Monitoramento das Produções

by Anna S. Heffron, Emma L. Mohr, David Baker, Amelia K. Haj, Connor R. Buechler, Adam Bailey, Dawn M. Dudley, Christina M. Newman, Mariel S. Mohns, Michelle Koenig, Meghan E. Breitbach, Mustafa Rasheed, Laurel M. Stewart, Jens Eickhoff, Richard S. Pinapati, Erica Beckman, Hanying Li, Jigar Patel, John C. Tan, David H. O’Connor

The specificity of the antibody response against Zika virus (ZIKV) is not well-characterized. This is due, in part, to the antigenic similarity between ZIKV and closely related dengue virus (DENV) serotypes. Since these and other similar viruses co-circulate, are spread by the same mosquito species, and can cause similar acute clinical syndromes, it is difficult to disentangle ZIKV-specific antibody responses from responses to closely-related arboviruses in humans. Here we use high-density peptide microarrays to profile anti-ZIKV antibody reactivity in pregnant and non-pregnant macaque monkeys with known exposure histories and compare these results to reactivity following DENV infection. We also compare cross-reactive binding of ZIKV-immune sera to the full proteomes of 28 arboviruses. We independently confirm a purported ZIKV-specific IgG antibody response targeting ZIKV nonstructural protein 2B (NS2B) that was recently reported in ZIKV-infected people and we show that antibody reactivity in pregnant animals can be detected as late as 127 days post-infection (dpi). However, we also show that these responses wane over time, sometimes rapidly, and in one case the response was elicited following DENV infection in a previously ZIKV-exposed animal. These results suggest epidemiologic studies assessing seroprevalence of ZIKV immunity using linear epitope-based strategies will remain challenging to interpret due to susceptibility to false positive results. However, the method used here demonstrates the potential for rapid profiling of proteome-wide antibody responses to a myriad of neglected diseases simultaneously and may be especially useful for distinguishing antibody reactivity among closely related pathogens.

by Diana Patricia Rojas, Gloria Abigail Barrera-Fuentes, Norma Pavia-Ruz, Mariel Salgado-Rodriguez, Azael Che-Mendoza, Pablo Manrique-Saide, Gonzalo M. Vazquez-Prokopec, M. Elizabeth Halloran, Ira M. Longini, Hector Gomez-Dantes

Dengue is the most prevalent mosquito-borne viral disease of humans and is caused by the four serotypes of dengue virus. To estimate the incidence of dengue and other arboviruses, we analyzed the baseline and first year follow-up of a prospective school-based cohort study and their families in three cities in the state of Yucatan, Mexico. Through enhanced surveillance activities, acute febrile illnesses in the participants were detected and yearly blood samples were collected to evaluate dengue infection incidence. A Cox model was fitted to identify hazard ratios of arboviral infections in the first year of follow-up of the cohort. The incidence of dengue symptomatic infections observed during the first year of follow-up (2015–2016) was 3.5 cases per 1,000 person-years (95% CI: 1.9, 5.9). The incidence of dengue infections was 33.9 infections per 1,000 person-years (95% CI: 31.7, 48.0). The majority of dengue infections and seroconversions were observed in the younger age groups (≤ 14 years old). Other arboviruses were circulating in the state of Yucatan during the study period. The incidence of symptomatic chikungunya infections was 8.6 per 1,000 person-years (95% CI: 5.8, 12.3) and the incidence of symptomatic Zika infections was 2.3 per 1,000 person-years (95% CI: 0.9, 4.5). Our model shows that having a dengue infection during the first year of follow-up was significantly associated with being female, living in Ticul or Progreso, and being dengue naïve at baseline. Age was not significantly associated with the outcome, it was confounded by prior immunity to dengue that increases with age. This is the first report of a cohort in Latin America that provides incidence estimates of the three arboviruses co-circulating in all age groups. This study provides important information for understanding the epidemiology of dengue and other arboviruses and better informing public health policies.

by Norma Pavía-Ruz, Gloria Abigail Barrera-Fuentes, Salha Villanueva-Jorge, Azael Che-Mendoza, Julio César Campuzano-Rincón, Pablo Manrique-Saide, Diana Patricia Rojas, Gonzalo M. Vazquez-Prokopec, M. Elizabeth Halloran, Ira M. Longini, Héctor Gómez-Dantés
Background The implementation of vector control interventions and potential introduction new tools requires baseline data to evaluate their direct and indirect effects. The objective of the study is to present the seroprevalence of dengue infection in a cohort of children 0 to 15 years old followed during 2015 to 2016, the risk factors and the role of enhanced surveillance strategies in three urban sites (Merida, Ticul and Progreso) in Yucatan, Mexico. Methods A cohort of school children and their family members was randomly selected in three urban areas with different demographic, social conditions and levels of transmission. We included results from 1,844 children aged 0 to 15 years. Serum samples were tested for IgG, NS1 and IgM. Enhanced surveillance strategies were established in schools (absenteeism) and cohort families (toll-free number). Results Seroprevalence in children 0 to 15 years old was 46.8 (CI 95% 44.1–49.6) with no difference by sex except in Ticul. Prevalence increased with age and was significantly lower in 0 to 5 years old (26.9%, 95% CI:18.4–35.4) compared with 6 to 8 years old (43.9%, 95% CI:40.1–47.7) and 9 to 15 years old (61.4%, 95% CI:58.0–64.8). Sharing the domestic space with other families increased the risk 1.7 times over the individual families that own or rented their house, while risk was significantly higher when kitchen and bathroom were outside. Complete protection with screens in doors and windows decreased risk of infection. Seroprevalence was significantly higher in the medium and high risk areas. Conclusions The prevalence of antibodies in children 0 to 15 years in three urban settings in the state of Yucatan describe the high exposure and the heterogenous transmission of dengue virus by risk areas and between schools in the study sites. The enhanced surveillance strategy was useful to improve detection of dengue cases with the coincident transmission of chikungunya and Zika viruses.

