Monitoramento das Produções

Title: Proposta de arcabouço regulatório e avaliação do risco sanitário e de biossegurança para a produção de vacina febre amarela de subunidade utilizando plataforma vegetal
Authors: Guimarães, Rosane Cuber
Abstract: O uso de células vegetais e plantas inteiras para sintetizar proteínas que são posteriormente processadas, reguladas e vendidas como medicamentos já é uma realidade mundial. Todavia, o arcabouço regulatório para a plataforma vegetal ainda não está bem estabelecido no mundo e muito menos no Brasil. Sendo uma vacina baseada na produção de antígenos em plantas e considerando que até 2016 nenhuma vacina produzida em plantas foi aprovada para uso em humanos, este estudo se propôs a analisar a produção de vacina de Febre Amarela de subunidade utilizando como inovação uma plataforma tecnológica vegetal, enfatizando o arcabouço regulatório e a legislação de Biossegurança. Neste trabalho foi realizada uma extensa revisão bibliográfica nas normas e diretrizes regulatórias e de biossegurança do Brasil que tratem da produção de biológicos em plataformas vegetais, foi feita uma comparação com a regulação de outros países como Estados Unidos, Canadá, União Europeia, Cuba e Argentina e uma análise das lacunas existentes na regulação brasileira. Utilizando como estudo de caso o desenvolvimento da vacina de Febre Amarela de subunidade, produzida em plataforma vegetal e através do uso da ferramenta HACCP, levantamos todos os pontos críticos de controle do processo de produção, com seus respectivos parâmetros críticos de processos, bem como os controles de qualidade recomendados, correlacionando-os com requisitos de BPF. Através do uso do software BioRAM fizemos uma análise dos riscos de biossegurança da produção da vacina de Febre Amarela de subunidade produzida em plataforma vegetal e nossos resultados demonstram um risco de biossegurança muito baixo para este processo. Além disso, desenvolvemos um Protocolo de Produção de Banco de Células Master e de Trabalho em BPF de forma a estabelecer e caracterizar os bancos de células de Agrobacterium tumefaciens. Sugerimos que algumas regulações sejam revisadas e concluímos que as entidades regulatórias brasileiras devem estabelecer um arcabouço regulatório conjunto e inter-relacionado que permita a produção industrial mantendo todavia a proteção à saúde humana e animal e a proteção ao meio ambiente.

Title: Dendritic cell apoptosis and the pathogenesis of dengue
Authors: Martins, Sharon de Toledo; Silveira, Guilherme Ferreira; Alves, Lysangela Ronalte; Santos, Claudia Nunes Duarte dos; Bordignon, Juliano

by Sébastien Marcombe, Bénédicte Fustec, Julien Cattel, Somesanith Chonephetsarath, Phoutmany Thammavong, Nothasin Phommavanh, Jean-Philippe David, Vincent Corbel, Ian W. Sutherland, Jeffrey C. Hertz, Paul T. Brey
Background The yellow fever mosquito Aedes aegypti is the major vector of dengue, yellow fever, Zika, and Chikungunya viruses. Worldwide vector control is largely based on insecticide treatments but, unfortunately, vector control programs are facing operational challenges due to mosquitoes becoming resistant to commonly used insecticides. In Southeast Asia, resistance of Ae. aegypti to chemical insecticides has been documented in several countries but no data regarding insecticide resistance has been reported in Laos. To fill this gap, we assessed the insecticide resistance of 11 Ae. aegypti populations to larvicides and adulticides used in public health operations in the country. We also investigated the underlying molecular mechanisms associated with resistance, including target site mutations and detoxification enzymes putatively involved in metabolic resistance. Methods and results Bioassays on adults and larvae collected in five provinces revealed various levels of resistance to organophosphates (malathion and temephos), organochlorine (DDT) and pyrethroids (permethrin and deltamethrin). Synergist bioassays showed a significant increased susceptibility of mosquitoes to insecticides after exposure to detoxification enzyme inhibitors. Biochemical assays confirmed these results by showing significant elevated activities of cytochrome P450 monooxygenases (P450), glutathione S-transferases (GST) and carboxylesterases (CCE) in adults. Two kdr mutations, V1016G and F1534C, were detected by qPCR at low and high frequency, respectively, in all populations tested. A significant negative association between the two kdr mutations was detected. No significant association between kdr mutations frequency (for both 1534C and 1016G) and survival rate to DDT or permethrin (P > 0.05) was detected. Gene Copy Number Variations (CNV) were detected for particular detoxification enzymes. At the population level, the presence of CNV affecting the carboxylesterase CCEAE3A and the two cytochrome P450 CYP6BB2 and CYP6P12 were significantly correlated to insecticide resistance. Conclusions These results suggest that both kdr mutations and metabolic resistance mechanisms are present in Laos but their impact on phenotypic resistance may differ in proportion at the population or individual level. Molecular analyses suggest that CNV affecting CCEAE3A previously associated with temephos resistance is also associated with malathion resistance while CNV affecting CYP6BB2 and CYP6P12 are associated with pyrethroid and possibly DDT resistance. The presence of high levels of insecticide resistance in the main arbovirus vector in Laos is worrying and may have important implications for dengue vector control in the country.

Title: A critical analysis of the neurodevelopmental and neurosensory outcomes after 2 years for children with in utero Zika virus exposure
Authors: Vouga, Manon; Pomar, Léo; Panchaud, Alice; Musso, Didier; Baud, David

by Binghua Zhu, Ligui Wang, Haiying Wang, Zhidong Cao, Lei Zha, Ze Li, Zhongyang Ye, Jinping Zhang, Hongbin Song, Yansong Sun
Introduction In order to improve the prediction accuracy of dengue fever incidence, we constructed a prediction model with interactive effects between meteorological factors, based on weekly dengue fever cases in Guangdong, China from 2008 to 2016. Methods Dengue fever data were derived from statistical data from the China National Notifiable Infectious Disease Reporting Information System. Daily meteorological data were obtained from the China Integrated Meteorological Information Sharing System. The minimum temperature for transmission was identified using data fitting and the Ross-Macdonald model. Correlations and interactive effects were examined using Spearman’s rank correlation and multivariate analysis of variance. A probit regression model to describe the incidence of dengue fever from 2008 to 2016 and forecast the 2017 incidence was constructed, based on key meteorological factors, interactive effects, mosquito-vector factors, and other important factors. Results We found the minimum temperature suitable for dengue transmission was ≥18°C, and as 97.91% of cases occurred when the minimum temperature was above 18 °C, the data were used for model training and construction. Epidemics of dengue are related to mean temperature, maximum/minimum and mean atmospheric pressure, and mean relative humidity. Moreover, interactions occur between mean temperature, minimum atmospheric pressure, and mean relative humidity. Our weekly probit regression prediction model is 0.72. Prediction of dengue cases for the first 41 weeks of 2017 exhibited goodness of fit of 0.60. Conclusion Our model was accurate and timely, with consideration of interactive effects between meteorological factors.

