Dengue virus type 4 in Niterói, Rio de Janeiro: the role of molecular techniques in laboratory diagnosis and entomological surveillance

Castro, Márcia Gonçalves de; Nogueira, Rita Maria Ribeiro; Filippis, Ana María Bispo de; Ferreira, Anielly Alves; Lima, Monique da Rocha Queiroz; Faria, Nieli Rodrigues da Costa; Nogueira, Fernanda de Bruycker; Simões, Jaqueline Bastos Santos; Nunes, Priscila Conrado Guerra; Sampaio, Simone Alves; Lourenço‐de‐Oliveira, Ricardo; Santos, Flávia Barreto dos

doi:10.1590/s0074-02762012000700017

Cited by 22 publications

(25 citation statements)

References 28 publications

(28 reference statements)

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“…Molecular detection of ZIKV ribonucleic acid (RNA) in mosquitoes can be challenging due to the limited number of primers and probes published [14][15][16][17][18][19][20][21][22][23][24][25][26][27], as well as the presence of viral genetic material integrated in mosquito genomes [28,29], which can reduce the specificity for RNA detections by quantitative reverse transcription polymerase chain reaction (RT-qPCR) [30]. Most of these ZIKV oligos were optimized in mammalian cells and samples and show often-unresolvable background when used to detect infection in mosquito tissues.…”

Section: Introductionmentioning

confidence: 99%

One-step RT-qPCR assay for ZIKV RNA detection in Aedes aegypti samples: a protocol to study infection and gene expression during ZIKV infection

et al. 2020

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Background: Zika virus (ZIKV) is transmitted to humans during the bite of an infected mosquito. In a scenario of globalization and climate change, the frequency of outbreaks has and will increase in areas with competent vectors, revealing a need for continuous improvement of ZIKV detection tools in vector populations. A simple, rapid and sensitive assay for viral detection is quantitative reverse transcription polymerase chain reaction (qRT-PCR), yet oligos optimized for ZIKV detection in mammalian cells and samples have repeatedly shown high background when used on mosquito ribonucleic acid (RNA). In this paper, we present a one-step qRT-PCR protocol that allows for the detection of ZIKV in mosquitoes and for the evaluation of gene expression from the same mosquito sample and RNA. This assay is a less expensive qRT-PCR approach than that most frequently used in the literature and has a much lower background, allowing confident detection. Methods: Our new oligo design to detect ZIKV RNA included in silico analysis of both viral and mosquito (Ae. aegypti and Ae. albopictus) genomes, targeting sequences conserved between Asian and African ZIKV lineages, but not matching Aedes genomes. This assay will allow researchers to avoid nonspecific amplification in insect samples due to viral integration into the mosquito genome, a phenomenon known to happen in wild and colonized populations of mosquitoes. Standard curves constructed with in vitro transcribed ZIKV RNA were used to optimize the sensitivity, efficiency and reproducibility of the assay. Results: Finally, the assay was used with success to detect both ZIKV RNA in infected mosquitoes and to detect expression of the Defensin A gene, an antimicrobial peptide (AMP) involved in Aedes aegypti immune response to virus infection. Conclusions: The experimental approach to detect ZIKV RNA in Aedes aegypti presented here has demonstrated to be specific, sensitive and reliable, and additionally it allows for the analysis of mosquito gene expression during ZIKV infection.

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Section: Introductionmentioning

confidence: 99%

One-step RT-qPCR assay for ZIKV RNA detection in Aedes aegypti samples: a protocol to study infection and gene expression during ZIKV infection

et al. 2020

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“…While DENVs isolated from patients is vital in dengue disease surveillance, the complementary data from mosquitoes, including viral sequences, mosquito infection rate and serotype/genotype prevalence, has the potential to provide additional information in understanding the transmission dynamics of the DENV. For this reason, virus surveillance in eld-collected mosquitoes is useful in tracking virus activity and in implementing control measures [2,3,4,5].…”

Section: Introductionmentioning

confidence: 99%

“…Detection of DENV in adult female Aedes aegypti mosquitoes remains a challenge due to the low infection rate (typically about 0.1%) observed in adult female mosquitoes [6]. However, recent advancements in molecular virus detection techniques, particularly nucleic acid ampli cation tests such as RT-PCR and real-time RT-PCR assays, have enabled researchers to directly detect DENV RNA in eld-collected mosquitoes [3,4,5,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21]. The sensitivity, speci city and speed of nucleic acid ampli cation tests provide an alternative to the traditional method (i.e.…”

Section: Introductionmentioning

confidence: 99%

Individual-based dengue virus surveillance in Aedes aegypti mosquitoes collected concurrently with suspected patients in Tarlac City, Philippines

Balingit

Carvajal

Saito-Obata

et al. 2020

Preprint

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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.

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“…While DENV isolation from patients is vital in dengue disease surveillance, the complementary data from mosquitoes, including viral sequences, mosquito infection rate and serotype/genotype prevalence, has the potential to provide additional information in understanding the transmission dynamics of the DENV. For this reason, virus surveillance in eld-collected mosquitoes is useful in tracking virus activity and in implementing control measures [2][3][4][5].…”

Section: Introductionmentioning

confidence: 99%

Individual-based dengue virus surveillance in Aedes aegypti mosquitoes collected concurrently with suspected patients in Tarlac City, Philippines

Balingit

Carvajal

Saito-Obata

et al. 2020

Preprint

View full text Add to dashboard Cite

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.

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Dengue virus type 4 in Niterói, Rio de Janeiro: the role of molecular techniques in laboratory diagnosis and entomological surveillance

Cited by 22 publications

References 28 publications

One-step RT-qPCR assay for ZIKV RNA detection in Aedes aegypti samples: a protocol to study infection and gene expression during ZIKV infection

One-step RT-qPCR assay for ZIKV RNA detection in Aedes aegypti samples: a protocol to study infection and gene expression during ZIKV infection

Individual-based dengue virus surveillance in Aedes aegypti mosquitoes collected concurrently with suspected patients in Tarlac City, Philippines

Individual-based dengue virus surveillance in Aedes aegypti mosquitoes collected concurrently with suspected patients in Tarlac City, Philippines

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