Monitoramento das Produções

by Elena Lorente, Alejandro Barriga, Eilon Barnea, Concepción Palomo, Juan García-Arriaza, Carmen Mir, Mariano Esteban, Arie Admon, Daniel López
Background Efficient adaptive antiviral cellular and humoral immune responses require previous recognition of viral antigenic peptides bound to human leukocyte antigen (HLA) class I and II molecules, which are exposed on the surface of infected and antigen presenting cells, respectively. The HLA-restricted immune response to Chikungunya virus (CHIKV), a mosquito-borne Alphavirus of the Togaviridae family responsible for severe chronic polyarthralgia and polyarthritis, is largely unknown. Methodology/Principal findings In this study, a high-throughput mass spectrometry analysis of complex HLA-bound peptide pools isolated from large amounts of human cells infected with a vaccinia virus (VACV) recombinant expressing CHIKV structural proteins was carried out. Twelve viral ligands from the CHIKV polyprotein naturally presented by different HLA-A, -B, and -C class I, and HLA-DR and -DP class II molecules were identified. Conclusions/Significance The immunoprevalence of the HLA class II but not the HLA class I-restricted cellular immune response against the CHIKV structural polyprotein was greater than that against the VACV vector itself. In addition, most of the CHIKV HLA class I and II ligands detected by mass spectrometry are not conserved compared to its closely related O'nyong-nyong virus. These findings have clear implications for analysis of both cytotoxic and helper immune responses against CHIKV as well as for the future studies focused in the exacerbated T helper response linked to chronic musculoskeletal disorders in CHIKV patients.

by Lu Liang, Pengfei Jia, Xiaoyue Tan, Jin Chen, Xiang Chen

Extreme weather events affect the development and survival of disease pathogens and vectors. Our aim was to investigate the potential effects of heat waves on the population dynamics of Asian tiger mosquito (Aedes albopictus), which is a major vector of dengue and Zika viruses. We modeled the population abundance of blood-fed mosquito adults based on a mechanistic population model of Ae. albopictus with the consideration of diapause. Using simulated heat wave events derived from a 35-year historical dataset, we assessed how the mosquito population responded to different heat wave characteristics, including the onset day, duration, and the average temperature. Two important observations are made: (1) a heat wave event facilitates the population growth in the early development phase but tends to have an overall inhibitive effect; and (2) two primary factors affecting the development are the unusual onset time of a heat wave and a relatively high temperature over an extended period. We also performed a sensitivity analysis using different heat wave definitions, justifying the robustness of the findings. The study suggests that particular attention should be paid to future heat wave events with an abnormal onset time or a lasting high temperature in order to develop effective strategies to prevent and control Ae. albopictus-borne diseases.

by Chris M. Stone, Samantha R. Schwab, Dina M. Fonseca, Nina H. Fefferman

Vector control is still our primary intervention for both prevention and mitigation of epidemics of many vector-borne diseases. Efficiently targeting control measures is important since control can involve substantial economic costs. Targeting is not always straightforward, as transmission of vector-borne diseases is affected by various types of host movement. Here we assess how taking daily commuting patterns into consideration can help improve vector control efforts. We examine three tropical urban centers (San Juan, Recife, and Jakarta) that have recently been exposed to Zika and/or dengue infections and consider whether the distribution of human populations and resulting commuting flows affects the optimal scale at which control interventions should be implemented. We developed a stochastic, spatial model and investigated four control scenarios. The scenarios differed in the spatial extent of their implementation and were: 1) a response at the level of an individual neighborhood; 2) a response targeted at a neighborhood in which infected humans were detected and the one with which it was most strongly connected by human movement; 3) a limited area-wide response where all neighborhoods within a certain radius of the focal area were included; and 4) a collective response where all participating neighborhoods implemented control. The relative effectiveness of the scenarios varied only slightly between different settings, with the number of infections averted over time increasing with the scale of implementation. This difference depended on the efficacy of control at the neighborhood level. At low levels of efficacy, the scenarios mirrored each other in infections averted. At high levels of efficacy, impact increased with the scale of the intervention. As a result, the choice between scenarios will not only be a function of the amount of effort decision-makers are willing to invest, but largely epend on the overall effectiveness of vector control approaches.

by Guido España, Yutong Yao, Kathryn B. Anderson, Meagan C. Fitzpatrick, David L. Smith, Amy C. Morrison, Annelies Wilder-Smith, Thomas W. Scott, T. Alex Perkins