by Tie-Long Xu, Yin Han, Wei Liu, Xing-Ya Pang, Bin Zheng, Yi Zhang, Xiao-Nong Zhou
Background Dengue fever is the most rapidly spreading mosquito-borne viral disease over the past 50 years, with a 30-fold increase in global incidence. Dengue vector control is a key component for the dengue control strategy, since no absolutely effective vaccine or drug is available yet. However, the rapid rise and spread of mosquito insecticide resistance have become major threats to the efficiency of insecticide-based vector control activities. Thus, innovative vector control tools are badly needed. This study aims to confirm the antivirus effectiveness of ivermectin on dengue virus type 2 (DENV-2) in Aedes albopictus (Skuse, 1894), then to explore its potential use in the combating to the dengue epidemics. Methods Aedes albopictus were first infected with DENV-2 in human whole blood, and at the fourth day after infectious blood feeding, they were divided into eight groups. Seven of them were held for six days with access to 0, 2, 4, 8, 16, 32 and 64 ng/ml ivermectin, respectively, and the last one was set as a historical control group, which was stored at -80°C until being detected at the same time with the other groups. Each mosquito was detected using real-time fluorescent RT-PCR kit. DENV-2 RNA concentration (copies/ml) and infection rate in each group were compared. Results Both of quantitatively and qualitatively inhibiting effects of ivermectin have been detected in this study. Generally, DENV-2 replicated well in Aedes albopictus without ivermectin intervention, whose virus loads exhibited significantly higher when the mosquitoes were holding from 4 days to 10 days after infectious blood feeding. In contrast, with the treatment of ivermectin, the infection rate was reduced by as much as 49.63%. The regression equation between infection rates (Y2) and ivermectin concentration log2 values (X2) was obtained as Y2 = 91.41–7.21*X2 with R2 = 0.89. Conclusion Ivermectin can directly or indirectly inhibit DENV-2 multiplication in Aedes albopictus. Moreover, the actual concentration for application in zooprophylaxis needs to be confirmed in the further field trials.

by Letícia B. Smith, Rakshit Tyagi, Shinji Kasai, Jeffrey G. Scott

Aedes aegypti poses a serious risk to human health due to its wide global distribution, high vector competence for several arboviruses, frequent human biting, and ability to thrive in urban environments. Pyrethroid insecticides remain the primary means of controlling adult A. aegypti populations during disease outbreaks. As a result of decades of use, pyrethroid resistance is a global problem. Cytochrome P450 monooxygenase (CYP)-mediated detoxification is one of the primary mechanisms of pyrethroid resistance. However, the specific CYP(s) responsible for resistance have not been unequivocally determined. We introgressed the resistance alleles from the resistant A. aegypti strain, Singapore (SP), into the genetic background of the susceptible ROCK strain. The resulting strain (CKR) was congenic to ROCK. Our primary goal was to determine which CYPs in SP are linked to resistance. To do this, we first determined which CYPs overexpressed in SP are also overexpressed in CKR, with the assumption that only the CYPs linked to resistance will be overexpressed in CKR relative to ROCK. Next, we determined whether any of the overexpressed CYPs were genetically linked to resistance (cis-regulated) or not (trans-regulated). We found that CYP6BB2, CYP6Z8, CYP9M5 and CYP9M6 were overexpressed in SP as well as in CKR. Based on the genomic sequences and polymorphisms of five single copy CYPs (CYP4C50, 6BB2, 6F2, 6F3 and 6Z8) in each strain, none of these genes were linked to resistance, except for CYP6BB2, which was partially linked to the resistance locus. Hence, overexpression of these four CYPs is due to a trans-regulatory factor(s). Knowledge on the specific CYPs and their regulators involved in resistance is critical for resistance management strategies because it aids in the development of new control chemicals, provides information on potential environmental modulators of resistance, and allows for the detection of resistance markers before resistance becomes fixed in the population.

by Sifat Sharmin, Kathryn Glass, Elvina Viennet, David Harley

Geographical mapping of dengue in resource-limited settings is crucial for targeting control interventions but is challenging due to the problem of zero-inflation because many cases are not reported. We developed a negative binomial generalised linear mixed effect model accounting for zero-inflation, spatial, and temporal random effects to investigate the spatial variation in monthly dengue cases in Bangladesh. The model was fitted to the district-level (64 districts) monthly reported dengue cases aggregated over the period 2000 to 2009 and Bayesian inference was performed using the integrated nested Laplace approximation. We found that mean monthly temperature and its interaction with mean monthly diurnal temperature range, lagged by two months were significantly associated with dengue incidence. Mean monthly rainfall at two months lag was positively associated with dengue incidence. Densely populated districts and districts bordering India or Myanmar had higher incidence than others. The model estimated that 92% of the annual dengue cases occurred between August and September. Cases were identified across the country with 94% in the capital Dhaka (located almost in the middle of the country). Less than half of the affected districts reported cases as observed from the surveillance data. The proportion reported varied by month with a higher proportion reported in high-incidence districts, but dropped towards the end of high transmission season.

by Lindsey A. Moser, Brendan T. Boylan, Fernando R. Moreira, Laurel J. Myers, Emma L. Svenson, Nadia B. Fedorova, Brett E. Pickett, Kristen A. Bernard

The recent emergence of Zika virus (ZIKV) in the Americas coincident with increased caseloads of microcephalic infants and Guillain-Barre syndrome has prompted a flurry of research on ZIKV. Much of the research is difficult to compare or repeat because individual laboratories use different virus isolates, growth conditions, and quantitative assays. Here we obtained three readily available contemporary ZIKV isolates and the prototype Ugandan isolate. We generated stocks of each on Vero mammalian cells (ZIKVmam) and C6/36 mosquito cells (ZIKVmos), determined titers by different assays side-by-side, compared growth characteristics using one-step and multi-step growth curves on Vero and C6/36 cells, and examined plaque phenotype. ZIKV titers consistently peaked earlier on Vero cells than on C6/36 cells. Contemporary ZIKV isolates reached peak titer most quickly in a multi-step growth curve when the amplifying cell line was the same as the titering cell line (e.g., ZIKVmam titered on Vero cells). Growth of ZIKVmam on mosquito cells was particularly delayed. These data suggest that the ability to infect and/or replicate in insect cells is limited after growth in mammalian cells. In addition, ZIKVmos typically had smaller, more homogenous plaques than ZIKVmam in a standard plaque assay. We hypothesized that the plaque size difference represented early adaptation to growth in mammalian cells. We plaque purified representative-sized plaques from ZIKVmos and ZIKVmam. ZIKVmos isolates maintained the initial phenotype while plaques from ZIKVmam isolates became larger with passaging. Our results underscore the importance of the cells used to produce viral stocks and the potential for adaptation with minimal cell passages. In addition, these studies provide a foundation to compare current and emerging ZIKV isolates in vitro and in vivo.

