina da Costa; Matos, Denise Cristina de Souza; Marcovistz, Rugimar; Galler, Ricardo

by Razvan C. Stan, Katia S. Françoso, Rubens P. S. Alves, Luís Carlos S. Ferreira, Irene S. Soares, Maristela M. de Camargo

Fever is a regulated increase of the body temperature resulting from both infectious and non-infectious causes. Fever is known to play a role in modulating immune responses to infection, but the potential of febrile temperatures in regulating antigen binding affinity to antibodies has not been explored. Here we investigated this process under in vitro conditions using Isothermal titration calorimetry and ELISA. We used selected malarial and dengue antigens against specific monoclonal antibodies, and observed a marked increase in the affinity of these antibody-antigen complexes at 40°C, compared to physiological (37°C) or pathophysiological temperatures (42°C). Induced thermal equilibration of the protein partners at these temperatures in vitro, prior to measurements, further increased their binding affinity. These results suggest another positive and adaptive role for fever in vivo, and highlight the favourable role of thermal priming in enhancing protein-protein affinity for samples with limited availability.

Title: Molecular and phenotypic analysis of a working seed lot of yellow fever virus 17DD vaccine strain produced from the secondary seed lot 102/84 with an additional passage in chicken embryos
Authors: Marchevsky, Renato S.; Leal, Maria da Luz; Homma, Akira; Coutinho, Evandro S. F.; Camacho, Luis A. B.; Jabor, Alfredo V.; Galler, Ricardo; Freire, Marcos S.

by Han-Hsuan Chung, I-Cheng Cheng, Yen-Chi Chen, Cheo Lin, Takashi Tomita, Hwa-Jen Teng
Background Knockdown resistance (kdr) to dichlorodiphenyltrichloroethane (DDT) and pyrethroids is known to link amino acid substitutions in the voltage-gated sodium channel (VGSC) in Aedes aegypti. Dengue fever primarily transmitted by Ae. aegypti is an annual public health issue in Taiwan. Accordingly, pyrethroid insecticides have been heavily used for decades to control mosquito populations in the summer and autumn. In Taiwan, an Ae. aegypti population with two VGSC mutations, V1016G and D1763Y, was described previously. Methodology/Principal finding Aedes aegypti (G0) were collected in Tainan and Kaohsiung in southern Taiwan. The VGSC gene polymorphisms of the kdr mutations and the intron flanked by exons 20 and 21 were verified. The first generation offspring (G1) were used to measure the resistance level to cypermethrin, a pyrethroid insecticide currently used in Taiwan. In addition to V1016G and D1763Y, we describe two new mutations, S989P and F1534C, which have not been reported in Taiwan. Moreover, we also identify two types (groups A and B) of introns between exons 20 and 21. Intriguingly, the kdr mutations S989P, V1016G and D1763Y are strictly located on the haplotype harboring the group A intron, whereas F1534C links to the group B intron. When those data were taken together, we proposed the following six haplotypes for VGSC genes in Taiwan today: (i)S989-intron A-V1016-F1534-D1763, (ii)S989-intron A-V1016G-F1534-D1763, (iii)S989P-intron A-V1016G-F1534-D1763, (iv)S989-intron A-V1016G-F1534-D1763Y, (v)S989-intron B-V1016-F1534-D1763 and (vi)S989-intron B-V1016-F1534C-D1763. Triple heterozygous mutations of either S989P/V1016G/F1534C or V1016G/F1534C/D1763Y can be found in one single Ae. aegypti mosquito. The proportions of the VGSC mutations were relevant to cypermethrin resistance. Notably, the presence of S989P and V1016G in the population could be a helpful reference to predict the resistance level to cypermethrin. This is the first study to demonstrate the coexistence of four kdr mutations in a population of Ae. aegypti. Conclusions/Significance Four kdr mutations (S989P, V1016G, F1534C and D1763Y) and two intron forms (Group A and B) were commonly found in local Ae. aegypti populations in Taiwan.

Title: Transmissão, vigilância, controle e prevenção da Febre Amarela - 2018A
Authors: Fundação Oswaldo Cruz. Vice-presidência de Ensino, Informação e Comunicação
Abstract: Apresenta os conceitos gerais sobre febre amarela, sendo indicado para os profissionais de saúde que atuam na atenção básica e em postos de vacinação. Mas é aberto para qualquer pessoa interessada. Ao final desse minicurso, o participante vai ser capaz de: reconhecer áreas de risco de transmissão da doença; identificar ciclos urbanos e silvestres; rever procedimentos de vigilância; definir os diferentes tipos de caso de febre amarela nos diferentes cenários epidemiológicos; encontrar informações atualizadas sobre a situação epidemiológica da doença e rever as medidas gerais de controle da febre amarela e formas de prevenção adicionais à vacina.