The tetravalent dengue vaccine CYD-TDV (Dengvaxia) is the first licensed vaccine against dengue, but recent findings indicate an elevated risk of severe disease among vaccinees without prior dengue virus (DENV) exposure. The World Health Organization currently recommends CYD-TDV only for individuals with serological confirmation of past DENV exposure. Our objective was to evaluate the potential health impact and cost-effectiveness of vaccination following serological screening. To do so, we used an agent-based model to simulate DENV transmission with and without vaccination over a 10-year timeframe. Across a range of values for the proportion of vaccinees with prior DENV exposure, we projected the proportion of symptomatic and hospitalized cases averted as a function of the sensitivity and specificity of serological screening. Scenarios about the cost-effectiveness of screening and vaccination were chosen to be representative of Brazil and the Philippines. We found that public health impact depended primarily on sensitivity in high-transmission settings and on specificity in low-transmission settings. Cost-effectiveness could be achievable from the perspective of a public payer provided that sensitivity and the value of a disability-adjusted life-year were both high, but only in high-transmission settings. Requirements for reducing relative risk and achieving cost-effectiveness from an individual perspective were more restricted, due to the fact that those who test negative pay for screening but receive no benefit. Our results predict that cost-effectiveness could be achieved only in high-transmission areas of dengue-endemic countries with a relatively high per capita GDP, such as Panamá (13,680 USD), Brazil (8,649 USD), México (8,201 USD), or Thailand (5,807 USD). In conclusion, vaccination with CYD-TDV following serological screening could have a positive impact in some high-transmission settings, provided that screening is highly specific (to minimize individual harm), at least moderately sensitive (to maximize population benefit), and sufficiently inexpensive (depending on the setting).

by Darong Yang, Nan L. Li, Dahai Wei, Baoming Liu, Fang Guo, Husni Elbahesh, Yunzhi Zhang, Zhi Zhou, Guo-Yun Chen, Kui Li

Infection by Zika virus (ZIKV) is linked to microcephaly and other neurological disorders, posing a significant health threat. Innate immunity is the first line of defense against invading pathogens, but relatively little is understood regarding host intrinsic mechanisms that guard against ZIKV. Here, we show that host tripartite motif-containing protein 56 (TRIM56) poses a barrier to ZIKV infection in cells of neural, epithelial and fibroblast origins. Overexpression of TRIM56, but not an E3 ligase-dead mutant or one lacking a short C-terminal portion, inhibited ZIKV RNA replication. Conversely, depletion of TRIM56 increased viral RNA levels. Although the C-terminal region of TRIM56 bears sequence homology to NHL repeat of TRIM-NHL proteins that regulate miRNA activity, knockout of Dicer, which abolishes production of miRNAs, had no demonstrable effect on ZIKV restriction imposed by TRIM56. Rather, we found that TRIM56 is an RNA-binding protein that associates with ZIKV RNA in infected cells. Moreover, a recombinant TRIM56 fragment comprising the C-terminal 392 residues captured ZIKV RNA in cell-free reactions, indicative of direct interaction. Remarkably, deletion of a short C-terminal tail portion abrogated the TRIM56-ZIKV RNA interaction, concomitant with a loss in antiviral activity. Altogether, our study reveals TRIM56 is an RNA binding protein that acts as a ZIKV restriction factor and provides new insights into the antiviral mechanism by which this E3 ligase tackles flavivirus infections.

by Jian-Wei Xu, Hui Liu, Didan Ai, Yan Yu, Bian Yu

Sustainable dengue intervention requires the participation of communities. Therefore, understanding the health beliefs, knowledge and perceptions of dengue among the local people can help to design locally appropriate strategies for effective interventions. A combination of qualitative semi-structured in-depth interviews (SDIs) and quantitative household questionnaire surveys (HHSs) was used to investigate the beliefs, knowledge and perceptions of dengue among the Shan people in Eastern Shan Special Region IV (ESSR4), Myanmar. The SDI was administered to 18 key informants, and the HHS was administered to 259 respondents. Only 14.7% (95% CI: 10.6–19.6%) of the HHS respondents could confirm that mosquitoes transmit dengue; 14.3% (95% CI: 10.3–19.1%) knew that piebald or Aedes mosquitoes transmit dengue; and 24.3% (95% CI: 19.2–30.0%) believed that dengue-transmitting mosquitoes mainly lived in small ponds. Merely ten (0.4%) of the 259 respondents of the HHS thought that dengue-transmitting mosquitoes bite in the day time. The people in the villages where there were outbreaks of dengue had more knowledge about dengue. This study demonstrates that the health beliefs of the Shan people were closely associated with their lifestyles, social and natural environments. To stay healthy, the Shan people clean their houses and surroundings regularly. However, their knowledge about dengue was not adequate for effective dengue control because it was mostly learned from previous dengue experiences and in a context that lacks systematic health education. Thus, in this setting, with a weak public health structure, more international support should be provided to promote the knowledge of the Shan people about dengue and to increase their sensitive awareness to dengue, which might be beneficial for social mobilization and community participation during future dengue prevention.

by Baoyin Yuan, Hyojung Lee, Hiroshi Nishiura
Background In summer 2014, an autochthonous outbreak of dengue occurred in Tokyo, Japan, in which Yoyogi Park acted as the focal area of transmission. Recognizing the outbreak, concerted efforts were made to control viral spread, which included mosquito control, public announcement of the outbreak, and a total ban on entering the park. We sought to assess the effectiveness of these control measures. Methodology/Principal findings We used a mathematical model to describe the transmission dynamics. Using dates of exposure and illness onset, we categorized cases into three groups according to the availability of these datasets. The infection process was parametrically modeled by generation, and convolution of the infection process and the incubation period was fitted to the data. By estimating the effective reproduction number, we determined that the effect of dengue risk communication together with mosquito control from 28 August 2014 was insufficiently large to lower the reproduction number to below 1. However, once Yoyogi Park was closed on 4 September, the value of the effective reproduction number began to fall below 1, and the associated relative reduction in the effective reproduction number was estimated to be 20%–60%. The mean incubation period was an estimated 5.8 days. Conclusions/Significance Regardless of the assumed number of generations of cases, the combined effect of mosquito control, risk communication, and park closure appeared to be successful in interrupting the chain of dengue transmission in Tokyo.