by Juthamas Phadungsombat, Marco Yung-Cheng Lin, Narinee Srimark, Atsushi Yamanaka, Emi E. Nakayama, Visal Moolasart, Patama Suttha, Tatsuo Shioda, Sumonmal Uttayamakul

Dengue is a mosquito-borne disease that has spread to over 100 countries. Dengue fever is caused by dengue virus (DENV), which belongs to the Flavivirus genus of the family Flaviviridae. DENV comprises 4 serotypes (DENV-1 to DENV-4), and each serotype is divided into distinct genotypes. Thailand is an endemic area where all 4 serotypes of DENV co-circulate. To understand the current genotype distribution of DENVs in Thailand, we enrolled 100 cases of fever with dengue-like symptoms at the Bamrasnaradura Infectious Diseases Institute during 2016–2017. Among them, 37 cases were shown to be dengue-positive by real-time PCR. We were able to isolate DENVs from 21 cases, including 1 DENV-1, 8 DENV-2, 4 DENV-3, and 8 DENV-4. To investigate the divergence of the viruses, RNA was extracted from isolated DENVs and viral near-whole genome sequences were determined. Phylogenetic analysis of the obtained viral sequences revealed that DENV-2 genotype Cosmopolitan was co-circulating with DENV-2 genotype Asian-I, the previously predominating genotype in Thailand. Furthermore, DENV-3 genotype III was found instead of DENV-3 genotype II. The DENV-2 Cosmopolitan and DENV-3 genotype III found in Thailand were closely related to the respective strains found in nearby countries. These results indicated that DENVs in Thailand have increased in genotypic diversity, and suggested that the DENV genotypic shift observed in other Asian countries also might be taking place in Thailand.

by Giel P. Göertz, Marit Lingemann, Corinne Geertsema, Marleen H. C. Abma-Henkens, Chantal B. F. Vogels, Constantianus J. M. Koenraadt, Monique M. van Oers, Gorben P. Pijlman
Background Chikungunya virus (CHIKV) is a re-emerging arthropod-borne (arbo)virus that causes chikungunya fever in humans and is predominantly transmitted by Aedes aegypti mosquitoes. The CHIKV replication machinery consists of four non-structural proteins (nsP1-4) that additionally require the presence of a number of host proteins for replication of the viral RNA. NsP3 is essential for CHIKV replication and has a conserved macro, central and C-terminal hypervariable domain (HVD). The HVD is intrinsically disordered and interacts with various host proteins via conserved short peptide motifs: A proline-rich (P-rich) motif that has affinity for SH3-domain containing proteins and duplicate FGDF motifs with affinity for G3BP and its mosquito homologue Rasputin. The importance of these motifs for infection of mammalian cells has previously been implicated. However, their role during CHIKV infection of mosquito cells and transmission by mosquitoes remains unclear. Methodology / Principal findings Here, we show that in-frame deletion of the P-rich motif is lethal for CHIKV replication in both mosquito and mammalian cells. However, while mutagenesis of the P-rich motif negatively affects replication both in mammalian and mosquito cells, it did not compromise the infection and transmission of CHIKV by Ae. aegypti mosquitoes. Mutagenesis of both FGDF motifs together completely inactivated CHIKV replication in both mammalian and mosquito cells. Importantly, mutation of a single FGDF motif attenuated CHIKV replication in mammalian cells, while replication in mosquito cells was similar to wild type. Surprisingly, CHIKV mutants containing only a single FGDF motif were efficiently transmitted by Ae. aegypti. Conclusions / Significance The P-rich motif in CHIKV nsP3 is dispensable for transmission by mosquitoes. A single FGDF motif is sufficient for infection and dissemination in mosquitoes, but duplicate FGDF motifs are required for the efficient infection from the mosquito saliva to a vertebrate host. These results contribute to understanding the dynamics of the alphavirus transmission cycle and may help the development of arboviral intervention strategies.

by Shengjie Lai, Michael A. Johansson, Wenwu Yin, Nicola A. Wardrop, Willem G. van Panhuis, Amy Wesolowski, Moritz U. G. Kraemer, Isaac I. Bogoch, Dylain Kain, Aidan Findlater, Marc Choisy, Zhuojie Huang, Di Mu, Yu Li, Yangni He, Qiulan Chen, Juan Yang, Kamran Khan, Andrew J. Tatem, Hongjie Yu

Due to worldwide increased human mobility, air-transportation data and mathematical models have been widely used to measure risks of global dispersal of pathogens. However, the seasonal and interannual risks of pathogens importation and onward transmission from endemic countries have rarely been quantified and validated. We constructed a modelling framework, integrating air travel, epidemiological, demographical, entomological and meteorological data, to measure the seasonal probability of dengue introduction from endemic countries. This framework has been applied retrospectively to elucidate spatiotemporal patterns and increasing seasonal risk of dengue importation from South-East Asia into China via air travel in multiple populations, Chinese travelers and local residents, over a decade of 2005–15. We found that the volume of airline travelers from South-East Asia into China has quadrupled from 2005 to 2015 with Chinese travelers increased rapidly. Following the growth of air traffic, the probability of dengue importation from South-East Asia into China has increased dramatically from 2005 to 2015. This study also revealed seasonal asymmetries of transmission routes: Sri Lanka and Maldives have emerged as origins; neglected cities at central and coastal China have been increasingly vulnerable to dengue importation and onward transmission. Compared to the monthly occurrence of dengue reported in China, our model performed robustly for importation and onward transmission risk estimates. The approach and evidence could facilitate to understand and mitigate the changing seasonal threat of arbovirus from endemic regions.