by Andrea Gloria-Soria, John Soghigian, David Kellner, Jeffrey R. Powell

The yellow fever mosquito (Aedes aegypti), is the primary vector of dengue, Zika, and chikungunya fever, among other arboviral diseases. It is also a popular laboratory model in vector biology due to its ease of rearing and manipulation in the lab. Established laboratory strains have been used worldwide in thousands of studies for decades. Laboratory evolution of reference strains and contamination among strains are potential severe problems that could dramatically change experimental outcomes and thus is a concern in vector biology. We analyzed laboratory and field colonies of Ae. aegypti and an Ae. aegypti-derived cell line (Aag2) using 12 microsatellites and ~20,000 SNPs to determine the extent of divergence among laboratory strains and relationships to their wild relatives. We found that 1) laboratory populations are less genetically variable than their field counterparts; 2) colonies bearing the same name obtained from different laboratories may be highly divergent; 3) present genetic composition of the LVP strain used as the genome reference is incompatible with its presumed origin; 4) we document changes in two wild caught colonies over ~16 generations of colonization; and 5) the Aag2 Ae. aegypti cell line has experienced minimal genetic changes within and across laboratories. These results illustrate the degree of variability within and among strains of Ae. aegypti, with implications for cross-study comparisons, and highlight the need of a common mosquito repository and the implementation of strain validation tools.

by Monica K. Borucki, Nicole M. Collette, Lark L. Coffey, Koen K. A. Van Rompay, Mona H. Hwang, James B. Thissen, Jonathan E. Allen, Adam T. Zemla

The question of how Zika virus (ZIKV) changed from a seemingly mild virus to a human pathogen capable of microcephaly and sexual transmission remains unanswered. The unexpected emergence of ZIKV’s pathogenicity and capacity for sexual transmission may be due to genetic changes, and future changes in phenotype may continue to occur as the virus expands its geographic range. Alternatively, the sheer size of the 2015–16 epidemic may have brought attention to a pre-existing virulent ZIKV phenotype in a highly susceptible population. Thus, it is important to identify patterns of genetic change that may yield a better understanding of ZIKV emergence and evolution. However, because ZIKV has an RNA genome and a polymerase incapable of proofreading, it undergoes rapid mutation which makes it difficult to identify combinations of mutations associated with viral emergence. As next generation sequencing technology has allowed whole genome consensus and variant sequence data to be generated for numerous virus samples, the task of analyzing these genomes for patterns of mutation has become more complex. However, understanding which combinations of mutations spread widely and become established in new geographic regions versus those that disappear relatively quickly is essential for defining the trajectory of an ongoing epidemic. In this study, multiscale analysis of the wealth of genomic data generated over the course of the epidemic combined with in vivo laboratory data allowed trends in mutations and outbreak trajectory to be assessed. Mutations were detected throughout the genome via deep sequencing, and many variants appeared in multiple samples and in some cases become consensus. Similarly, amino acids that were previously consensus in pre-outbreak samples were detected as low frequency variants in epidemic strains. Protein structural models indicate that most of the mutations associated with the epidemic transmission occur on the exposed surface of viral proteins. At the macroscale level, consensus data was organized into large and interactive databases to allow the spread of individual mutations and combinations of mutations to be visualized and assessed for temporal and geographical patterns. Thus, the use of multiscale modeling for identifying mutations or combinations of mutations that impact epidemic transmission and phenotypic impact can aid the formation of hypotheses which can then be tested using reverse genetics.

by Jacqueline K. Lim, Yaro Seydou, Mabel Carabali, Ahmed Barro, Desire Lucien Dahourou, Kang Sung Lee, Teguewende Nikiema, Suk Namkung, Jung-Seok Lee, Mee Young Shin, Emmanuel Bonnet, Therese Kagone, Losseni Kaba, Tansy Edwards, Paul-André Somé, Jae Seung Yang, Neal Alexander, In-Kyu Yoon, Valéry Ridde
Background In Africa, the magnitude of dengue virus (DENV) transmission is largely unknown. In Burkina Faso, several outbreaks have been reported and data are often based on findings from outbreak investigations. Methods To better understand dengue epidemiology and clinical characteristics in Burkina Faso, a fever surveillance study was conducted among patients aged 1–55 years, who presented with non-malarial febrile illness at five primary healthcare facilities in Ouagadougou, Burkina Faso from December 2014 to February 2017, encompassing a 3-month dengue outbreak in September-November 2016. Acute and convalescent blood samples were collected within an interval of 10–21 days between visits. Acute samples were tested with dengue rapid diagnostic tests (RDT) and a selected subset with RT-PCR, and all acute/convalescent samples with IgM/IgG ELISA. Results Among 2929 non-malarial febrile patients, 740 (25%) were dengue–positive based on RT-PCR and/or IgM/IgG ELISA; 428 out of 777 patients (55%) and 312 out of 2152 (14%) were dengue-positive during outbreak and non-outbreak periods, respectively. There were 11% (316/2929) and 4% (129/2929) patients showing positive for NS1 and IgM, on the RDT, respectively. DENV 2 predominated during the outbreak, whereas DENV 3 predominated before the outbreak. Only 25% of dengue-positive cases were clinically diagnosed with suspected dengue. The odds of requiring observation for ≤3 days (versus routine outpatient care) were 11 times higher among dengue-positive cases than non-dengue cases. In adjusted analyses, dengue-positivity was associated with rash and retro-orbital pain (OR = 2.6 and 7.4, respectively) during the outbreak and with rash and nausea/vomiting (OR = 1.5 and 1.4, respectively) during the non-outbreak period. Conclusion Dengue virus is an important pathogen in Burkina Faso, accounting for a substantial proportion of non-malarial fevers both during and outside outbreak, but is only infrequently suspected by clinicians. Additional longitudinal data would help to further define characteristics of dengue for improved case detection and surveillance.