by Riyadi Adrizain, Djatnika Setiabudi, Alex Chairulfatah
Background Dengue virus infection (DVI) among children is a leading cause of hospitalization in endemic areas. Hospitalized patients are at risk of receiving unnecessary antibiotics. Methods A retrospective medical review analysis study was conducted to evaluate the prevalence, indication, and choice of antibiotics given to hospitalized patients less than 15 years of age with DVI in two different hospital settings (teaching and private hospitals) in the Municipality of Bandung. Epidemiological, clinical, and laboratory data were obtained using a pre-tested standardized questionnaire from patients’ medical records admitted from January 1 to December 31, 2015. Results There were 537 (17.5%) out of 3078 cases who received antibiotics. Among 176 cases admitted to the teaching hospitals, presumed bacterial upper respiratory tract infection (URTI) and typhoid fever were found in 1 (0.6%) case and 6 (0.3%) cases. In private hospitals among 2902 cases, presumed bacterial URTI was found in 324 (11.2%) cases, typhoid fever in 188 (6.5%) cases and urinary tract infection (UTI) in 18 (0.6%) cases. The prevalence of URTI and typhoid fever were significantly lower in the teaching hospitals compared to the private hospitals (p

Title: Doratoxylon apetalum, an indigenous medicinal plant from Mascarene Islands, is a potent inhibitor of Zika and Dengue Virus infection in human cells
Authors: Haddad, Juliano G.; Koishi, Andrea Cristine; Gaudry, Arnaud; Nunes Duarte dos Santos, Claudia; Viranaicken, Wildriss; Desprès, Philippe; El Kalamouni, Chaker

by Melisa Berenice Bonica, Silvina Goenaga, María Laura Martin, Mariel Feroci, Victoria Luppo, Evangelina Muttis, Cintia Fabbri, María Alejandra Morales, Delia Enria, María Victoria Micieli, Silvana Levis

The importance of Zika virus (ZIKV) has increased noticeably since the outbreak in the Americas in 2015, when the illness was associated with congenital disorders. Although there is evidence of sexual transmission of the virus, the mosquito Aedes aegypti is believed to be the main vector for transmission to humans. This species of mosquito has not only been found naturally infected with ZIKV, but also has been the subject of study in many vector competence assays that employ different strains of ZIKV around the world. In Argentina, the first case was reported in February 2016 and a total of 278 autochthonous cases have since been confirmed, however, ZIKV virus has not been isolated from any mosquito species yet in Argentina. In order to elucidate if Argentinian Ae. aegypti populations could be a possible vector of ZIKV, we conducted vector competence studies that involved a local strain of ZIKV from Chaco province, and a Venezuelan strain obtained from an imported case. For this purpose, Ae. aegypti adults from the temperate area of Argentina (Buenos Aires province) were fed with infected blood. Body, legs and saliva were harvested and tested by plaque titration on plates of Vero cells for ZIKV at 7, 11 and 14 days post infection (DPI) in order to calculate infection, transmission, and dissemination rates, respectively. Both strains were able to infect mosquitoes at all DPIs, whereas dissemination and transmission were observed at all DPIs for the Argentinian strain but only at 14 DPI for the Venezuelan strain. This study proves the ability of Ae. aegypti mosquitoes from Argentina to become infected with two different strains of ZIKV, both belonging to the Asian lineage, and that the virus can disseminate to the legs and salivary glands.

Title: Association between MBL2 haplotypes and dengue severity in children from Rio de Janeiro, Brazil
Authors: Ornelas, Alice Maria de Magalhães; Xavier-de-Carvalho, Caroline; Alvarado-Arnez, Lucia Elena; Ribeiro-Alves, Marcelo; Rossi, Átila Duque; Tanuri, Amilcar; Aguiar, Renato Santana; Moraes, Milton Ozório; Cardoso, Cynthia Chester

by Sadie J. Ryan, Stephanie J. Mundis, Alex Aguirre, Catherine A. Lippi, Efraín Beltrán, Froilán Heras, Valeria Sanchez, Mercy J. Borbor-Cordova, Rachel Sippy, Anna M. Stewart-Ibarra, Marco Neira

