Adaptation of a Human Diagnostic Kit to Detect Dengue, Zika, and Chikungunya Viruses in Mosquito Samples (Aedes aegypti and Aedes albopictus): A Contribution to Public Health in the International Triple Border (Brazil, Paraguay, and Argentina)
Robson Michael DelaiAndré de Souza LeandroCaroline Amaral MartinsAndressa Faria Rahyn FitzAçucena Veleh RivasAline Cristiane Cechinel Assing BatistaIsabela Carvalho dos SantosMarcelo FruehwirthLeonardo F. FerreiraRita de Cássia Pontello RampazzoLarissa Rafaela de Paula FerreiraDaniela Dib Gonçalves
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Objective: The objective of this work was to adapt a diagnostic kit developed for humans to identify Dengue (DENV1, DENV2, DENV3, DENV4), Zika (ZIKV) and Chikungunya virus (CHIKV) in females of Aedes aegypti and Aedes albopictus and to verify if the occurrence of mosquitoes infected with these three arboviruses are being found in regions with high occurrence of these diseases in humans. Materials and Methods: For this purpose, live mosquitoes were captured between January and June 2020 using 3,476 traps permanently installed in the field were used. After capture, the species were identified, then the females were placed in a pool of 2 to 10 specimens and sent to the laboratory for detection of DENV1, DENV2, DENV3, DENV4, ZIKV and CHIKV by RT-PCR using a commercial human kit for arboviruses. Results: Of the 76 mosquito pools collected, six (7.9%) pools tested positive for the DENV2 virus. The DENV-positive mosquitoes were collected in regions with a high incidence of reported cases of Dengue or in adjacent areas. Conclusion: The absence of kits for the detection of these arboviruses in Aedes is a limiting factor and the adequacy of commercial kits, already used for the diagnosis of arboviruses in humans, the results presented demonstrate that it is possible to identify the presence of DENV2 in mosquitoes with the respective kit, reinforcing the use of RT-qPCR as a robust diagnostic tool for epidemiological surveillance allowing managers to receive timely results for decision-making regarding prevention and control actions.Keywords:
Aedes albopictus
Zika Virus
ABSTRACT 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 that more closely mimic the situation 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. AUTHOR SUMMARY Mosquito-borne viruses usually only infect humans through the bite of a mosquito that carries the virus. Viruses transmitted by the ‘yellow fever mosquito’ Aedes aegypti , including dengue virus, Zika virus, yellow fever virus and chikungunya virus, are causing an ever-increasing number of human disease cases globally. Mosquito-borne viruses have to infect and replicate inside the mosquito before they are transmitted to humans, and the presence of other infectious agents can change the efficiency of virus transmission. Mosquitoes are known to be infected with ‘insect-specific viruses’ that only infect mosquitoes and cannot cause human disease. We have shown here that in laboratory cell cultures derived from the Aedes aegypti mosquito, pre-existing infection with an insect-specific virus called Phasi Charoen-like virus does not affect the infection and growth of the mosquito-borne viruses dengue virus, Zika virus, Sindbis virus or vesicular stomatitis virus. Compared to previous research, our research is more reflective of conditions that mosquito-borne viruses encounter in nature, and our results provide important new insights into whether and how insect-specific viruses affect mosquito-borne virus transmission. Ultimately, this information could inform ongoing research into whether insect-specific viruses could be used to prevent the transmission of mosquito-borne viruses to reduce global disease burdens.
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Zika Virus
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Dengue, Chikungunya, and Zika: The Causes and Threats of Emerging and Re-emerging Arboviral Diseases
The recent emergence and re-emergence of viral infections transmitted by vectors, Zika, chikungunya, dengue, and others, is a cause for international concern. Here, we provide a summary of the current understanding of the transmission, clinical features, diagnosis, global burden, and the likelihood of future epidemics by these viruses. Arboviruses transmitted by mosquitoes are challenging to diagnose and can have surprising clinical complications. Dengue, chikungunya, and Zika are the most important diseases caused by arboviruses worldwide, especially in tropical and subtropical regions. These are transmitted to humans by day-biting Aedes aegypti and Aedes albopictus mosquitoes. In India, the increase in the incidence of dengue and chikungunya cases is primarily linked to the dissemination of Aedes aegypti. A rapid and accurate diagnosis is paramount for effectively controlling dengue outbreaks. As there is no vaccination or specific treatment available for these viruses, vector control is the only comprehensive solution available.