by Chun-Yin Yeh, Bing-Ze Lu, Wei-Jie Liang, Yu-Chen Shu, Kun-Ta Chuang, Po-Lin Chen, Wen-Chien Ko, Nai-Ying Ko
Background Hepatic dysfunction and coagulopathy are common in acute dengue illness. We analyzed the trajectories of the above parameters in the survivors and fatal patients in the outbreak in Tainan, 2015. Methods A retrospective study was conducted using data from a tertiary hospital between January and December 2015. Multilevel modeling (MLM) was used to identify the changes in aminotransferase (AST), alanine aminotransferase (ALT), activated partial thromboplastin time (aPTT), and platelet counts from Day 0 to Day 7 of the onset of dengue infection. The machine-learning algorithm was used by purity measure assumption to calculate the accuracy of serum transaminases and coagulation variables to discriminate between the fatal and survival groups. Results There were 4,069 dengue patients, of which 0.9% died in one week after illness onset (i.e., early mortality). Case fatality rate was the highest for those aged ≥70 years. Both AST and ALT values of the fatal group were significantly higher than those of the survivor group from Day 3 (AST median, 624 U/L vs. 60 U/L, p 0.001; ALT median, 116 U/L vs. 29 U/L, p = 0.01) of illness onset and peaked on Day 6 (AST median, 9805 U/L vs. 90 U/L, p 0.001; ALT median, 1504 U/L vs. 49 U/L, p 0.001). AST ≥ 203 U/L, ALT ≥ 55 U/L, AST2/ALT criteria ≥337.35, or AST/platelet count ratio index (APRI) ≥ 19.18 on Day 3 of dengue infection had a high true positive rate, 90%, 78%, 100%, or 100%, respectively, of early mortality. The platelet counts of the fatal group declined significantly than those of the survivor group since Day 3 of illness onset (median, 19 x103/μl vs. 91 x103/μl, p 0.01), and aPTT values of the fatal group significantly prolonged longer since Day 5 (median, 68.7 seconds vs. 40.1 seconds, p 0.001). Conclusions AST, ALT, and platelet counts should be monitored closely from Day 0 to Day 3 of dengue infection, and aPTT be followed up on Day 5 of infection to identify the individuals at risk for early mortality.

by Shivanand Hegde, Pornjarim Nilyanimit, Elena Kozlova, Enyia R. Anderson, Hema P. Narra, Sanjeev K. Sahni, Eva Heinz, Grant L. Hughes
Background Symbiotic bacteria are pervasive in mosquitoes and their presence can influence many host phenotypes that affect vectoral capacity. While it is evident that environmental and host genetic factors contribute in shaping the microbiome of mosquitoes, we have a poor understanding regarding how bacterial genetics affects colonization of the mosquito gut. The CRISPR/Cas9 gene editing system is a powerful tool to alter bacterial genomes facilitating investigations into host-microbe interactions but has yet to be applied to insect symbionts. Methodology/Principal findings To investigate the role of bacterial genetic factors in mosquito biology and in colonization of mosquitoes we used CRISPR/Cas9 gene editing system to mutate the outer membrane protein A (ompA) gene of a Cedecea neteri symbiont isolated from Aedes mosquitoes. The ompA mutant had an impaired ability to form biofilms and poorly infected Ae. aegypti when reared in a mono-association under gnotobiotic conditions. In adult mosquitoes, the mutant had a significantly reduced infection prevalence compared to the wild type or complement strains, while no differences in prevalence were seen in larvae, suggesting genetic factors are particularly important for adult gut colonization. We also used the CRISPR/Cas9 system to integrate genes (antibiotic resistance and fluorescent markers) into the symbionts genome and demonstrated that these genes were functional in vitro and in vivo. Conclusions/Significance Our results shed insights into the role of ompA gene in host-microbe interactions in Ae. aegypti and confirm that CRISPR/Cas9 gene editing can be employed for genetic manipulation of non-model gut microbes. The ability to use this technology for site-specific integration of genes into the symbiont will facilitate the development of paratransgenic control strategies to interfere with arboviral pathogens such Chikungunya, dengue, Zika and Yellow fever viruses transmitted by Aedes mosquitoes.

by Frida Jakobsen, Thang Nguyen-Tien, Long Pham- Thanh, Vuong Nghia Bui, Hung Nguyen-Viet, Son Tran- Hai, Åke Lundkvist, Anh Bui- Ngoc, Johanna F. Lindahl

Urban livestock provides an important source of food and income, but it may increase the risks for disease transmission. Vectors, such as mosquitoes, might increase and thereby cause an enhanced transmission of infectious diseases, such as dengue fever; considered the most important mosquito-borne viral disease globally. This cross-sectional study evaluated the awareness of dengue fever and investigated how the presence of dengue vectors is affected by the keeping of livestock in urban households in the city of Hanoi, Vietnam.
From February to March 2018, during the season of lowest occurrence of dengue in Hanoi, 140 households were interviewed, of which 69 kept livestock. A general trend was observed; respondents living in the Dan Phuong district, a peri-urban district, had better knowledge and practice regarding dengue as compared to the urban Ha Dong district. In total, 3899 mosquitoes were collected and identified, of which 52 (1.33%) were Aedes species. A significant difference between the two districts was observed, with more households in Ha Dong having Aedes spp. mosquitoes (p = 0.02) and a higher incidence of dengue fever (p = 0.001). There was no significant association between livestock-rearing and the presence of Aedes spp. mosquitoes (p = 0.955), or between livestock-rearing and the incidence of dengue fever (p = 0.08).
In conclusion, this study could not find any indication that households keeping livestock were at higher risk of dengue virus infections in Hanoi during the season of lowest occurrence of dengue, but clearly indicated the need of more information provided to urban inhabitants, particularly on personal protection.

by Megan B. Vogt, Anismrita Lahon, Ravi P. Arya, Jennifer L. Spencer Clinton, Rebecca Rico-Hesse

One of the most important clinical signs of dengue virus infection is the reduction of white blood cells and platelets in human peripheral blood (leukopenia and thrombocytopenia, respectively), which may significantly impair the clearance of dengue virus by the immune system. The cause of thrombocytopenia and leukopenia during dengue infection is still unknown, but may be related to severe suppression of bone marrow populations including hematopoietic stem cells and megakaryocytes, the progenitors of white blood cells and platelets respectively. Here, we explored the possibility that bone marrow suppression, including ablation of megakaryocyte populations, is caused by dengue virus infection of megakaryocytes. We used three different models to measure dengue virus infection and replication: in vitro, in a human megakaryocyte cell line with viral receptors, ex vivo, in primary human megakaryocytes, and in vivo, in humanized mice. All three systems support dengue virus infection and replication, including virus strains from serotypes 1, 2, and 3, and clinical signs, in vivo; all assays showed viral RNA and/or infectious viruses 7–14 days post-infection. Although we saw no significant decrease in cell viability in vitro, there was significant depletion of mature megakaryocytes in vivo. We conclude that megakaryocytes can produce dengue viruses in the bone marrow niche, and a reduction of cell numbers may affect bone marrow homeostasis.