Insecticide resistance (IR) can undermine efforts to control vectors of public health importance. Aedes aegypti is the main vector of resurging diseases in the Americas such as yellow fever and dengue, and recently emerging chikungunya and Zika fever, which have caused unprecedented epidemics in the region. Vector control remains the primary intervention to prevent outbreaks of Aedes-transmitted diseases. In many high-risk regions, like southern Ecuador, we have limited information on IR. In this study, Ae. aegypti IR was measured across four cities in southern Ecuador using phenotypic assays and genetic screening for alleles associated with pyrethroid IR. Bottle bioassays showed significant inter-seasonal variation in resistance to deltamethrin, a pyrethroid commonly used by the Ministry of Health, and alpha-cypermethrin, as well as between-city differences in deltamethrin resistance. There was also a significant difference in phenotypic response to the organophosphate, Malathion, between two cities during the second sampling season. Frequencies of the resistant V1016I genotype ranged from 0.13 to 0.68. Frequencies of the resistant F1534C genotype ranged from 0.63 to 1.0, with sampled populations in Machala and Huaquillas at fixation for the resistant genotype in all sampled seasons. In Machala and Portovelo, there were statistically significant inter-seasonal variation in genotype frequencies for V1016I. Resistance levels were highest in Machala, a city with hyperendemic dengue transmission and historically intense insecticide use. Despite evidence that resistance alleles conferred phenotypic resistance to pyrethroids, there was not a precise correspondence between these indicators. For the F1534C gene, 17.6% of homozygous mutant mosquitoes and 70.8% of heterozygotes were susceptible, while for the V1016I gene, 45.6% homozygous mutants and 55.6% of heterozygotes were susceptible. This study shows spatiotemporal variability in IR in Ae. aegypti populations in southern coastal Ecuador, and provides an initial examination of IR in this region, helping to guide vector control efforts for Ae. aegypti.

by Yasuhiro Kawai, Eri Nakayama, Kenta Takahashi, Satoshi Taniguchi, Ken-ichi Shibasaki, Fumihiro Kato, Takahiro Maeki, Tadaki Suzuki, Shigeru Tajima, Masayuki Saijo, Chang-Kweng Lim

We investigated the growth properties and virulence in mice of three Zika virus (ZIKV) strains of Asian/American lineage, PRVABC59, ZIKV/Hu/Chiba/S36/2016 (ChibaS36), and ZIKV/Hu/NIID123/2016 (NIID123), belonging to the three distinct subtypes of this lineage. The American-subtype strain, PRVABC59, showed the highest growth potential in vitro, whereas the Southeast Asian-subtype strain, NIID123, showed the lowest proliferative capacity. Moreover, PRVABC59- and NIID123-infected mice showed the highest and lowest viremia levels and infectious virus levels in the testis, respectively, and the rate of damaged testis in PRVABC59-infected mice was higher than in mice infected with the other two strains. Lastly, ZIKV NS1 antigen was detected in the damaged testes of mice infected with PRVABC59 and the Pacific-subtype strain, ChibaS36, at 2 weeks post-inoculation and in the epididymides of PRVABC59-infected mice at 6 weeks post-inoculation. Our results indicate that PRVABC59 and ChibaS36 exhibit increased abilities to grow in vitro and in vivo and to induce testis damage in mice.

by Sunil Kumar Dubey, Jatin Shrinet, Sujatha Sunil
Background RNA interference is among the most important mechanisms that serve to restrict virus replication within mosquitoes, where microRNAs (miRNAs) are important in regulating viral replication and cellular functions. These miRNAs function by binding to complementary sequences mostly in the untranslated regions of the target. Chikungunya virus (CHIKV) genome consists of two open reading frames flanked by 5′ and 3′ untranslated regions on the two sides. A recent study from our laboratory has shown that Aedes miRNAs are regulated during CHIKV infection. The present study was undertaken to further understand the role of these miRNAs in CHIKV replication. Methods/Findings We observe that miR-2944b-5p binds to the 3′ untranslated region of CHIKV and the binding is abated when the binding sites are abolished. Loss-of-function studies of miR-2944b-5p using antagomirs, both in vitro and in vivo, reveal an increase in CHIKV viral replication, thereby directly implying a role of miR-2944b-5p in CHIKV replication. We further showed that the mitochondrial membrane potential of the mosquito cells is maintained by this miRNA during CHIKV replication, and cellular factor vps-13 plays a contributing role. Conclusions Our study has opened new avenues to understand vector-virus interactions and provides novel insights into CHIKV replication in Aedes aegypti. Furthermore, our study has shown miR-2944b-5p to be playing role, where one of its target vps-13 also contributes, in maintaining mitochondrial membrane potential in Aedes aegypti.

Title: From Mosquitos to Humans: Genetic Evolution of Zika Virus
Authors: Wang, Lulan; Valderramos, Stephanie G.; Wu, Aiping; Ouyang, Songying; Li, Chunfeng; Brasil, Patricia; Bonaldo, Myrna; Coates, Thomas; Nielsen-Saines, Karin; Jiang, Taijiao; Aliyari, Roghiyh; Cheng, Genhong