by Cíntia Bittar, Rafael R. G. Machado, Manuela T. Comelis, Larissa M. Bueno, Eliana Morielle-Versute, Matheus R. Beguelini, Renato P. de Souza, Maurício L. Nogueira, Paula Rahal

Viruses are important agents of emerging zoonoses and are a substantial public health issue. Among emerging viruses, an important group are arboviruses, which are characterized by being maintained in nature in cycles involving hematophagous arthropod vectors and a wide range of vertebrate hosts. Recently, bats have received increasing attention as an important source for the emergence of zoonoses and as possible viral reservoirs. Among the arboviruses, there are many representatives of the genera Flavivirus and Alphavirus, which are responsible for important epidemics such as Dengue virus, Zika virus and Chikungunya virus. Due to the importance of analyzing potential viral reservoirs for zoonosis control and expanding our knowledge of bat viruses, this study aimed to investigate the presence of viruses of the Alphavirus and Flavivirus genera in bats. We analyzed serum, liver, lungs and intestine from 103 bats sampled in northeast and southern Brazil via Nested-PCR and the hemagglutination inhibition test. All samples tested in this study were negative for arboviruses, suggesting that no active or past infection was present in the captured bats. These data indicate that the bats examined herein probably do not constitute a reservoir for these viruses in the studied areas. Further studies are needed to clarify the role of bats as reservoirs and sources of infection of these viral zoonoses.

by Leigh R. Bowman, Joacim Rocklöv, Axel Kroeger, Piero Olliaro, Ronald Skewes
Background Aedes-borne arboviruses continue to precipitate epidemics worldwide. In Dominican Republic, the appearance of Zika virus cases that closely followed a large dengue epidemic provided an opportunity to study the different transmission drivers behind these two flaviviruses. Retrospective datasets were used to collect information on the populations at risk and descriptive statistics were used to describe the outbreaks on a national scale. Methodology/ Principal findings Expectedly, box plots showed that 75% of dengue was reported in those aged

by Clarence C. Tam, Megan O’Driscoll, Anne-Frieda Taurel, Joshua Nealon, Sri Rezeki Hadinegoro

Understanding the heterogeneous nature of dengue transmission is important for prioritizing and guiding the implementation of prevention strategies. However, passive surveillance data in endemic countries are rarely adequately informative. We analyzed data from a cluster-sample, cross-sectional seroprevalence study in 1–18 year-olds to investigate geographic differences in dengue seroprevalence and force of infection in Indonesia. We used catalytic models to estimate the force of infection in each of the 30 randomly selected sub-districts. Based on these estimates, we determined the proportion of sub-districts expected to reach seroprevalence levels of 50%, 70% and 90% by year of age. We used population averaged generalized estimating equation models to investigate individual- and cluster-level determinants of dengue seropositivity. Dengue force of infection varied substantially across Indonesia, ranging from 4.3% to 30.0% between sub-districts. By age nine, 60% of sub-districts are expected to have a seroprevalence ≥70%, rising to 83% by age 11. Higher odds of seropositivity were associated with higher population density (OR = 1.54 per 10-fold rise in population density, 95% CI: 1.03–2.32) and with City (relative to Regency) administrative status (OR = 1.92, 95% CI: 1.32–2.79). Our findings highlight the substantial variation in dengue endemicity within Indonesia and the importance of understanding spatial heterogeneity in dengue transmission intensity for optimal dengue prevention strategies including future implementation of dengue vaccination programmes.

Title: Repercussões na qualidade de vida de indivíduos com Artralgia crônica pós Chikungunya
Authors: Cerqueira, Hélvia Maia de Lima; Ribeiro, Iago Barbosa; Cerqueira, Erenilde Marques de; Lima, Maricélia Maia de; Lima, João Batista Oliveira; Alcântara, Luiz Carlos Junior; Lima, Maria Aparecida Oliveira; Rios, Mara Luiza Anunciação; Falcão, Melissa Barreto; Cunha, Rivaldo Venâncio da
Abstract: Um dos graves problemas de saúde pública que recentemente emergiu no cenário epidemiológico brasileiro foi a entrada do vírus Chikungunya (CHIKV) em 2014 no município de Feira de Santana, BA, que deixou um grande contingente de pessoas convivendo com dor articular crônica incapacitante e com repercussões na qualidade de vida (QV) dos indivíduos acometidos pela doença.

by Mehdi R. M. Bidokhti, Debashis Dutta, Lepakshe S. V. Madduri, Shawna M. Woollard, Robert Norgren Jr., Luis Giavedoni, Siddappa N. Byrareddy

Due to the large geographical overlap of populations exposed to Zika virus (ZIKV) and human immunodeficiency virus (HIV), understanding the disease pathogenesis of co-infection is urgently needed. This warrants the development of an animal model for HIV-ZIKV co-infection. In this study, we used adult non-pregnant macaques that were chronically infected with simian immunodeficiency virus/chimeric simian human immunodeficiency virus (SIV/SHIV) and then inoculated with ZIKV. Plasma viral loads of both SIV/SHIV and ZIKV co-infected animals revealed no significant changes as compared to animals that were infected with ZIKV alone or as compared to SIV/SHIV infected animals prior to ZIKV inoculation. ZIKV tissue clearance of co-infected animals was similar to animals that were infected with ZIKV alone. Furthermore, in co-infected macaques, there was no statistically significant difference in plasma cytokines/chemokines levels as compared to prior to ZIKV inoculation. Collectively, these findings suggest that co-infection may not alter disease pathogenesis, thus warranting larger HIV-ZIKV epidemiological studies in order to validate these findings.