by Kasen K. Riemersma, Lark L. Coffey

Chikungunya virus (Togaviridae, Alphavirus; CHIKV) is a mosquito-borne global health threat that has been transmitted transiently in the southeastern United States. A primary CHIKV mosquito vector, Aedes aegypti, was recently established in the populous state of California, but the vector competence of Californian mosquitoes is unknown. Explosive CHIKV epidemics since 2004 have been associated with the acquisition of mosquito-adaptive mutations that enhance transmission by Ae. aegypti or Ae. albopictus. As a highly mutable RNA virus, CHIKV has the potential for extensive and rapid genetic diversification in vertebrate hosts and mosquito vectors. We previously demonstrated that expansion of CHIKV diversity in cell culture allows for greater adaptability to novel selection pressures, and that CHIKV fidelity variants are able to diversify more than wildtype (WT) CHIKV in mice. The evolution of intra-vector CHIKV populations and the correlation between CHIKV population diversity and infectivity and transmissibility in mosquitoes has not yet been studied. Here, we address these gaps in knowledge via experimental infection of Ae. aegypti from California with WT and fidelity variant CHIKV. We show that Ae. aegypti from California are highly competent vectors for CHIKV. We also report that CHIKV fidelity variants diversify more than WT in mosquitoes and exhibit attenuated infectivity at the level of the midgut. Furthermore, we demonstrate that intra-vector populations of CHIKV are subjected to purifying selection in mosquito bodies, and sequences of non-coding CHIKV regions are highly conserved. These findings will inform public health risk assessment for CHIKV in California and improve our understanding of constraints to CHIKV evolution in mosquitoes.

by Moshe E. Jasper, Qiong Yang, Perran A. Ross, Nancy Endersby-Harshman, Nicholas Bell, Ary A. Hoffmann

With Wolbachia-based arbovirus control programs being scaled and operationalised around the world, cost effective and reliable detection of Wolbachia in field samples and laboratory stocks is essential for quality control. Here we validate a modified loop-mediated isothermal amplification (LAMP) assay for routine scoring of Wolbachia in mosquitoes from laboratory cultures and the field, applicable to any setting. We show that this assay is a rapid and robust method for highly sensitive and specific detection of wAlbB Wolbachia infection within Aedes aegypti under a variety of conditions. We test the quantitative nature of the assay by evaluating pooled mixtures of Wolbachia-infected and uninfected mosquitoes and show that it is capable of estimating infection frequencies, potentially circumventing the need to perform large-scale individual analysis for wAlbB infection status in the course of field monitoring. These results indicate that LAMP assays are useful for routine screening particularly under field conditions away from laboratory facilities.

by Muhammad Shafique, Sergio Lopes, Dyna Doum, Vanney Keo, Ly Sokha, BunLeng Sam, Chan Vibol, Neal Alexander, John Bradley, Marco Liverani, Jeffrey Hii, Leang Rithea, Siddhi Aryal, John Hustedt
Background In Cambodia dengue vector control activities are focused on larviciding with temephos and pyrethroid based adulticide sprays to which Aedes have been shown to be increasingly resistant.A cluster randomized trial assessed the impact of using biological control tools (guppy fish, pyriproxyfen (PPF), and Communication for Behavioral Impact (COMBI) activities in combination), which would be used in a value comparison to traditional chemical control tools. Given these new intervention methods, a qualitative assessment was designed in order to represent the quality of understanding, acceptance, and implementation by participants. Methodology/Principal findings A total of 103 participants in 12 Focus Group Discussions (FGDs) and nine In-Depth Interviews (IDIs) were included in the study. The majority of participants in intervention villages (50 out of 80) preferred guppy fish over other vector control methods due to ease of use and rearing, quick reproduction and propensity to eat larvae. A substantial number of participants (11 out of 40) in intervention villages with PPF favored it due to long-lasting effectiveness, lack of smell and easy maintenance. Participants showed high demand for both interventions and were willing to pay between 100–500 riel (0.03–0.13 USD). Nearly all participants perceived that the interventions resulted in a reduction in Aedes mosquitos (both adults and immatures) and dengue cases. The presence of larvae in the water despite the use of PPF was a source of concern for some participants, although this was overcome in some cases with proper health education through health volunteers. Interpersonal communication through health volunteers was the most favorite method of transmitting prevention messages. Conclusions/Significance The community led COMBI strategy resulted in high acceptance and perceived effectiveness of the interventions in target villages. Health volunteers are an effective and accepted channel of communication to engage communities, disseminate information and promote behavioral change at the household and community level. If shown effective through corresponding entomological surveys, the interventions should be continued and further strengthened to ensure they are accessible, available and affordable.

by Hideaki Shimizu, Akatsuki Saito, Junko Mikuni, Emi E. Nakayama, Hiroo Koyama, Teruki Honma, Mikako Shirouzu, Shun-ichi Sekine, Tatsuo Shioda

Dengue is a mosquito-borne viral infection that has spread globally in recent years. Around half of the world’s population, especially in the tropics and subtropics, is at risk of infection. Every year, 50–100 million clinical cases are reported, and more than 500,000 patients develop the symptoms of severe dengue infection: dengue haemorrhagic fever and dengue shock syndrome, which threaten life in Asia and Latin America. No antiviral drug for dengue is available. The dengue virus (DENV) non-structural protein 5 (NS5), which possesses the RNA-dependent RNA polymerase (RdRp) activity and is responsible for viral replication and transcription, is an attractive target for anti-dengue drug development. In the present study, 16,240 small-molecule compounds in a fragment library were screened for their capabilities to inhibit the DENV type 2 (DENV2) RdRp activities in vitro. Based on in cellulo antiviral and cytotoxity assays, we selected the compound RK-0404678 with the EC50 value of 6.0 μM for DENV2. Crystallographic analyses revealed two unique binding sites for RK-0404678 within the RdRp, which are conserved in flavivirus NS5 proteins. No resistant viruses emerged after nine rounds of serial passage of DENV2 in the presence of RK-0404678, suggesting the high genetic barrier of this compound to the emergence of a resistant virus. Collectively, RK-0404678 and its binding sites provide a new framework for antiviral drug development.

