Background Vector control measures are critical for the prevention and reduction of dengue virus (DENV) transmission. Effective vector control is reliant not only on knowledge of mosquito abundance, but also on the timely and accurate detection of mosquito-borne infection. Mosquito-based virus surveillance programs typically rely on pool-based mosquito testing, although whether individual-based mosquito testing is a feasible alternative to this has not been widely studied. Applying an individual-based mosquito testing approach, we conducted a 1-month surveillance study of DENV in adult Aedes aegypti mosquitoes in homes of suspected dengue patients during the 2015 peak dengue season in Tarlac City, Philippines to more accurately assess the mosquito infection rate and identify the DENV serotypes and genotypes concurrently co-circulating in mosquitoes and patients there. Methods We performed a one-step multiplex real-time reverse transcription-polymerase chain reaction (RT-PCR) assay for the simultaneous detection and serotyping of DENV in patients and individual female Ae. aegypti mosquitoes. Additionally, we performed sequencing and phylogenetic analyses to further characterize the detected DENV serotypes in mosquitoes and patients at the genotype level. Results We collected a total of 583 adult Ae. aegypti mosquitoes, of which we individually tested 359 female mosquitoes for the presence of DENV. Ten (2.8%) of the 359 female mosquitoes were positive for the presence of DENV. We detected DENV-1, DENV-2, and DENV-4 in the field-collected mosquitoes, which was consistent with the serotypes concurrently found in infected patients. Sequencing and phylogenetic analyses of the detected DENV serotypes based on the partial sequence of the evelope (E) gene revealed three genotypes concurrently present in the sampled mosquitoes and patients during the study period, namely DENV-1 genotype IV, DENV-2 Cosmopolitan genotype, and DENV-4 genotype II. Conclusions We demonstrated the utility of a one-step multiplex real-time RT-PCR assay for the individual-based DENV surveillance of mosquitoes. Our findings reinforce the importance of detecting and monitoring virus activity in local mosquito populations, which are critical for dengue prevention and control.

Mosquito-borne flavivirus infections, including dengue virus and Zika virus, are major public health threats globally. While the plaque reduction neutralization test (PRNT) is considered the gold standard for determining neutralizing antibody levels to flaviviruses, the assay is time-consuming and laborious. This study, therefore, aimed to develop an enzyme-linked immunosorbent assay (ELISA)-based microneutralization test (EMNT) for the detection of neutralizing antibodies to mosquito-borne flaviviruses. The inhibition of viral growth due to neutralizing antibodies was determined colorimetrically by using EMNT. Given the significance of Fcγ-receptors (FcγR) in antibody-mediated neutralization and antibody-dependent enhancement (ADE) of flavivirus infection, non-FcγR and FcγR-expressing cell lines were used in the EMNT to allow the detection of the sum of neutralizing and immune-enhancing antibody activity as the neutralizing titer. Using anti-flavivirus monoclonal antibodies and clinical samples, the utility of EMNT was evaluated by comparing the end-point titers of the EMNT and the PRNT. The correlation between EMNT and PRNT titers was strong, indicating that EMNT was robust and reproducible. The new EMNT assay combines the biological functional assessment of virus neutralization activity and the technical advantages of ELISA and, is simple, reliable, practical, and could be automated for high-throughput implementation in flavivirus surveillance studies and vaccine trials.

The RT-qPCR method remains the gold standard and first-line diagnostic method for the detection of SARS-CoV-2 and flaviviruses, especially in the early stage of viral infection. Rapid and accurate viral detection is a starting point in the containment of the COVID-19 pandemic and flavivirus outbreaks. However, the shortage of diagnostic reagents and supplies, especially in resource-limited countries that experience co-circulation of SARS-CoV-2 and flaviviruses, are limitations that may result in lesser availability of RT-qPCR-based diagnostic tests. In this study, the utility of RNA-free extraction methods was assessed for the direct detection of SARS-CoV-2 and DENV-2 in heat-inactivated or chemical-inactivated samples. The findings demonstrate that direct real-time RT-qPCR is a feasible option in comparison to conventional real-time RT-qPCR based on viral genome extraction-based methods. The utility of heat-inactivation and direct real-time RT-qPCR for SARS-CoV-2, DENV-2 viral RNA detection was demonstrated by using clinical samples of SARS-CoV-2 and DENV-2 and spiked cell culture samples of SARS-CoV-2 and DENV-2. This study provides a simple alternative workflow for flavivirus and SARS-CoV-2 detection that includes heat inactivation and viral RNA extraction-free protocols, with aims to reduce the risk of exposure during processing of SARS-CoV-2 biological specimens and to overcome the supply-chain bottleneck, particularly in resource limited settings with flavivirus co-circulation.