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Aedes albopictus
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There are reports about the susceptibility of Aedes mosquitoes to ZIKV from various countries, however, no such information is available from Indian sub-continent, although, high level of group cross-reactivity of ZIKV with other flaviviruses has been reported. During outbreak situations, many cases of Dengue (DEN) and Chikungunya (CHIK) are reported. In such scenario, vector mosquitoes are likely to get co-infection/secondary-infection with one or other virus. The present study was carried out to determine the susceptibility of Indian strain of Aedes aegypti to Zika virus (ZIKV) strain (MR-766) and the effect of co-infection/super-infection with either dengue virus (serotype-2) (DENV) or chikungunya virus (CHIKV) on ZIKV replication.Ae. aegypti mosquitoes used in this study were reared for many generations since 1980 at laboratory colony maintained at the ICMR-National Institute of Virology, Pune, India. Transmissibility of ZIKV from infected mosquitoes to suckling mice was also studied. Mosquitoes were experimentally infected with ZIKV and super-infected with either DENV or CHIKV via membrane-feeding route and incubated for 14 days at 28±2°C and humidity of 85±5 per cent. Replication of these viruses in mosquitoes was confirmed using real-time reverse transcription-polymerase chain reaction and immunofluorescence assay. Twenty infected mosquitoes were allowed to feed upon four suckling CD1 mice for about 30 min. Transmission of the ZIKV by infected mosquitoes to suckling mice was confirmed by the appearance of clinical signs and the presence of viral RNA in different organs.Concomitant infection of mosquitoes with all the three viruses showed simultaneous propagation of all three viruses, confirmed by real time RT-PCR and IFA. Infection of mosquitoes with CHIKV followed by ZIKV showed positivity in individual head squashes (7%) for both viruses using IFA; only 8.3 per cent showed dual positivity with primary infection of ZIKV followed by DENV; 8.3 per cent dual infection positivity was observed when infected with DENV followed by ZIKV; 5 per cent showed dual infection was observed when infected with ZIKV followed by CHIKV. Ae. aegypti was found to be susceptible to ZIKV strain as ZIKV could be detected from the second post-infection day (PID) in infected mosquitoes. Transmission of ZIKV to mice by the bite of infected Ae. aegypti establishes this species as a potential vector.From super-infection experiments, it was concluded that ZIKV might have a relative advantage in replication dynamics over DENV. Vertical transmission was not observed for ZIKV in experimentally infected mosquitoes (n=920 larvae). Further studies are required to understand the possibility of silently circulating ZIKV in India, which remain non-detected because of lack of surveillance.
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Mechanical transmission is an understudied mode of arbovirus transmission that occurs when a biting insect transmits virus among hosts by the direct transfer of virus particles contaminating its mouthparts. Multiple arboviruses have been shown to be capable of utilizing this transmission route, but most studies were conducted 40 to 70 years ago using dated methodologies. To gain a better understanding of this phenomenon, we used molecular techniques to evaluate the efficiency of mechanical transmission by Aedes aegypti mosquitoes for two evolutionarily divergent arboviruses, chikungunya virus (CHIKV) and dengue virus (DENV). Viral RNA and/or infectious DENV could be detected on 13.8% of mosquito proboscises sampled immediately after an infectious bloodmeal, but positivity rates declined within hours. CHIKV RNA and/or infectious virus was detected on 38.8% of proboscises immediately after feeding but positivity rates dropped to 2.5% within 4 hours. RNA copy numbers were low for both viruses, and we were unable to demonstrate mechanical transmission of CHIKV using an established animal model, suggesting that this mode of transmission is unlikely under natural conditions.
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Infections caused by arboviruses and transmitted by Aedes species mosquitoes are a serious health concern. India is endemic for diseases like Dengue, Chikungunya and recently Zika has been reported from few states. Vector control is the only way to contain these diseases, however, data regarding vectors from central India is lacking; to fulfill the lacuna we conducted this study.Entomological surveys were conducted from November 2017 to December 2018 for Aedes species in Dengue endemic areas of central India. The mosquitoes were identified, pooled and tested for the presence of Dengue, Chikungunya and Zika viruses by RT-PCR. The PCR products were sequenced to identify serotypes and genotypes of viruses.A total of 2991 adults of Aedes specimens were collected and tested. Ae. aegypti (94.6%) was found to be the most abundant species. Highest mosquito density was recorded in the monsoon periods. Dengue (n=5) and Chikungunya (n=4) virus were detected from pools of female Ae. aegypti. One pool of male Ae. aegypti was positive for Dengue virus-3 and Chikungunya virus. Zika virus was not detected from any pool.The findings suggest that Ae. aegypti is the principal vector of Dengue and Chikungunya, which is capable to transmit these viruses vertically. The findings have epidemiological importance and will be helpful to program managers.