by Kavita Yadav, Sunil Dhiman, BN Acharya, Rama Rao Ghorpade, Devanathan Sukumaran
Background Reduced susceptibility of mosquito vectors to currently used insecticides hampers control interventions. Recently pyriproxyfen, an insect growth regulator has been demonstrated to effectively reduce the reproductive potential in vector mosquitoes. Methods Pyriproxyfen (PPF), in different concentrations (0.75%, 0.075%, 0.0075%, and 0.00075%) was applied on papers and Indian wild type Aedes aegypti female mosquitoes (N ≥ 20 for each treatment) were exposed onto it as per WHO guidelines, to study the reproductive disruption. PPF concentration on treated papers was quantitatively cross-determined using HPLC method. Reduction in fecundity, fertility and adult emergence in exposed female Ae. aegypti was determined. Abnormal development in ovary and eggs of exposed females was studied microscopically after different time intervals. Results Eggs laid, eggs hatched, pupae formed and adults emerged per female exposed in both before blood meal and after blood meal groups declined significantly from lowest to highest concentration of PPF (F ≥ 5.2; p 0.02). Adult emergence inhibition in females exposed to PPF before and after blood meal groups ranged from 58.8% [OR = 0.18 (95% CI = 0.09–0.36)] to 79.2% [OR = 0.04 (95% CI = 0.02–0.10)] and 64.4% [OR = 0.12 (95% CI = 0.05–0.28)] to 77.2% [OR = 0.05 (95% CI = 0.02–0.14)] respectively in different concentrations. The probit model used suggested that FI50 (50% fertility inhibition) and EI50 (50% emergence inhibition) were 0.002% (p = 0.8) and 0.0001% (p = 0.99) for females exposed before blood meal, while 0.01% (p = 0.6) and

by Nur Alia Johari, Kenny Voon, Shen Yung Toh, Lokman Hakim Sulaiman, Ivan Kok Seng Yap, Patricia Kim Chooi Lim

Dengue fever is endemic in Malaysia, contributing to significant economic and health burden in the country. Aedes aegypti and Ae. albopictus are the main vectors of the dengue virus (DENV), which circulates in sylvatic and human transmission cycles and has been present in Malaysia for decades. The study investigated the presence and distribution of DENV in urban localities in the Klang Valley, Peninsular Malaysia. A total of 364 Ae. aegypti and 1,025 Ae. albopictus larvae, and 10 Ae. aegypti and 42 Ae. albopictus adult mosquitoes were screened for the presence of DENV. In total, 31 (2.2%) samples were positive, of which 2 Ae. albopictus larvae were co-infected with two serotypes, one with DENV-2 and DENV-3 and the other with DENV-3 and DENV-4. Phylogenetic analysis determined that the isolates belonged to DENV-1 genotype I (1 Ae. aegypti adult), DENV-2 (1 Ae. albopictus larva), DENV-3 genotype V (3 Ae. aegypti larvae and 10 Ae. albopictus larvae) and DENV-4 genotype IV (6 Ae. aegypti larvae and 12 Ae. albopictus larvae), a sylvatic strain of DENV-4 which was most closely related with sylvatic strains isolated from arboreal mosquitoes and sentinel monkeys in Peninsular Malaysia in the 1970s. All four DENV serotypes were co-circulating throughout the study period. The detection of a sylvatic strain of DENV-4 in Ae. aegypti and Ae. albopictus mosquitoes in urban areas in Peninsular Malaysia highlights the susceptibility of these vectors to infection with sylvatic DENV. The infectivity and vector competence of these urban mosquitoes to this strain of the virus needs further investigation, as well as the possibility of the emergence of sylvatic virus into the human transmission cycle.

by Víctor Hugo Peña-García, Rebecca C. Christofferson

Chikungunya virus (CHIKV) emerged in Colombia in 2014 into a population presumed fully susceptible. This resulted in a quick and intense spread across Colombia, resulting in an epidemic that affected an estimated 450,000 people. The reported Colombian cases accounted for over 49% of all the cases reported to the PAHO. Eco-environmental factors are known to be associated with the spread of arboviruses such as CHIKV, and likely contribute to the differences in transmission profiles that were observed across several municipalities. To determine the association of eco-environmental factors and CHIKV, the basic reproduction number (R0) in 85 municipalities, which accounted for 65.6% of reported CHIKV cases in Colombia, was estimated. Estimates of R0 ranged from 1 to 9, where over 76% of municipalities had R0 values between 1 and 2. When we looked at the distribution of R0, the cumulative proportions were 20% with R0>2, 14% with R0>3, and 9% with R0>4. Next, we determined that there were different patterns of correlation between environmental and/or ecological variables and R0 when we considered different R0 lower-thresholds. Broadly, we found that temperature-related variables are significantly and positively correlated to R0 regardless of the lower threshold, while other variables like duration of outbreak and size of the urban area are inversely related to R0. Specifically, we conclude that high values of temperature-related variables where R0 > 1 will result in a fast growth of cases in a shorter time period (with faster cessation of outbreak transmission) but will result overall in a fewer total cases compared to outbreak areas (R0 > 1, but classified as lower). Thus, in the absence of vector control, a less explosive outbreak may be more advantageous for the virus in terms of transmission.

by Perran A. Ross, Meng-Jia Lau, Ary A. Hoffmann

Modified Aedes aegypti mosquitoes are being mass-reared for release in disease control programs around the world. Releases involving female mosquitoes rely on them being able to seek and feed on human hosts. To facilitate the mass-production of mosquitoes for releases, females are often provided blood through artificial membrane feeders. When reared across generations there is a risk that mosquitoes will adapt to feeding on membranes and lose their ability to feed on human hosts. To test adaptation to membrane feeding, we selected replicate populations of Ae. aegypti for feeding on either human arms or membrane feeders for at least 8 generations. Membrane-selected populations suffered fitness costs, likely due to inbreeding depression arising from bottlenecks. Membrane-selected females had higher feeding rates on membranes than human-selected ones, suggesting adaptation to membrane feeding, but they maintained their attraction to host cues and feeding ability on humans despite a lack of selection for these traits. Host-seeking ability in small laboratory cages did not differ between populations selected on the two blood sources, but membrane-selected females were compromised in a semi-field enclosure where host-seeking was tested over a longer distance. Our findings suggest that Ae. aegypti may adapt to feeding on blood provided artificially, but this will not substantially compromise field performance or affect experimental assessments of mosquito fitness. However, large population sizes (thousands of individuals) during mass rearing with membrane feeders should be maintained to avoid bottlenecks which lead to inbreeding depression.