by Chia-Hsien Lin, Karin Linda Schiøler, Claus Thorn Ekstrøm, Flemming Konradsen
Background Aedes aegypti carries several viruses of public health importance, including the dengue virus. Dengue is the most rapidly spreading mosquito-borne viral disease in the world. Prevention and control of dengue mainly rely on mosquito control as there is no antiviral treatment or a WHO-approved vaccine. To reduce the Ae. aegypti population, studying the characteristics of their habitats is necessary. Aedes aegypti prefer breeding in artificial water holding containers in peridomestic or domestic settings. Their juveniles (1st - 4th instar larvae and pupae) have a tendency to cluster in certain types of containers. To inform control strategies, it is important to assess whether the pupae subgroup has a distinct distribution by container type as compared to the overall group of juveniles. The objective of this study was to assess for distinct predictors (location, season, and function) of Ae. aegypti juveniles and pupae numbers in water holding containers by applying hurdle model analyses. Methodology The field component of this study was carried out from November 2013 to July 2015 in Southern Taiwan where annual autochthonous dengue has been reported for decades. Water holding containers with stagnant water were identified in a predefined urban area in Kaohsiung City (KH) and a rural area in Pingtung County. Given that mosquito survey data often include many containers with zero Ae. aegypti, a negative binomial hurdle model was applied to model the association between location, seasonal and functional characteristics of the water holding containers and the number of Ae. aegypti in each container. Results The results showed that Ae. aegypti were almost exclusively present in the urban area. In this area, the negative binomial hurdle model predicted significantly more juveniles as well as pupae Ae. aegypti in water holding containers during the wet season when compared to the dry season. Notably, the model predicted more juveniles in containers located on private property compared to those on government property, irrespective of season. As for pupae, the model predicted higher amounts in indoor containers used for water storage compared to outdoor water storage containers, irrespective of season. However, for the specific category ‘other water receptacle’, higher amounts of pupae were predicted in outdoor compared to indoor in water receptacles, such as flower pot saucers and water catchment buckets. Conclusions The difference in predictors for juveniles and the pupae subgroup was identified and it may be of importance to the control strategies of the authorities in KH. At present the authorities focus control activities on all water holding containers found on government property. To improve the ongoing control efforts in KH, the focus of control activities maintained by the KH authorities should be expanded to indoor water storage containers and outdoor water receptacles on both private and government properties to adequately address habitats harboring greater numbers of pupae. In addition, it is proposed to increase community engagement in managing water in all types of water holding containers located on privately owned properties (indoor and outdoor), especially during wet season.

by Jih-Jin Tsai, Ching-Kuan Liu, Wen-Yang Tsai, Li-Teh Liu, Jasmine Tyson, Ching-Yi Tsai, Ping-Chang Lin, Wei-Kung Wang

Dengue virus (DENV) is the leading cause of arboviral diseases in humans worldwide. In this study, we investigated the seroprevalence of DENV infection in two districts of Kaohsiung City, a metropolis in southern Taiwan, where major dengue outbreaks have occurred in the past three decades. We enrolled 1,088 participants from the Sanmin and Nanzih districts after the dengue outbreak of 2015, the largest in Taiwan since World War II, and found an overall DENV seroprevalence of 12.4% (95% confidence interval: 10.5–13.4%) based on the InBios DENV IgG ELISA kit. The ratios of clinically inapparent to symptomatic infections were 2.86 and 4.76 in Sanmin and Nanzih districts, respectively. Consistent with higher case numbers during recent outbreaks, the DENV seroprevalence was higher in Sanmin district (16.4%) than in Nanzih district (6.9%), suggesting district differences in seroprevalence and highlighting the importance of screening the DENV immune status of each individual before using the currently available DENV vaccine, Dengvaxia. In the two districts, the seroprevalence rates increased from 2.1% (in the 30–39-year age group) to 17.1% (60–69) and 50% (70–79). The pattern of a sharp and significant increase in seroprevalence in the 70–79-year age group correlated with a dramatic increase in the proportion of clinically severe DENV infections among total dengue cases in that age group. This differed from observations in the Americas and Southeast Asia and suggested that a large proportion of monotypically immune individuals together with other risk factors may contribute to clinically severe dengue among the elderly in Taiwan.

by Leah C. Katzelnick, Ana Coello Escoto, Benjamin D. McElvany, Christian Chávez, Henrik Salje, Wensheng Luo, Isabel Rodriguez-Barraquer, Richard Jarman, Anna P. Durbin, Sean A. Diehl, Derek J. Smith, Stephen S. Whitehead, Derek A. T. Cummings

The gold-standard method for quantifying neutralizing antibody responses to many viruses, including dengue virus (DENV), is the plaque reduction neutralization test (PRNT, also called the immunofocus reduction neutralization test). The PRNT conducted on 96-well plates is high-throughput and requires a smaller volume of antiserum than on 6- or 24-well plates, but manual plaque counting is challenging and existing automated plaque counters are expensive or difficult to optimize. We have developed Viridot (Viridot package), a program for R with a user interface in shiny, that counts viral plaques of a variety of phenotypes, estimates neutralizing antibody titers, and performs other calculations of use to virologists. The Viridot plaque counter includes an automatic parameter identification mode (misses

by Yu-Hsuan Wu, Chin-Kai Tseng, Chun-Kuang Lin, Chih-Ku Wei, Jin-Ching Lee, Kung-Chia Young
Background Zika virus (ZIKV) infection causes diseases ranging from acute self-limiting febrile illness to life-threatening Guillain–Barré Syndrome and other neurological disorders in adults. Cumulative evidence suggests an association between ZIKV infection and microcephaly in newborn infants. Given the host-range restrictions of the virus, a susceptible animal model infected by ZIKV must be developed for evaluation of vaccines and antivirals. In this study, we propose a convenient mouse model for analysis of neurological disorders caused by ZIKV. Methodology Six-day-old immunocompetent ICR suckling mice were used in the experiment. Different inoculum virus concentrations, challenge routes, and challenge times were assessed. Viremic dissemination was determined in the liver, spleen, kidney, and brain through Western blot assay, plaque assay, absolute quantification real-time PCR, and histological observation. Azithromycin, a well-characterized anti-ZIKV compound, was used to evaluate the ICR suckling mouse model for antiviral testing. Conclusions Signs of illness and neurological disease and high mortality rate were observed in mice injected with ZIKV intracerebrally (102 to 105) and intraperitoneally (103 to 105). Viremic dissemination was observed in the liver, spleen, kidney, and brain. ZIKV transmitted, rapid replicated, and induced monocyte infiltration into the brain approximately 5 to 6 days post inoculum. Azithromycin conferred protection against ZIKV-caused neurological and life-threatening diseases. The developed model of ZIKV infection and disease can be used for screening drugs against ZIKV and discovering the underlying mechanism of ZIKV pathogenesis.