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Dengue fever, chikungunya, and Zika are diseases caused by viruses transmitted by Aedes aegypti and Aedes albopictus. In Brazil, the number of human infections is high, but few studies are performed in mosquito vectors. This study aimed to investigate the presence of Zika, Dengue and Chikungunya viruses in Ae. aegypti and Ae. albopictus from the municipalities of Alto Alegre, Caxias, Codó, and São Mateus do Maranhão, located in the state of Maranhão, Northeast Brazil. The mosquitoes were collected with a mechanical aspirator, identified, triturated, and then submitted to RNA extraction and RT-qPCR. The positive samples were confirmed by virus isolation and genome sequencing. Three hundred and forty-eight Ae. aegypti (176 males and 172 females) and 12 Ae. albopictus (eight males and four females) were collected and tested. Ae. aegypti was the only vector positive in two municipalities—Codó, with detection of Chikungunya virus (CHIKV) belonging to the East-Central-South African genotype, and in Caxias, with detection of Dengue virus (DENV)-2 belonging to the Asian/American genotype. The detection of CHIKV and DENV-2 is evidence that those viruses are maintained in arthropod vectors, and shows the epidemiological risk in the area for chikungunya cases and a possible increase of severe dengue cases, associated with the occurrence of dengue hemorrhagic fever.
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dengue fever (DF). Meanwhile, chikungunya virus causes Chikungunya fever (CF). These diseases involve three organisms, namely virus, mosquito Aedes sp., and human. The transmission of dengue and chikungunya virus is related to the population of Ae. aegypti. Banyumas regency is one of the regions with many cases of dengue and chikungnya virus infections, particularly in Purwokerto, Sokaraja, and Cilongok sub-district. Up to this time, there is no medicine and vaccine provided to treat these viruses effectively. Thus, detection of virus inside vector will be effectively performed in order to predict the transmission risk of dengue and chikungunya virus. This research aimed to know the molecular detection of dengue and chikungunya virus on adult Ae.aegypti mosquito in Sokaraja Region, Banyumas Regency. Survey was done by a cross-sectional method in Sokaraja sub-district from May 2019 – March 2019. Furthermore, technical sampling that used was purposive sampling method of adult Ae.aegypti using BG-Sentital Trap, followed by molecular detection of dengue virus using Two-step RT-PCR and chikungunya gene virus using RT-PCR. Molecular detection of DENV and CHIKV of mosquitoes which collected from Sokaraja region showed negative result
Aedes albopictus
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The mosquito Aedes aegypti carries several arthropod-borne viruses (arboviruses) that are pathogenic to humans, including dengue and Zika viruses. Interestingly, A. aegypti is also naturally infected with insect-only viruses, such as cell-fusing agent virus. Although interactions between cell-fusing agent virus and dengue virus have been documented in mosquito cells in culture, whether wild strains of cell-fusing agent virus interfere with arbovirus transmission by live mosquitoes was unknown. We used an experimental approach to demonstrate that cell-fusing agent virus infection reduces the propagation of dengue and Zika viruses in A. aegypti mosquitoes. These results support the idea that insect-only viruses in nature can modulate the ability of mosquitoes to carry arboviruses of medical significance and that they could possibly be manipulated to reduce arbovirus transmission.
Arbovirus Infections
Zika Virus
Flavivirus
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Objective: The objective of this work was to adapt a diagnostic kit developed for humans to identify Dengue (DENV1, DENV2, DENV3, DENV4), Zika (ZIKV) and Chikungunya virus (CHIKV) in females of Aedes aegypti and Aedes albopictus and to verify if the occurrence of mosquitoes infected with these three arboviruses are being found in regions with high occurrence of these diseases in humans. Materials and Methods: For this purpose, live mosquitoes were captured between January and June 2020 using 3,476 traps permanently installed in the field were used. After capture, the species were identified, then the females were placed in a pool of 2 to 10 specimens and sent to the laboratory for detection of DENV1, DENV2, DENV3, DENV4, ZIKV and CHIKV by RT-PCR using a commercial human kit for arboviruses. Results: Of the 76 mosquito pools collected, six (7.9%) pools tested positive for the DENV2 virus. The DENV-positive mosquitoes were collected in regions with a high incidence of reported cases of Dengue or in adjacent areas. Conclusion: The absence of kits for the detection of these arboviruses in Aedes is a limiting factor and the adequacy of commercial kits, already used for the diagnosis of arboviruses in humans, the results presented demonstrate that it is possible to identify the presence of DENV2 in mosquitoes with the respective kit, reinforcing the use of RT-qPCR as a robust diagnostic tool for epidemiological surveillance allowing managers to receive timely results for decision-making regarding prevention and control actions.
Aedes albopictus
Zika Virus
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