by Anthony C. Fredericks, Tiffany A. Russell, Louisa E. Wallace, Andrew D. Davidson, Ana Fernandez-Sesma, Kevin Maringer
Background Aedes aegypti is a vector mosquito of major public health importance, transmitting arthropod-borne viruses (arboviruses) such as chikungunya, dengue, yellow fever and Zika viruses. Wild mosquito populations are persistently infected at high prevalence with insect-specific viruses that do not replicate in vertebrate hosts. In experimental settings, acute infections with insect-specific viruses have been shown to modulate arbovirus infection and transmission in Ae. aegypti and other vector mosquitoes. However, the impact of persistent insect-specific virus infections, which arboviruses encounter more commonly in nature, has not been investigated extensively. Cell lines are useful models for studying virus-host interactions, however the available Ae. aegypti cell lines are poorly defined and heterogenous cultures. Methodology/Principle findings We generated single cell-derived clonal cell lines from the commonly used Ae. aegypti cell line Aag2. Two of the fourteen Aag2-derived clonal cell lines generated harboured markedly and consistently reduced levels of the insect-specific bunyavirus Phasi Charoen-like virus (PCLV) known to persistently infect Aag2 cells. In contrast to studies with acute insect-specific virus infections in cell culture and in vivo, we found that pre-existing persistent PCLV infection had no major impact on the replication of the flaviviruses dengue virus and Zika virus, the alphavirus Sindbis virus, or the rhabdovirus vesicular stomatitis virus. We also performed a detailed characterisation of the morphology, transfection efficiency and immune status of our Aag2-derived clonal cell lines, and have made a clone that we term Aag2-AF5 available to the research community as a well-defined cell culture model for arbovirus-vector interaction studies. Conclusions/Significance Our findings highlight the need for further in vivo studies that more closely recapitulate natural arbovirus transmission settings in which arboviruses encounter mosquitoes harbouring persistent rather than acute insect-specific virus infections. Furthermore, we provide the well-characterised Aag2-derived clonal cell line as a valuable resource to the arbovirus research community.

by Kaiming Tan, Gabriel B. Faierstein, Pingxi Xu, Rosângela M. R. Barbosa, Garrison K. Buss, Walter S. Leal

Insect repellents are widely used as the first line of defense against mosquito bites and transmission of disease-causing agents. However, the cost of daily applications of even the most affordable and the gold standard of insect repellents, DEET, is still high for low-income populations where repellents are needed the most. An Indian clove-based homemade recipe has been presented as a panacea. We analyzed this homemade repellent and confirmed by behavioral measurements and odorant receptor responses that eugenol is the active ingredient in this formulation. Prepared as advertised, this homemade repellent is ineffective, whereas 5x more concentrated extracts from the brand most enriched in eugenol showed moderate repellency activity against Culex quinquefasciatus and Aedes aegypti. DEET showed higher performance when compared to the 5x concentrated formulation and is available in the same market at a lower price than the cost of the ingredients to prepare the homemade formulation.

Title: GloPID-R report on chikungunya, o'nyong-nyong and Mayaro virus, part 3: Epidemiological distribution of Mayaro virus
Authors: Pezzi, L.; Rodriguez-Morales, A. J.; Reusken, C. B.; Ribeiro, Guilherme de Sousa; LaBeaud, A. D.; Oliveira, Ricardo Lourenço de; Brasil, P.; Lecuit, M.; Failloux, A. B.; Gallian, P.; Jaenisch, T.; Simon, F.; Siqueira, A. M.; Freitas, M. G. Rosa; Rua, A. Vega; Weaver, S. C.; Drexler, J. F.; Vasilakis, N.; Lamballerie, X. de; Boyer, S.; Busch, M.; Diallo, M.; Diamond, M. S.; Drebot, M. A.; Kohl, A.; Neytsab, J.; Nga, L. F. P.; Riosad, M.; Sall, A.; Simmons, G.
Description: Ribeiro, Guilherme de Sousa. Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, Brasil. a Unité des Virus Émergents (UVE: Aix-Marseille Univ-IRD 190-Inserm 1207-IHU Méditerranée Infection), Marseille, France
b EA7310, Laboratoire de Virologie, Université de Corse-Inserm, Corte, France
c Public Health and Infection Research Group, Faculty of Health Sciences, Universidad Tecnologica de Pereira, Pereira, Colombia
d Centre for Infectious Disease Control, National Institute for Public Health and the Environment (RIVM), Bilthoven, the Netherlands
e Department Viroscience, Erasmus University Medical Center, Rotterdam, the Netherlands
f Gonçalo Moniz Institute, Oswaldo Cruz Foundation, Federal University of Bahia, Salvador, Brazil
g Department of Pediatrics, Division of Infectious Diseases, Stanford University School of Medicine, Stanford, USA
h Instituto Oswaldo Cruz-Fiocruz, Laboratório de Mosquitos Transmissores de Hematozoários, Rio de Janeiro, Brazil
i Instituto Nacional de Infectologia Evandro Chagas - Oswaldo Cruz Foundation, Rio de Janeiro, Brazil
j Institut Pasteur, Biology of Infection Unit, Inserm U1117, Paris Descartes University, Departement of Infectious Diseases and Tropical Medicine, Necker-Enfants Malades
University Hospital, APHP, IHU Imagine, Paris, France
k Department of Virology, Institut Pasteur, Arboviruses and Insect Vectors Unit, Paris, France
l Établissement Français du Sang Alpes Méditerranée, Marseille, France
m Section Clinical Tropical Medicine, Department of Infectious Diseases, Heidelberg University Hospital, Heidelberg, Germany
n Laveran Military Teaching Hospital, Marseille, France
o Laboratory of Vector Control Research, Environment and Health Unit, Institut Pasteur de la Guadeloupe, Guadeloupe
p Institute for Human Infections and Immunity and Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, USA
q Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, Berlin Institute of Health, Institute of Virology, 10117,
Berlin, Germany
r German Centre for Infection Research (DZIF), Germany
s Department of Pathology, Institute of Human Infection and Immunity, University of Texas Medical Branch, Galveston, USA
t Medical Entomology Platform, Institut Pasteur du Cambodge, Phnom Penh, Cambodia
u Blood Systems Research Institute, San Francisco, Department of Laboratory Medicine, University of California, San Francisco, USA
v Unité d'Entomologie Médicale, Institut Pasteur de Dakar, Dakar, Senegal
w Department of Medicine, The Andrew M. and Jane M. Bursky Center for Human Immunology and Immunotherapy Programs, Washington University School of Medicine,
St. Louis, USA
x Department of Molecular Microbiology, The Andrew M. and Jane M. Bursky Center for Human Immunology and Immunotherapy Programs, Washington University School
of Medicine, St. Louis, USA
y Department of Pathology and Immunology, The Andrew M. and Jane M. Bursky Center for Human Immunology and Immunotherapy Programs, Washington University
School of Medicine, St. Louis, USA
z Zoonotic Diseases and Special Pathogens, National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Canada
aa MRC-University of Glasgow Centre for Virus Research, Glasgow, UK
ab KU Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, Leuven, Belgium
ac Singapore Immunology Network, Agency for Science, Technology and Research (A*STAR), Singapore
ad Division of Emerging and Transfusion Transmitted Diseases, Laboratory of Emerging Pathogens, Office of Blood Research and Review, Center for Biologics Evaluation and
Research, U.S. Food and Drug Administration, Silver Spring, USA
ae Blood Systems Research Institute, San Francisco, USA
af Department of Pathology and Laboratory Medicine, University of California, San Francisco, San Francisco, USA