by Mengli Chen, Yuzhe Du, Shaoying Wu, Yoshiko Nomura, Guonian Zhu, Boris S. Zhorov, Ke Dong
Background Multiple mutations in the voltage-gated sodium channel have been associated with knockdown resistance (kdr) to DDT and pyrethroid insecticides in a major human disease vector Aedes aegypti. One mutation, V1016G, confers sodium channel resistance to pyrethroids, but a different substitution in the same position V1016I alone had no effect. In pyrethroid-resistant Ae. aegypti populations, V1016I is often linked to another mutation, F1534C, which confers sodium channel resistance only to Type I pyrethroids including permethrin (PMT), but not to Type II pyrethroids including deltamethrin (DMT). Mosquitoes carrying both V1016G and F1534C exhibited a greater level of pyrethroid resistance than those carrying F1534C alone. More recently, a new mutation T1520I co-existing with F1534C was detected in India. However, whether V1016I or T1520I enhances pyrethroid resistance of sodium channels carrying F1534C remains unknown. Methodology/Principal findings V1016I, V1016G, T1520I and F1534C substitutions were introduced alone and in various combinations into AaNav1-1, a sodium channel from Aedes aegypti. The mutant channels were then expressed in Xenopus oocytes and examined for channel properties and sensitivity to pyrethroids using the two-electrode voltage clamping technique. The results showed that V1016I or T1520I alone did not alter the AaNav1-1 sensitivity to PMT or DMT. However, the double mutant T1520I+F1534C was more resistant to PMT than F1534C, but remained sensitive to DMT. In contrast, the double mutant V1016I+F1534C was resistant to DMT and more resistant to PMT than F1534C. Furthermore, V1016I/G and F1534C channels, but not T1520I, were resistant to dichlorodiphenyltrichloroethane (DDT). Cryo-EM structures of sodium channels suggest that T1520I allosterically deforms geometry of the pyrethroid receptor site PyR1 in AaNav1-1. The small deformation does not affect binding of DDT, PMT or DMT, but in combination with F1534C it increases the channel resistance to PMT and DDT. Conclusions/Significance Our data corroborated the previously proposed sequential selection of kdr mutations in Ae. aegypti. We proposed that mutation F1534C first emerged in response to DDT/pyrethroids providing a platform for subsequent selection of mutations V1016I and T1520I that confer greater and broader spectrum of pyrethroid resistance.

by Robert C. Reiner Jr., Steven T. Stoddard, Gonzalo M. Vazquez-Prokopec, Helvio Astete, T. Alex Perkins, Moises Sihuincha, Jeffrey D. Stancil, David L. Smith, Tadeuz J. Kochel, Eric S. Halsey, Uriel Kitron, Amy C. Morrison, Thomas W. Scott

During the last 50 years, the geographic range of the mosquito Aedes aegypti has increased dramatically, in parallel with a sharp increase in the disease burden from the viruses it transmits, including Zika, chikungunya, and dengue. There is a growing consensus that vector control is essential to prevent Aedes-borne diseases, even as effective vaccines become available. What remains unclear is how effective vector control is across broad operational scales because the data and the analytical tools necessary to isolate the effect of vector-oriented interventions have not been available. We developed a statistical framework to model Ae. aegypti abundance over space and time and applied it to explore the impact of citywide vector control conducted by the Ministry of Health (MoH) in Iquitos, Peru, over a 12-year period. Citywide interventions involved multiple rounds of intradomicile insecticide space spray over large portions of urban Iquitos (up to 40% of all residences) in response to dengue outbreaks. Our model captured significant levels of spatial, temporal, and spatio-temporal variation in Ae. aegypti abundance within and between years and across the city. We estimated the shape of the relationship between the coverage of neighborhood-level vector control and reductions in female Ae. aegypti abundance; i.e., the dose-response curve. The dose-response curve, with its associated uncertainties, can be used to gauge the necessary spraying effort required to achieve a desired effect and is a critical tool currently absent from vector control programs. We found that with complete neighborhood coverage MoH intra-domicile space spray would decrease Ae. aegypti abundance on average by 67% in the treated neighborhood. Our framework can be directly translated to other interventions in other locations with geolocated mosquito abundance data. Results from our analysis can be used to inform future vector-control applications in Ae. aegypti endemic areas globally.

by Athanase Badolo, Aboubacar Sombié, Patricia M. Pignatelli, Aboubakar Sanon, Félix Yaméogo, Dimitri W. Wangrawa, Antoine Sanon, Hirotaka Kanuka, Philip J. McCall, David Weetman
Background Recent outbreaks of dengue and other Aedes aegypti-borne arboviruses highlight the importance of a rapid response for effective vector control. Data on insecticide resistance and underlying mechanisms are essential for outbreak preparedness, but are sparse in much of Africa. We investigated the levels and heterogeneity of insecticide resistance and mechanisms of Ae. aegypti from contrasting settings within and around Ouagadougou, Burkina Faso. Methodology/Principal findings Bioassays were performed on larvae and adults to diagnose prevalence of resistance, and to assess levels where resistance was detected. Investigation of resistance mechanisms was performed using synergist bioassays, knockdown resistance (kdr) target site mutation genotyping and quantitative PCR expression analysis of candidate P450 genes.Larval dose-response assays indicated susceptibility to the organophosphates tested. Adult females were also susceptible to organophosphates, but resistance to carbamates was suspected in urban and semi-urban localities. Females from all localities showed resistance to pyrethroids but resistance prevalence and level were higher in urban and especially in semi-urban areas, compared to the rural population. Environment was also associated with susceptibility: adults reared from larvae collected in tires from the semi-urban site were significantly less resistant to pyrethroids than those collected from large outdoor drinking water containers (‘drums’). Susceptibility to both pyrethroids tested was largely restored by pre-exposure to Piperonyl Butoxide (PBO), suggesting a strong metabolic basis to resistance.The 1534C kdr mutation was nearly fixed in semi-urban and urban areas but was far less common in the rural area, where the 1016I kdr mutation frequency was also significantly lower. P450 gene analysis detected limited over-expression of single candidates but significantly elevated average expression in the semi-urban site compared to both a susceptible laboratory colony, and females from the other collection sites. Conclusions/Significance Our results reveal pyrethroid resistance and paired kdr mutations in both urban and semi-urban sites at levels that are unprecedented for mainland Africa. The combination of target site and metabolic mechanisms is common in Ae. aegypti populations from other continents but is a worrying finding for African populations. However, organophosphate insecticides are still active against both larvae and adults of Ae. aegypti, providing useful insecticidal options for control and resistance management.