Background: Vector control measures are critical in the prevention and reduction of dengue virus (DENV) transmission. In this context, effective vector control is reliant not only on knowledge of mosquito abundance, but also on the timely and accurate detection of mosquito infection. Mosquito-based virus surveillance programs commonly rely on pool-based mosquito testing, but whether individual-based mosquito testing could represent a feasible alternative is not largely studied. Applying an individual-based mosquito testing approach, we conducted a one-month DENV surveillance of adult Aedes aegypti mosquitoes around households of suspected dengue patients during the 2015 dengue peak season in Tarlac City, Philippines to more accurately assess the mosquito infection rate, and to identify the DENV serotypes and genotypes concurrently co-circulating in mosquitoes and patients. Methods: We performed a one-step multiplex real-time RT-PCR assay for the simultaneous detection and serotyping of DENV in patients and in individual female Ae. aegypti mosquito. Additionally, we performed sequencing and phylogenetic analyses to further characterize the detected DENVs in mosquitoes and patients at the genotype level. Results: We collected a total of 583 adult Ae. aegypti mosquitoes, of which we tested 359 female mosquitoes individually for the presence of the DENV. Ten mosquitoes (2.8%) from amongst 359 female mosquitoes were confirmed to be positive for the presence of the DENV. We detected DENV-1, DENV-2, and DENV-4 in the field-collected mosquitoes, which were consistent with the serotypes concurrently infecting patients. Sequencing and phylogenetic analyses of the detected DENVs based on the partial envelope (E) gene revealed three genotypes concurrently present in the sampled mosquitoes and patients during the study period, namely: DENV-1 genotype IV, DENV-2 Cosmopolitan genotype and DENV-4 genotype II. Conclusions: In this study, we demonstrate the utility of a one-step multiplex real-time RT-PCR assay in individual-based DENV surveillance of mosquitoes. Our findings reinforce the importance of detecting and monitoring virus activity in local mosquito populations, which is critical for dengue prevention and control activities.

Background: Vector control measures are critical in the prevention and reduction of dengue virus (DENV) transmission. In this context, an effective vector control is reliant not only on the knowledge of mosquito abundance, but also on the timely and accurate detection of mosquito infection. Mosquito-based virus surveillance programs typically rely on a pool-based mosquito testing, although whether individual-based mosquito testing could represent a feasible alternative is not largely studied. Applying an individual-based mosquito testing approach, we conducted a one-month DENV surveillance of adult Aedes (Ae.) aegypti mosquitoes around households of suspected dengue patients during the 2015 dengue peak season in Tarlac City, Philippines to more accurately assess the mosquito infection rate and identify the DENV serotypes and genotypes concurrently co-circulating in mosquitoes and patients.Methods: We performed a one-step multiplex real-time reverse transcription-polymerase chain reaction (RT-PCR) assay for the simultaneous DENV detection and serotyping in patients and individual female Ae. aegypti mosquito. Additionally, we performed sequencing and phylogenetic analyses to further characterize the detected DENVs in mosquitoes and patients at the genotype level.Results: We collected a total of 583 adult Ae. aegypti mosquitoes; of which, we individually tested 359 female mosquitoes for the presence of DENV. Ten (2.8%) among the 359 female mosquitoes were positive for the presence of DENV. We detected DENV-1, DENV-2, and DENV-4 in the field-collected mosquitoes, which were consistent with the serotypes concurrently infecting patients. Sequencing and phylogenetic analyses of the detected DENVs based on the partial envelope (E) gene revealed three genotypes concurrently present in the sampled mosquitoes and patients during the study period, namely: DENV-1 genotype IV, DENV-2 Cosmopolitan genotype, and DENV-4 genotype II.Conclusions: In this study, we demonstrate the utility of the one-step multiplex real-time RT-PCR assay in the individual-based DENV surveillance of mosquitoes. Our findings reinforce the significance of detecting and monitoring virus activity in local mosquito populations, which is critical for dengue prevention and control activities.

Background Dengue virus (DENV) infection continues to be a major public health concern throughout tropical and subtropical regions of the world where Aedes aegypti mosquitoes, its primary vector, dwell. In the context of DENV transmission, effective control is reliant not only on knowledge of mosquito abundance, but also on mosquito infection. In the 2015 dengue season, we conducted a one-month entomological surveillance of adult Aedes aegypti mosquitoes around households of suspected dengue patients in Tarlac City, Philippines to assess the DENV infection rate in the local mosquito population, and to identify the DENV genotypes and serotypes concurrently co-circulating in mosquitoes and patients. Methods We performed a one-step multiplex real-time RT-PCR assay for the simultaneous detection and serotyping of DENV in patients and in individual female Aedes aegypti mosquito. Consequently, we performed sequencing and phylogenetic analyses to further characterize the detected DENVs in mosquitoes and patients at the genotype level. Results We collected a total of 583 adult Aedes aegypti mosquitoes, of which we tested 359 female mosquitoes individually for the presence of the DENV. Ten mosquitoes (2.8%) from amongst 359 female mosquitoes were confirmed to be positive for the presence of the DENV. We detected DENV-1, DENV-2, and DENV-4 in the field-collected mosquitoes, which were consistent with the serotypes concurrently infecting patients. Sequencing and phylogenetic analyses of the detected DENVs based on the partial envelope ( E ) gene revealed three genotypes concurrently present in the sampled mosquitoes and patients during the study period, namely: DENV-1 genotype IV, DENV-2 Cosmopolitan genotype and DENV-4 genotype II. Notably, we observed a random geographic distribution of DENVs in the study area suggesting the occurrence of active DENV transmission within and outside the vicinities of Tarlac City. Conclusions In this study, we demonstrate the utility of an individual-based DENV surveillance in field-collected mosquitoes and the importance of incorporating mosquito virus data in phylogenetic studies. Analyzing virus sequences from vector and host could potentially improve our understanding of the dynamics of DENV transmission.