Title: GloPID-R report on chikungunya, o'nyong-nyong and Mayaro virus, part 2: Epidemiological distribution of o'nyong-nyong virus
Authors: Pezzi, L.; LaBeaud, A. D.; Reusken, C. B.; Drexler, J. F.; Vasilakis, N.; Diallo, M.; Simon, F.; Jaenisch, T.; Gallian, P.; Sall, A.; Failloux, A. B.; Weaver, S. C.; Lamballerie, X. de; Boyero, S.; Brasil, P.; Busch, M.; Diamond, M.S.; Drebot, M.A.; Kohl, A.; Lecuit, M.; Oliveira, Ricardo Lourenço de; Neytsy, J.; Ng, Lfp; Ribeiro, Guilherme de Sousa; Rios, M.; Rodriguez-Morales, A. J.; Freitas, M. G. Rosa; Simmons, G.; Siqueira, A. M.; Ruaae, A. Vega
Description: Ribeiro, Guilherme de Sousa. Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, Brasil. a Unité des Virus Émergents (UVE: Aix-Marseille Univ-IRD 190-Inserm 1207-IHU Méditerranée Infection), Marseille, France
b EA7310, Laboratoire de Virologie, Université de Corse-Inserm, Corte, France
c Department of Pediatrics, Division of Infectious Diseases, Stanford University School of Medicine, Stanford, USA
d Centre for Infectious Disease Control, National Institute for Public Health and the Environment (RIVM), Bilthoven, the Netherlands
e Department Viroscience, Erasmus University Medical Center, Rotterdam, the Netherlands
f Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, And Berlin Institute of Health, Institute of Virology,
10117, Berlin, Germany
g German Centre for Infection Research (DZIF), Germany
h Department of Pathology, Institute of Human Infection and Immunity, University of Texas Medical Branch, Galveston, USA
i Unité D'Entomologie Médicale, Institut Pasteur de Dakar, Dakar, Senegal
j Laveran Military Teaching Hospital, Marseille, France
k Section Clinical Tropical Medicine, Department of Infectious Diseases, Heidelberg University Hospital, Heidelberg, Germany
l Établissement Français Du Sang Alpes Méditerranée, Marseille, France
m Department of Virology, Institut Pasteur, Arboviruses and Insect Vectors Unit, Paris, France
n Institute for Human Infections and Immunity and Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, USA
o Medical Entomology Platform, Institut Pasteur Du Cambodge, Phnom Penh, Cambodia
p Instituto Nacional de Infectologia Evandro Chagas - Oswaldo Cruz Foundation, Rio de Janeiro, Brazil
q Blood Systems Research Institute, San Francisco, and Department of Laboratory Medicine, University of California, San Francisco, USA
r Department of Medicine, and the Andrew M. and Jane M. Bursky Center for Human Immunology and Immunotherapy Programs, Washington University School of
Medicine, St. Louis, USA
s Department of Molecular Microbiology, And the Andrew M. and Jane M. Bursky Center for Human Immunology and Immunotherapy Programs, Washington University
School of Medicine, St. Louis, USA
t Department of Pathology and Immunology, And the Andrew M. and Jane M. Bursky Center for Human Immunology and Immunotherapy Programs, Washington University
School of Medicine, St. Louis, USA
u Zoonotic Diseases and Special Pathogens, National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Canada
v MRC-University of Glasgow Centre for Virus Research, Glasgow, UK
w Institut Pasteur, Biology of Infection Unit, Inserm U1117, Paris Descartes University, Departement of Infectious Diseases and Tropical Medicine, Necker-Enfants Malades
University Hospital, APHP, IHU Imagine, Paris, France
x Instituto Oswaldo Cruz-Fiocruz, Laboratório de Mosquitos Transmissores de Hematozoários, Rio de Janeiro, Brazil
y KU Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, Leuven, Belgium
z Singapore Immunology Network, Agency for Science, Technology and Research (A*STAR), Singapore
aa Gonçalo Moniz Institute, Oswaldo Cruz Foundation, And Federal University of Bahia, Salvador, Brazil
ab Division of Emerging and Transfusion Transmitted Diseases, Laboratory of Emerging Pathogens, Office of Blood Research and Review, Center for Biologics Evaluation and
Research, U.S. Food and Drug Administration, Silver Spring, USA
ac Public Health and Infection Research Group, Faculty of Health Sciences, Universidad Tecnologica de Pereira, Pereira, Colombia

by Trung Tuan Vu, Hannah Clapham, Van Thi Thuy Huynh, Long Vo Thi, Dui Le Thi, Nhu Tuyet Vu, Giang Thi Nguyen, Trang Thi Xuan Huynh, Kien Thi Hue Duong, Vi Thuy Tran, Huy Le Anh Huynh, Duyen Thi Le Huynh, Thuy Le Phuong Huynh, Thuy Thi Van Nguyen, Nguyet Minh Nguyen, Tai Thi Hue Luong, Nguyen Thanh Phong, Chau Van Vinh Nguyen, Gerald Gough, Bridget Wills, Lauren B. Carrington, Cameron P. Simmons
Background Dengue is the most prevalent arboviral disease of humans. Virus neutralizing antibodies are likely to be critical for clinical immunity after vaccination or natural infection. A number of human monoclonal antibodies (mAbs) have previously been characterized as able to neutralize the infectivity of dengue virus (DENV) for mammalian cells in cell-culture systems. Methodology/Principle findings We tested the capacity of 12 human mAbs, each of which had previously been shown to neutralize DENV in cell-culture systems, to abrogate the infectiousness of dengue patient viremic blood for mosquitoes. Seven of the twelve mAbs (1F4, 14c10, 2D22, 1L12, 5J7, 747(4)B7, 753(3)C10), almost all of which target quaternary epitopes, inhibited DENV infection of Ae. aegypti. The mAbs 14c10, 747(4)B7 and 753(3)C10 could all inhibit transmission of DENV in low microgram per mL concentrations. An Fc-disabled variant of 14c10 was as potent as its parent mAb. Conclusions/Significance The results demonstrate that mAbs can neutralize infectious DENV derived from infected human cells, in the matrix of human blood. Coupled with previous evidence of their ability to prevent DENV infection of mammalian cells, such mAbs could be considered attractive antibody classes to elicit with dengue vaccines, or alternatively, for consideration as therapeutic candidates.