by Alden S. Estep, Neil D. Sanscrainte, Christy M. Waits, Sarah J. Bernard, Aaron M. Lloyd, Keira J. Lucas, Eva A. Buckner, Rajeev Vaidyanathan, Rachel Morreale, Lisa A. Conti, James J. Becnel

Recent outbreaks of locally transmitted dengue and Zika viruses in Florida have placed more emphasis on integrated vector management plans for Aedes aegypti (L.) and Aedes albopictus Skuse. Adulticiding, primarily with pyrethroids, is often employed for the immediate control of potentially arbovirus-infected mosquitoes during outbreak situations. While pyrethroid resistance is common in Ae. aegypti worldwide and testing is recommended by CDC and WHO, resistance to this class of products has not been widely examined or quantified in Florida. To address this information gap, we performed the first study to quantify both pyrethroid resistance and genetic markers of pyrethroid resistance in Ae. aegypti and Ae. albopictus strains in Florida. Using direct topical application to measure intrinsic toxicity, we examined 21 Ae. aegypti strains from 9 counties and found permethrin resistance (resistance ratio (RR) = 6-61-fold) in all strains when compared to the susceptible ORL1952 control strain. Permethrin resistance in five strains of Ae. albopictus was very low (RR

Title: Seja bem vindo vizinho: a viagem de Heitor Praguer Fróes pelos Estados Unidos e a reconstrução da campanha contra a febre amarela nas Américas (1943-1947)
Authors: Magalhães, Rodrigo Cesar da Silva; Maio, Marcos Chor
Abstract: O presente artigo analisa as articulações que levaram à proposição da Campanha Continental para a Erradicação do Aedes aegypti, aprovada e implementada pela Organização Sanitária Pan-Americana (OSP) a partir de 1947, em um contexto marcado por uma crescente cooperação dos países latino-americanos na área da saúde. A análise parte da viagem que o médico brasileiro Heitor Praguer Fróes realizou aos Estados Unidos em 1943, relacionando-a com o papel de destaque desempenhado por ele na proposição da Campanha Continental. A viagem de Fróes e os seus desdobramentos no que concerne à luta contra a febre amarela nas Américas nos oferecem uma possibilidade de revisitar a “Política da Boa Vizinhança”, dialogando com a literatura existente sobre o tema a partir de suas implicações sobre o campo da saúde pública no continente.

Title: Soroprevalência de Zika, Dengue e Chikugunya em uma Comunidade Rural do Estado da Bahia
Authors: Costa, Bernardo Gratival Gouvea; Francisco, Marcos Vinicius Lima de Oliveira; Santos, Cleiton Silva; Casaes, Ane Caroline; Santos, Yuri Tabajara Pereira Costa dos; Jesus, Kelvin Edson Marques de; Silva, Thairone Moura da; Fialho, Thainá Rodrigues de Souza; Santos, Ronald Alves dos; Macedo, Michael Nascimento; Almeida, Breno Lima de; Retallack, Hanna; Alcântara, Luiz Carlos Júnior; Rajan, Jayant; Oliveira, Ricardo Riccio; Siqueira, Isadora Cristina de
Abstract: Doenças transmitidas por vetores levam a óbito cerca de 1 milhão de indivíduos por ano. Há um grande risco de infecção por vírus transmitidos por mosquitos Aedes em cidades nas quais esses encontram condições favoráveis para proliferação . Dessas arboviroses , a doença aguda pelo vírus Zika (ZIKV) , dengue (DENV) e febre de chikungunya (CHIKV) vêm tornando - se preocupação no cenário mundial , devido a fatores , como sua rápida propagação geográfica e os altos custos gerados aos sistemas de saúde.

Title: Prevalence of Zika, dengue and Chikungunya virus infection in pregnant women and surveillance of congenital Zika infection in Salvador, Brazil
Authors: Oliveira, João Vitor; Duarte, A; Santos, Cleiton; Pessoa, L; Magalhães Filho, C; Lima, J; Carvalho, D; Xavier, T; Figueiredo, E; Giovanetti, Marta; Almeida, B; Goes, Jacqueline; Lima, F; Alcantara Júnior, Luis Carlos; Siqueira, Isadora Cristina de

by Suhendro Suwarto, Riyanti Astrid Diahtantri, Mohammad Jauharsyah Hidayat, Bing Widjaya

A prominent histopathological feature of fatal dengue cases is hepatic steatosis. However, the association between hepatic steatosis and dengue severity is unknown. We conducted a study to determine the associations of nonalcoholic fatty liver disease (NAFLD) with laboratory markers of dengue severity and length of hospital stay (LOS). A retrospective study was conducted at a private hospital in Jakarta, Indonesia, from December 2011 to December 2016. Bivariate analysis was performed to analyze the associations of laboratory markers of dengue severity and LOS with the presence or absence of NAFLD in no-plasma-leakage (no leakage) and plasma-leakage (leakage) groups. There were 267 dengue-infected patients included in this study. Of these patients, 115 (43.1%) were classified as belonging to the no leakage group, and 152 (56.9%) were classified as belonging to the leakage group. Of the 115 patients belonging to the no leakage group, 53 (46.1%) did not have NAFLD, and 62 (53.9%) had NAFLD. Of the 152 patients belonging to the leakage group, 85 (55.9%) did not have NAFLD, and 67 (44.1%) had NAFLD. Leakage group patients with NAFLD experienced significantly higher hemoconcentration severity (p = 0.04), lower platelet count (p = 0.004) and higher LOS (p = 0.042) than did leakage group patients without NAFLD. The presence of NAFLD in dengue-infected patients with plasma leakage was associated with more severe hemoconcentration, thrombocytopenia and prolonged hospital stay.