by Josef Zapletal, Himanshu Gupta, Madhav Erraguntla, Zach N. Adelman, Kevin M. Myles, Mark A. Lawley

Mosquito-borne pathogens continue to be a significant burden within human populations, with Aedes aegypti continuing to spread dengue, chikungunya, and Zika virus throughout the world. Using data from a previously conducted study, a linear regression model was constructed to predict the aquatic development rates based on the average temperature, temperature fluctuation range, and larval density. Additional experiments were conducted with different parameters of average temperature and larval density to validate the model. Using a paired t-test, the model predictions were compared to experimental data and showed that the prediction models were not significantly different for average pupation rate, adult emergence rate, and juvenile mortality rate. The models developed will be useful for modeling and estimating the upper limit of the number of Aedes aegypti in the environment under different temperature, diurnal temperature variations, and larval densities.

by Benjamin A. McKenzie, Alan E. Wilson, Sarah Zohdy
Background Much work has been done in recent years to determine the vector competence of Aedes albopictus (Skuse) for Zika virus (ZIKV). If competent, Ae. albopictus could become an important vector in the spread of ZIKV to areas which until now have been considered safe from the virus. Despite much speculation about Ae. albopictus’ competence for ZIKV, there have been, to date, no quantitative syntheses of Ae. albopictus’ competence, nor have the potentially confounding differences between studies been addressed. Methodology/ principle findings This study represents a quantitative meta-analysis of the literature surrounding this topic by examining infection rates (IR) and transmission rates (TR) among sample populations of Ae. albopictus at 7 and 14 days post infection (dpi) across 15 journal articles comprising 23 studies. Our analyses examined potentially confounding variables in the studies contained therein, including: geographic origin of viral strain or mosquito population tested, whether sympatry of the tested viral strain and mosquito population was important, and freshness of blood meal. Our results suggest 1) Ae albopictus is a competent vector for ZIKV and 2) that origin of Ae. albopictus population and origin of viral strain had significant effects on the competence of Ae. albopictus to transmit ZIKV. Conclusions/ significance These results indicate a need to further explore the effects of methodology on vector competence studies and to examine in more detail the geographic variation in the competence of Ae. albopictus for ZIKV as well as the underlying causes of said variation. The ability of Ae. albopictus to carry and transmit ZIKV also points to a need to create new vector control strategies in case of a ZIKV outbreak in an area where Ae. albopictus is prominent. Finally, this study represents a potential template for future meta-analyses in the field of vector competence, where this type of study has been under-utilized despite the abundance of relevant studies.

Title: Association between microcephaly, Zika virus infection, and other risk factors in Brazil: final report of a case-control study
Authors: Araújo, Thalia Velho Barreto de; Ximenes, Ricardo Arraes de Alencar; Miranda-Filho, Demócrito de Barros; Souza, Wayner Vieira; Montarroyos, Ulisses Ramos; Melo, Ana Paula Lopes de; Valongueiro, Sandra; Albuquerque, Maria de Fátima Pessoa Militão de; Braga, Cynthia; Filho, Sinval Pinto Brandão; Cordeiro, Marli Tenório; Vazquez, Enrique; Cruz, Danielle di Cavalcanti Souza; Henriques, Claudio Maierovitch Pessanha; Bezerra, Luciana Caroline Albuquerque; Castanha, Priscila Mayrelle da Silva; Dhalia, Rafael; Marques-Júnior, Ernesto Torres Azevedo; Martelli, Celina Maria Turchi; Rodrigues, Laura Cunha