by Fabio Antonio Venancio, Maria Eulina Quilião Bernal, Maria da Conceição de Barros Vieira Ramos, Neuma Rocha Chaves, Marcos Vinicius Hendges, Mattheus Marques Rodrigues de Souza, Márcio José de Medeiros, Cláudia Du Bocage Santos Pinto, Everton Falcão de Oliveira

by Pattamaporn Kittayapong, Suwannapa Ninphanomchai, Wanitch Limohpasmanee, Chitti Chansang, Uruyakorn Chansang, Piti Mongkalangoon
Background Important arboviral diseases, such as dengue, chikungunya, and Zika virus infections, are transmitted mainly by the Aedes aegypti vector. So far, controlling this vector species with current tools and strategies has not demonstrated sustainable and significant impacts. Our main objective was to evaluate whether open field release of sterile males, produced from combining the sterile insect technique using radiation with the insect incompatible technique through Wolbachia-induced incompatibility (SIT/IIT), could suppress natural populations of Ae. aegypti in semi-rural village settings in Thailand. Methodology/Principal findings Irradiated Wolbachia-infected Aedes aegypti males produced by the SIT/IIT approach were completely sterile and were able to compete with the wild fertile ones. Open field release of these sterile males was conducted in an ecologically isolated village in Chachoengsao Province, eastern Thailand. House-to-house visit and media reports resulted in community acceptance and public awareness of the technology. During intervention, approximately 100–200 sterile males were released weekly in each household. After 6 months of sterile male release, a significant reduction (p

Title: Development and assessment of the feasibility of a Zika family support programme: a study protocol
Authors: Duttine, Antony; Smythe, Tracey; Sá, Miriam Ribeiro Calheiro de; Ferrite, Silvia; Moreira, Maria Elisabeth; Kuper, Hannah

by I Made Susila Utama, Nurhayati Lukman, Dewi Dian Sukmawati, Bachti Alisjahbana, Anggraini Alam, Dewi Murniati, I Made Gede Dwi Lingga Utama, Dwiyanti Puspitasari, Herman Kosasih, Ida Laksono, Muhammad Karyana, Mulya Rahma Karyanti, M. M. D. E. A. H. Hapsari, Ninny Meutia, C Jason Liang, Wahyu Nawang Wulan, Chuen-Yen Lau, Ketut Tuti Merati Parwati
Background Dengue virus (DENV) infection is a major cause of acute febrile illness in Indonesia. Diagnostic inaccuracy may occur due to its varied and non-specific presentation. Characterization of DENV epidemiology, clinical presentation, and virology will facilitate appropriate clinical management and public health policy. Methodology/Principal findings A multicenter observational cohort study was conducted in Indonesia to assess causes of acute fever requiring hospitalization. Clinical information and specimens were collected at enrollment, 14–28 days, and 3 months from 1,486 children and adults. Total of 468 (31.9%) cases of DENV infection were confirmed by reference laboratory assays. Of these, 414 (88.5%) were accurately diagnosed and 54 had been misdiagnosed as another infection by sites. One hundred initially suspected dengue cases were finally classified as ‘non-dengue’; other pathogens were identified in 58 of those cases. Mortality of DENV infection was low (0.6%). Prior DENV exposure was found in 92.3% of subjects >12 years. DENV circulated year-round in all cities, with higher incidence from January to March. DENV-3 and DENV-1 were the predominant serotypes. This study identified DENV-1 with TS119(C→T) substitution in the serotyping primer annealing site, leading to failure of serotype determination. Conclusions/Significance DENV is a common etiology of acute febrile illness requiring hospitalization in Indonesia. Diagnostic accuracy at clinical sites merits optimization since misdiagnosis of DENV infection and over-estimation of dengue can negatively impact management and outcomes. Mutation at the annealing site of the serotyping primer may confound diagnosis. Clinicians should consider following diagnostic algorithms that include DENV confirmatory testing. Policy-makers should prioritize development of laboratory capacity for diagnosis of DENV.

by Sara Buezo Montero, Paolo Gabrieli, Francesco Severini, Leonardo Picci, Marco Di Luca, Federico Forneris, Luca Facchinelli, Marta Ponzi, Fabrizio Lombardo, Bruno Arcà
Background Aedes mosquitoes are vectors of arboviral diseases of great relevance for public health. The recent outbreaks of dengue, Zika, chikungunya and the rapid worldwide spreading of Aedes albopictus emphasize the need for improvement of vector surveillance and control. Host antibody response to mosquito salivary antigens is emerging as a relevant additional tool to directly assess vector-host contact, monitor efficacy of control interventions and evaluate risk of arboviral transmission. Methodology/principal findings Groups of four BALB/c mice were immunized by exposure to bites of either Aedes albopictus or Aedes aegypti. The 34k2 salivary proteins from Ae. albopictus (al34k2) and Ae. aegypti (ae34k2) were expressed in recombinant form and Ae. albopictus salivary peptides were designed through B-cell epitopes prediction software. IgG responses to salivary gland extracts, peptides, al34k2 and ae34k2 were measured in exposed mice. Both al34k2 and ae34k2, with some individual and antigen-specific variation, elicited a clearly detectable antibody response in immunized mice. Remarkably, the two orthologous proteins showed very low level of immune cross-reactivity, suggesting they may eventually be developed as species-specific markers of host exposure. The al34k2 immunogenicity and the limited immune cross-reactivity to ae34k2 were confirmed in a single human donor hyperimmune to Ae. albopictus saliva. Conclusions/significance Our study shows that exposure to bites of Ae. albopictus or Ae. aegypti evokes in mice species-specific IgG responses to al34k2 or ae34k2, respectively. Deeper understanding of duration of antibody response and validation in natural conditions of human exposure to Aedes mosquitoes are certainly needed. However, our findings point to the al34k2 salivary protein as a promising potential candidate for the development of immunoassays to evaluate human exposure to Ae. albopictus. This would be a step forward in the establishment of a serological toolbox for the simultaneous assessment of human exposure to Aedes vectors and the pathogens they transmit.