by Nicole L. Achee, John P. Grieco, Hassan Vatandoost, Gonçalo Seixas, Joao Pinto, Lee Ching-NG, Ademir J. Martins, Waraporn Juntarajumnong, Vincent Corbel, Clement Gouagna, Jean-Philippe David, James G. Logan, James Orsborne, Eric Marois, Gregor J. Devine, John Vontas

Title: Neurovirulence of yellow fever 17DD vaccine virus to rhesus monkeys
Authors: Marchevsky, Renato S.; Freire, Marcos S.; Coutinho, Evandro S. F.; Galler, Ricardo

Title: Yellow fever
Authors: Monath, Thomas P.; Vasconcelos, Pedro F. C.

Title: The role of amniocentesis in the diagnosis of congenital zika syndrome
Authors: Pereira Jr., José Paulo; Maykin, Melanie M.; Vasconcelos, Zilton; Avvad-Portari, Elyzabeth; Zin, Andrea A.; Tsui, Irena; Brasil, Patricia; Nielsen-Saines, Karin; Moreira, Maria E.; Gaw, Stephanie L.

by Jih-Jin Tsai, Wei-Liang Liu, Ping-Chang Lin, Bo-Yi Huang, Ching-Yi Tsai, Pin-Hsing Chou, Fu-Chun Lee, Chia-Fong Ping, Pei-Yu Alison Lee, Li-Teh Liu, Chun-Hong Chen
Background Dengue fever, a mosquito-borne disease, is caused by dengue virus (DENV) which includes four major serotypes (DENV-1, -2, -3, and -4). Some serotypes cause more severe diseases than the other; severe dengue is associated with secondary infections by a different serotype. Timely serotyping can provide early warning of dengue epidemics to improve management of patients and outbreaks. A mobile insulated isothermal PCR (iiPCR) system is available to allow molecular detection of pathogens near points of need. Methodology/Principle findings In this study, side-by-side comparison with the CDC DENV-1-4 Real Time RT-PCR (qRT-PCR) was performed to evaluate the performance of four singleplex DENV-1–4 serotyping reverse transcription-iiPCR (RT-iiPCR) reagents for DENV subtyping on the mobile PCR system. The four RT-iiPCRs did not react with Zika virus and chikungunya virus; tests with serial dilutions of the four DENV serotypes made in human serum showed they had detection endpoints comparable to those of the reference method, indicating great analytical sensitivity and specificity. Clinical performance of the RT-iiPCR reagents was evaluated by testing 40 serum samples each (around 20 target serotype-positive and 20 DENV-negative); all four reagents had high agreement (97.5–100%) with the reference qRT-PCR. Moreover, testing of mosquitoes separately infected experimentally with each serotype showed that the four reagents detected specifically their target DENV serotypes in mosquito. Conclusions/Significance With analytical and clinical performance comparable to the reference qRT-PCR assay, the four index RT-iiPCR reagents on the field-deployable PCR system can serve as a useful tool for DENV detection near points of needs.

by Damariz Marin-Palma, Cherilyn M. Sirois, Silvio Urcuqui-Inchima, Juan C. Hernandez
Introduction The triggering of severe dengue has been associated with an exacerbated inflammatory process characterized by the production of pro-inflammatory cytokines such as IL-1β/IL-18, which are the product of inflammasome activation. Furthermore, alteration in the levels of high-density (HDL) and low-density lipoproteins (LDL) has been observed; and HDL are known to have immunomodulatory properties, including the regulation of inflammasomes. While HDL would be expected to counteract hyperactivation of the inflammasome, the relationship between HDL and dengue severity, has not previously been explored. Methodology We conducted a cross-sectional study of 30 patients with dengue and 39 healthy controls matched by sex and age. Lipid profile and levels of C-reactive protein were quantified. Serum levels of IL-1β, IL-6, IL-10, IL-18, and TNF-α, were assessed by ELISA. Expression of inflammasome-related genes in PBMC was quantified by qPCR. Results Dengue patients presented an alteration in the parameters of the lipid profile, with a significant decrease in HDL levels, which was more pronounced in dengue patients with warning signs. Moreover, a decrease in the expression of the inflammasome-related genes NLRP1, NLRC4, caspase-1, IL-1β and IL-18 was observed, as well as an increase in serum levels of C-reactive protein and IL-10 in dengue patients versus healthy donors. Significant positive correlations between LDL levels and the relative expression of NLRP3, NLRC4, IL-1β and IL-18, were found. Conclusion The results suggest that there is a relationship between the alteration of LDL and HDL with the imbalance in the inflammatory response, which could be associated with the severity of dengue.