by Eric P. Caragata, Marcele N. Rocha, Thiago N. Pereira, Simone B. Mansur, Heverton L. C. Dutra, Luciano A. Moreira
Background Wolbachia’s ability to restrict arbovirus transmission makes it a promising tool to combat mosquito-transmitted diseases. Wolbachia-infected Aedes aegypti are currently being released in locations such as Brazil, which regularly experience concurrent outbreaks of different arboviruses. A. aegypti can become co-infected with, and transmit multiple arboviruses with one bite, which can complicate patient diagnosis and treatment. Methodology/principle findings Using experimental oral infection of A. aegypti and then RT-qPCR, we examined ZIKV/DENV-1 and ZIKV/DENV-3 co-infection in Wolbachia-infected A. aegypti and observed that Wolbachia-infected mosquitoes experienced lower prevalence of infection and viral load than wildtype mosquitoes, even with an extra infecting virus. Critically, ZIKV/DENV co-infection had no significant impact on Wolbachia’s ability to reduce viral transmission. Wolbachia infection also strongly altered expression levels of key immune genes Defensin C and Transferrin 1, in a virus-dependent manner. Conclusions/significance Our results suggest that pathogen interference in Wolbachia-infected A. aegypti is not adversely affected by ZIKV/DENV co-infection, which suggests that Wolbachia-infected A. aegypti will likely prove suitable for controlling mosquito-borne diseases in environments with complex patterns of arbovirus transmission.

by Xiaodong Huang, Wenbiao Hu, Laith Yakob, Gregor J. Devine, Elizabeth A. McGraw, Cassie C. Jansen, Helen M. Faddy, Francesca D. Frentiu
Background Chikungunya virus (CHIKV) is an emerging mosquito-borne pathogen circulating in tropical and sub-tropical regions. Although autochthonous transmission has not been reported in Australia, there is a potential risk of local CHIKV outbreaks due to the presence of suitable vectors, global trade, frequent international travel and human adaptation to changes in climate. Methodology/Principal findings A time series seasonal decomposition method was used to investigate the seasonality and trend of monthly imported CHIKV cases. This pattern was compared with the seasonality and trend of monthly overseas arrivals. A wavelet coherence analysis was applied to examine the transient relationships between monthly imported CHIKV cases and southern oscillation index (SOI) in time-frequency space. We found that the number and geographical distribution of countries of acquisition for CHIKV in travellers to Australia has increased in recent years. The number of monthly imported CHIKV cases displayed an unstable increased trend compared with a stable linear increased trend in monthly overseas arrivals. Both imported CHIKV cases and overseas arrivals showed substantial seasonality, with the strongest seasonal effects in each January, followed by each October and July. The wavelet coherence analysis identified four significant transient relationships between monthly imported CHIKV cases and 6-month lagged moving average SOI, in the years 2009–2010, 2012, 2014 and 2015–2016. Conclusion/Significance High seasonal peaks of imported CHIKV cases were consistent with the high seasonal peaks of overseas arrivals into Australia. Our analysis also indicates that El Niño Southern Oscillation (ENSO) variation may impact CHIKV epidemics in endemic regions, in turn influencing the pattern of imported cases.

Title: Clinical and Laboratory Signs as Dengue Markers During an Outbreak in Rio de Janeiro
Authors: Passos, S. R. L.; Bedoya, S. J.; Hökerberg, Y. H. M.; Maia, S. C.; Georg, I.; Nogueira, Rita Maria R.; Souza, R. V.; Marzochi, K. B. F.

by Lais Ceschini Machado, Mariana Carolina de Morais-Sobral, Tulio de Lima Campos, Mylena Ribeiro Pereira, Maria de Fátima Pessoa Militão de Albuquerque, Clément Gilbert, Rafael Freitas Oliveira Franca, Gabriel Luz Wallau

Chikungunya virus (CHIKV) is an RNA virus from the Togaviridae family transmitted by mosquitoes in both sylvatic and urban cycles. In humans, CHIKV infection leads to a febrile illness, denominated Chikungunya fever (CHIKF), commonly associated with more intense and debilitating outcomes. CHIKV arrived in Brazil in 2014 through two independent introductions: the Asian/Caribbean genotype entered through the North region and the African ECSA genotype was imported through the Northeast region. Following their initial introduction, both genotypes established their urban cycle among large naive human populations causing several outbreaks in the Americas. Here, we sequenced CHIKV genomes from a recent outbreak in the Northeast region of Brazil, employing an in-house developed Next-Generation Sequencing (NGS) protocol capable of directly detecting multiple known CHIKV genotypes from clinical positive samples. Our results demonstrate that both Asian/Caribbean and ECSA genotypes expanded their ranges, reaching cocirculation in the Northeast region of Brazil. In addition, our NGS data supports the findings of simultaneous infection by these two genotypes, suggesting that coinfection might be more common than previously thought in highly endemic areas. Future efforts to understand CHIKV epidemiology should thus take into consideration the possibility of coinfection by different genotypes in the human population.