by Chia-Hsien Lin, Karin Linda Schiøler, Claus Thorn Ekstrøm, Flemming Konradsen
Background Aedes aegypti carries several viruses of public health importance, including the dengue virus. Dengue is the most rapidly spreading mosquito-borne viral disease in the world. Prevention and control of dengue mainly rely on mosquito control as there is no antiviral treatment or a WHO-approved vaccine. To reduce the Ae. aegypti population, studying the characteristics of their habitats is necessary. Aedes aegypti prefer breeding in artificial water holding containers in peridomestic or domestic settings. Their juveniles (1st - 4th instar larvae and pupae) have a tendency to cluster in certain types of containers. To inform control strategies, it is important to assess whether the pupae subgroup has a distinct distribution by container type as compared to the overall group of juveniles. The objective of this study was to assess for distinct predictors (location, season, and function) of Ae. aegypti juveniles and pupae numbers in water holding containers by applying hurdle model analyses. Methodology The field component of this study was carried out from November 2013 to July 2015 in Southern Taiwan where annual autochthonous dengue has been reported for decades. Water holding containers with stagnant water were identified in a predefined urban area in Kaohsiung City (KH) and a rural area in Pingtung County. Given that mosquito survey data often include many containers with zero Ae. aegypti, a negative binomial hurdle model was applied to model the association between location, seasonal and functional characteristics of the water holding containers and the number of Ae. aegypti in each container. Results The results showed that Ae. aegypti were almost exclusively present in the urban area. In this area, the negative binomial hurdle model predicted significantly more juveniles as well as pupae Ae. aegypti in water holding containers during the wet season when compared to the dry season. Notably, the model predicted more juveniles in containers located on private property compared to those on government property, irrespective of season. As for pupae, the model predicted higher amounts in indoor containers used for water storage compared to outdoor water storage containers, irrespective of season. However, for the specific category ‘other water receptacle’, higher amounts of pupae were predicted in outdoor compared to indoor in water receptacles, such as flower pot saucers and water catchment buckets. Conclusions The difference in predictors for juveniles and the pupae subgroup was identified and it may be of importance to the control strategies of the authorities in KH. At present the authorities focus control activities on all water holding containers found on government property. To improve the ongoing control efforts in KH, the focus of control activities maintained by the KH authorities should be expanded to indoor water storage containers and outdoor water receptacles on both private and government properties to adequately address habitats harboring greater numbers of pupae. In addition, it is proposed to increase community engagement in managing water in all types of water holding containers located on privately owned properties (indoor and outdoor), especially during wet season.

by Francis Mulwa, Joel Lutomiah, Edith Chepkorir, Samwel Okello, Fredrick Eyase, Caroline Tigoi, Michael Kahato, Rosemary Sang
Background Kenya has experienced outbreaks of chikungunya in the past years with the most recent outbreak occurring in Mandera in the northern region in May 2016 and in Mombasa in the coastal region from November 2017 to February 2018. Despite the outbreaks in Kenya, studies on vector competence have only been conducted on Aedes aegypti. However, the role played by other mosquito species in transmission and maintenance of the virus in endemic areas remains unclear. This study sought to determine the possible role of rural Aedes bromeliae and Aedes vittatus in the transmission of chikungunya virus, focusing on Kilifi and West Pokot regions of Kenya. Methods Four day old female mosquitoes were orally fed on chikungunya virus-infected blood at a dilution of 1:1 of the viral isolate and blood (106.4 plaque-forming units [PFU]/ml) using artificial membrane feeder (Hemotek system) for 45 minutes. The engorged mosquitoes were picked and incubated at 29–30°C ambient temperature and 70–80% humidity in the insectary. At days 5, 7 and 10 post-infection, the mosquitoes were carefully dissected to separate the legs and wings from the body and their proboscis individually inserted in the capillary tube containing minimum essential media (MEM) to collect salivary expectorate. The resultant homogenates and the salivary expectorates were tested by plaque assay to determine virus infection, dissemination and transmission potential of the mosquitoes. Results A total of 515 female mosquitoes (311 Ae. bromeliae and 204 Ae. vittatus) were exposed to the East/Central/South Africa (ECSA) lineage of chikungunya virus. Aedes vittatus showed high susceptibility to the virus ranging between 75–90% and moderate dissemination and transmission rates ranging from 35–50%. Aedes bromeliae had moderate susceptibility ranging between 26–40% with moderate dissemination and transmission rates ranging from 27–55%. Conclusion This study demonstrates that both Ae. vittatus and Ae. bromeliae populations from West Pokot and Kilifi counties in Kenya are competent vectors of chikungunya virus. Based on these results, the two areas are at risk of virus transmission in the event of an outbreak. This study underscores the need to institute vector competence studies for populations of potential vector species as a means of evaluating risk of transmission of the emerging and re-emerging arboviruses in diverse regions of Kenya.

by Cho Naing, Norah Htet Htet, Wong Siew Tung, Arun Kumar Basavaraj, Joon Wah Mak

Individual studies have assessed the association between TNF-α-308G>A and TNF-α-238 G>A polymorphisms and severity of dengue infection. However, the results are inconclusive and most studies had small sample sizes. The objective of this study was to summarize the evidence of association between TNF-α-308 G>A and TNF-α-238 G>A and severity of dengue infection.
This study follows the preferred reporting items for systematic reviews and meta- analyses of genetic association studies, recommended by PLOS One. We calculated pooled odds ratio and its 95% confidence interval (CI) to estimate the association between TNF-α-308 G>A or TNF-α-238 G>A and the risk of severe dengue infections. To determine the information size required for this meta-analysis study, a trial sequential analysis (TSA) was done. Eight studies (640 cases and 1275 controls), which assessed the association of TNF-α-308 G>A or TNF-α-238 G>A and the risk of DHF were included. Overall, we found no significant association between TNF-α-308 G>A and the DHF risk in the allelic model (OR, 0.91; 95% CI, 0.51–1.63), the recessive model (OR,1.32;95%CI,0.73–2.37), the dominant model (OR,0.93;95%CI:0.59–1.47) or the additive model (OR,1.43,95;95%CI:0.79–2.59). There was also no significant association between TNF-α-238 G>A and DHF risk under the allele contrast model (OR:1.51;95%CI:0.88–2.58), the recessive model (OR,1.48,95% CI:0.33–6.58), the dominant model (OR,1.48;95%CI:0.56–3.92), or the additive model (OR:1.5;95%CI:0.34–6.69). On subgroup analysis, neither the Asian population nor the non-Asian population showed significant association between TNF-α-308 G>A/TNF-α-238 G>A and the DHF risk under any genetic models. Leave-one-out meta-analysis showed stability of the results. TSA plots suggested that the sample size in this meta-analysis study was below the required information size.
The findings suggest an inclusive evidence of the association between TNF-α-308/ TNF-α-238 G>A and the risk of developing severe dengue infection. Large studies with evidence of Hardy-Weinberg equilibrium, assessing gene-gene interactions are recommended.