by Li Wei Ang, Tun-Linn Thein, Yixiang Ng, Irving Charles Boudville, Po Ying Chia, Vernon Jian Ming Lee, Yee-Sin Leo
Objectives Since the 1990s, Singapore has experienced periodic dengue epidemics of increasing frequency and magnitude. In the aftermath of the 2004–2005 dengue epidemic, hospitals refined their admission criteria for dengue cases to right-site dengue case management and reduce the burden of healthcare utilization and negative outcomes. In this study, we describe the national trends of hospital admissions for dengue and disease severity in terms of length of stay (LOS), admission to the intensive care unit (ICU) and death in hospital, and case fatality rate (CFR) in Singapore. Methods We conducted a retrospective study of notified cases and laboratory confirmed dengue patients admitted to all public and private hospitals between 2003 and 2017. Case notifications for dengue and hospitalization records were extracted from national databases. Results The proportion of dengue cases hospitalized was lower in recent years; 28.9% in the 2013–2014 epidemic, compared to 93.2% in the 2004–2005 epidemic, and 58.1% in the 2007 epidemic. Median LOS remained stable over the years; overall LOS was 3 to 4 days and ICU stay was 2 to 3 days. Less than 2% of hospitalized patients were admitted to the ICU. Overall CFR was low and remained below 0.5%. The proportions of dengue cases hospitalized and patients admitted to the ICU were highest in the elderly aged 65 years and older. Conclusions While the proportion of dengue cases hospitalized saw a drastic decline due to more selective admission criteria, there was no concomitant increase in adverse outcomes, suggesting that admission criteria were appropriate to focus on severe dengue cases. Further studies are needed to optimize dengue management in older adults who are more likely to be hospitalized with greater disease severity, given the higher proportions of hospitalizations and severe disease among older adults.

Title: Scientific and technological contributions of Latin America and Caribbean countries to the Zika virus outbreak
Authors: Machado-Silva, Alice; Guindalini, Camila; Fonseca, Fernanda Lopes; Silva, Marcus Vinícius Pereira da; Fonseca, Bruna de Paula

by Vagner Fonseca, Pieter J. K. Libin, Kristof Theys, Nuno R. Faria, Marcio R. T. Nunes, Maria I. Restovic, Murilo Freire, Marta Giovanetti, Lize Cuypers, Ann Nowé, Ana Abecasis, Koen Deforche, Gilberto A. Santiago, Isadora C. de Siqueira, Emmanuel J. San, Kaliane C. B. Machado, Vasco Azevedo, Ana Maria Bispo-de Filippis, Rivaldo Venâncio da Cunha, Oliver G. Pybus, Anne-Mieke Vandamme, Luiz C. J. Alcantara, Tulio de Oliveira

In recent years, an increasing number of outbreaks of Dengue, Chikungunya and Zika viruses have been reported in Asia and the Americas. Monitoring virus genotype diversity is crucial to understand the emergence and spread of outbreaks, both aspects that are vital to develop effective prevention and treatment strategies. Hence, we developed an efficient method to classify virus sequences with respect to their species and sub-species (i.e. serotype and/or genotype). This ArboTyping tool provides an easy-to-use software implementation of this new method and was validated on a large dataset assessing the classification performance with respect to whole-genome sequences and partial-genome sequences. Available online: http://krisp.ukzn.ac.za/app/.

by Rachel Sippy, Diego Herrera, David Gaus, Ronald E. Gangnon, Jonathan A. Patz, Jorge E. Osorio

Season is a major determinant of infectious disease rates, including arboviruses spread by mosquitoes, such as dengue, chikungunya, and Zika. Seasonal patterns of disease are driven by a combination of climatic or environmental factors, such as temperature or rainfall, and human behavioral time trends, such as school year schedules, holidays, and weekday-weekend patterns. These factors affect both disease rates and healthcare-seeking behavior. Seasonality of dengue fever has been studied in the context of climatic factors, but short- and long-term time trends are less well-understood. With 2009–2016 medical record data from patients diagnosed with dengue fever at two hospitals in rural Ecuador, we used Poisson generalized linear modeling to determine short- and long-term seasonal patterns of dengue fever, as well as the effect of day of the week and public holidays. In a subset analysis, we determined the impact of school schedules on school-aged children. With a separate model, we examined the effect of climate on diagnosis patterns. In the first model, the most important predictors of dengue fever were annual sinusoidal fluctuations in disease, long-term trends (as represented by a spline for the full study duration), day of the week, and hospital. Seasonal trends showed single peaks in case diagnoses, during mid-March. Compared to the average of all days, cases were more likely to be diagnosed on Tuesdays (risk ratio (RR): 1.26, 95% confidence interval (CI) 1.05–1.51) and Thursdays (RR: 1.25, 95% CI 1.02–1.53), and less likely to be diagnosed on Saturdays (RR: 0.81, 95% CI 0.65–1.01) and Sundays (RR: 0.74, 95% CI 0.58–0.95). Public holidays were not significant predictors of dengue fever diagnoses, except for an increase in diagnoses on the day after Christmas (RR: 2.77, 95% CI 1.46–5.24). School schedules did not impact dengue diagnoses in school-aged children. In the climate model, important climate variables included the monthly total precipitation, an interaction between total precipitation and monthly absolute minimum temperature, an interaction between total precipitation and monthly precipitation days, and a three-way interaction between minimum temperature, total precipitation, and precipitation days. This is the first report of long-term dengue fever seasonality in Ecuador, one of few reports from rural patients, and one of very few studies utilizing daily disease reports. These results can inform local disease prevention efforts, public health planning, as well as global and regional models of dengue fever trends.

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