by Jih-Jin Tsai, Ping-Chang Lin, Ching-Yi Tsai, Ying-Hui Wang, Li-Teh Liu

To determine the prevalence of asymptomatic dengue virus-infected blood donors during the largest dengue outbreak in Taiwan history occurred in 2015, we examined the evidence of dengue virus (DENV) infection by the detection of DENV RNA genome using real-time reverse transcription-polymerase chain reaction (real-time RT-PCR), DENV NS1 antigen using rapid diagnosis test (RDT) and anti-dengue antibody using IgM/IgG capture enzyme-linked immunosorbent assay (capture ELISA) and RDT in eight thousand serum samples from blood donations to the blood centers of the Taiwan Blood Services Foundation (TBSF) in Kaohsiung City and Tainan City during the largest dengue outbreak in Taiwan history occurred in 2015. Only one serum sample was positive for DENV RNA detection by using dengue-specific real-time RT-PCR, the virus was DENV-2 determined by serotype-specific real-time RT-PCR and sequencing, and the DENVs in the serum were confirmed as being infectious by a plaque assay. The recipient of this blood did not develop any dengue fever symptom on follow-up. None of the samples was NS1 RDT-reactive. Seventeen IgM-positive samples were identified. There was a low prevalence of asymptomatic confirmed or probable DENV-infected blood donors in our study (0.013% and 0.21%, respectively), and no symptomatic transfusion-transmitted dengue (TT dengue) was developed during the largest dengue outbreak in Taiwan history in highly endemic areas and periods.

by Hui-Ying Ko, Yao-Tsun Li, Day-Yu Chao, Yun-Cheng Chang, Zheng-Rong T. Li, Melody Wang, Chuan-Liang Kao, Tzai-Hung Wen, Pei-Yun Shu, Gwong-Jen J. Chang, Chwan-Chuen King

Purifying selection during dengue viral infection has been suggested as the driving force of viral evolution and the higher complexity of the intra-host quasi-species is thought to offer an adaptive advantage for arboviruses as they cycle between arthropod and vertebrate hosts. However, very few studies have been performed to investigate the viral genetic changes within (intra-host) and between (inter-host) humans in a spatio-temporal scale. Viruses of different serotypes from various countries imported to Taiwan cause annual outbreaks. During 2001–2003, two consecutive outbreaks were caused by dengue virus serotype 2 (DENV-2) and resulted in a larger-scale epidemic with more severe dengue cases in the following year. Phylogenetic analyses showed that the viruses from both events were similar and related to the 2001 DENV-2 isolate from the Philippines. We comprehensively analyzed viral sequences from representative dengue patients and identified three consensus genetic variants, group Ia, Ib and II, with different spatio-temporal population dynamics. The phylodynamic analysis suggested group Ib variants, characterized by lower genetic diversity, transmission rate, and intra-host variant numbers, might play the role of maintenance variants. The residential locations among the patients infected by group Ib variants were in the outer rim of case clusters throughout the 2001–2003 period whereas group Ia and II variants were located in the centers of case clusters, suggesting that group Ib viruses might serve as “sheltered overwintering” variants in an undefined ecological niche. Further deep sequencing of the viral envelope (E) gene directly from individual patient serum samples confirmed the emergence of variants belonging to three quasi-species (group Ia, Ib, and II) and the ancestral role of the viral variants in the latter phase of the 2001 outbreak contributed to the later, larger-scale epidemic beginning in 2002. These findings enhanced our understanding of increasing epidemic severity over time in the same epidemic area. It also highlights the importance of combining phylodynamic and deep sequencing analysis as surveillance tools for detecting dynamic changes in viral variants, particularly searching for and monitoring any specific viral subpopulation. Such subpopulations might have selection advantages in both fitness and transmissibility leading to increased epidemic severity.

by Stephen J. Thomas, Liane Agulto, Kim Hendrickx, Martin Erpicum, Kay M. Tomashek, M. Cristina Cassetti, Catherine Laughlin, Alexander Precioso, Alexander C. Schmidt, Federico Narvaez, João Bosco Siqueira, Hasitha Tissera, Robert Edelman

Dengue virus infections are a major cause of febrile illness that significantly affects individual and societal productivity and drives up health care costs principally in the developing world. Two dengue vaccine candidates are in advanced clinical efficacy trials in Latin America and Asia, and another has been licensed in more than fifteen countries but its uptake has been limited. Despite these advances, standardized metrics for comparability of protective efficacy between dengue vaccines remain poorly defined. The Dengue Illness Index (DII) is a tool that we developed thru refinement of previous similar iterations in an attempt to improve and standardize the measurement of vaccine and drug efficacy in reducing moderate dengue illness. The tool is designed to capture an individual’s overall disease experience based on how the totality of their symptoms impacts their general wellness and daily functionality. We applied the DII to a diary card, the Dengue Illness Card (DIC), which was examined and further developed by a working group. The card was then refined with feedback garnered from a Delphi methodology-based query that addressed the adequacy and applicability of the tool in clinical dengue research. There was overall agreement that the tool would generate useful data and provide an alternative perspective to the assessment of drug or vaccine candidates, which in the case of vaccines, are assessed by their reduction in any virologically confirmed dengue of any severity with a focus on the more severe. The DIC needs to be evaluated in the field in the context of vaccine or drug trials, prospective cohort studies, or during experimental human infection studies. Here, we present the final DIC resulting from the Delphi process and offer its further development or use to the dengue research community.