Multi-Gene Detection and Identification of Mosquito-Borne RNA Viruses Using an Oligonucleotide Microarray

Grubaugh, Nathan D.; McMenamy, Scott S.; Turell, Michael J.; Lee, John S.

doi:10.1371/journal.pntd.0002349

Cited by 11 publications

(7 citation statements)

References 54 publications

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“…Although a wide array of assays exist to detect emerging pathogens (e.g. [8][9][10]), novel and inexpensive methods for sample collection and for pathogen detection are needed to use them most effectively.…”

Section: Introductionmentioning

confidence: 99%

Xenosurveillance: A Novel Mosquito-Based Approach for Examining the Human-Pathogen Landscape

et al. 2015

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BackgroundGlobally, regions at the highest risk for emerging infectious diseases are often the ones with the fewest resources. As a result, implementing sustainable infectious disease surveillance systems in these regions is challenging. The cost of these programs and difficulties associated with collecting, storing and transporting relevant samples have hindered them in the regions where they are most needed. Therefore, we tested the sensitivity and feasibility of a novel surveillance technique called xenosurveillance. This approach utilizes the host feeding preferences and behaviors of Anopheles gambiae, which are highly anthropophilic and rest indoors after feeding, to sample viruses in human beings. We hypothesized that mosquito bloodmeals could be used to detect vertebrate viral pathogens within realistic field collection timeframes and clinically relevant concentrations.Methodology/Principal FindingsTo validate this approach, we examined variables influencing virus detection such as the duration between mosquito blood feeding and mosquito processing, the pathogen nucleic acid stability in the mosquito gut and the pathogen load present in the host’s blood at the time of bloodmeal ingestion using our laboratory model. Our findings revealed that viral nucleic acids, at clinically relevant concentrations, could be detected from engorged mosquitoes for up to 24 hours post feeding by qRT-PCR. Subsequently, we tested this approach in the field by examining blood from engorged mosquitoes from two field sites in Liberia. Using next-generation sequencing and PCR we were able to detect the genetic signatures of multiple viral pathogens including Epstein-Barr virus and canine distemper virus.Conclusions/SignificanceTogether, these data demonstrate the feasibility of xenosurveillance and in doing so validated a simple and non-invasive surveillance tool that could be used to complement current biosurveillance efforts.

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Section: Introductionmentioning

confidence: 99%

Xenosurveillance: A Novel Mosquito-Based Approach for Examining the Human-Pathogen Landscape

et al. 2015

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“…Subsequent detection is done by either agarose gel electrophoresis (followed by ethidium bromide staining) or hybridization with molecular probes (Houng et al, 2001; Scaramozzino et al, 2001; de Morais Bronzoni et al, 2005; Chao et al, 2007; Naze et al, 2009; Yeh et al, 2010; Grubaugh et al, 2013a; Grubaugh et al, 2013b). …”

Section: Introductionmentioning

confidence: 99%

High-throughput multiplexed xMAP Luminex array panel for detection of twenty two medically important mosquito-borne arboviruses based on innovations in synthetic biology

Glushakova

Bradley

et al. 2015

Journal of Virological Methods

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Mosquito-borne arboviruses are emerging world-wide as important human and animal pathogens. This makes assays for their accurate and rapid identification essential for public health, epidemiological, ecological studies. Over the past decade, many mono- and multiplexed assays targeting arboviruses nucleic acids have been reported. None has become established for the routine identification of multiple viruses in a “single tube” setting. With increasing multiplexing, the detection of viral RNAs is complicated by noise, false positives and negatives. In this study, an assay was developed that avoids these problems by combining two new kinds of nucleic acids emerging from the field of synthetic biology. The first is a “self-avoiding molecular recognition system” (SAMRS), which enables high levels of multiplexing. The second is an “artificially expanded genetic information system” (AEGIS), which enables clean PCR amplification in nested PCR formats. A conversion technology was used to place AEGIS component into amplicon, improving their efficiency of hybridization on Luminex beads. When Luminex “liquid microarrays” are exploited for downstream detection, this combination supports single-tube PCR amplification assays that can identify 22 mosquito-borne RNA viruses from the genera Flavivirus, Alphavirus, Orthobunyavirus. The assay differentiates between closely-related viruses, as dengue, West Nile, Japanese encephalitis, and the California serological group. The performance and the sensitivity of the assay were evaluated with dengue viruses and infected mosquitoes; as few as 6–10 dengue virions can be detected in a single mosquito.

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“…Previously published microarray or diagnostic chip approaches [20] lack the ease of operation of the ArrayStrip platform used here and cover only part of the range of viruses that we can identify [12, 14, 19, 46]. …”

Section: Discussionmentioning

confidence: 99%

A Novel Pan-FlavivirusDetection and Identification Assay Based on RT-qPCR and Microarray

Vina-Rodrı́guez

Sachse

Ziegler

et al. 2017

BioMed Research International

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The genus Flavivirus includes arthropod-borne viruses responsible for a large number of infections in humans and economically important animals. While RT-PCR protocols for specific detection of most Flavivirus species are available, there has been also a demand for a broad-range Flavivirus assay covering all members of the genus. It is particularly challenging to balance specificity at genus level with equal sensitivity towards each target species. In the present study, a novel assay combining a SYBR Green-based RT-qPCR with a low-density DNA microarray has been developed. Validation experiments confirmed that the RT-qPCR exhibited roughly equal sensitivity of detection and quantification for all flaviviruses tested. These PCR products are subjected to hybridization on a microarray carrying 84 different oligonucleotide probes that represent all known Flavivirus species. This assay has been used as a screening and confirmation tool for Flavivirus presence in laboratory and field samples, and it performed successfully in international External Quality Assessment of NAT studies. Twenty-six Flavivirus strains were tested with the assay, showing equivalent or superior characteristics compared with the original or even with species-specific RT-PCRs. As an example, test results on West Nile virus detection in a panel of 340 mosquito pool samples from Greece are presented.

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Multi-Gene Detection and Identification of Mosquito-Borne RNA Viruses Using an Oligonucleotide Microarray

Cited by 11 publications

References 54 publications

Xenosurveillance: A Novel Mosquito-Based Approach for Examining the Human-Pathogen Landscape

Xenosurveillance: A Novel Mosquito-Based Approach for Examining the Human-Pathogen Landscape

High-throughput multiplexed xMAP Luminex array panel for detection of twenty two medically important mosquito-borne arboviruses based on innovations in synthetic biology

A Novel Pan-FlavivirusDetection and Identification Assay Based on RT-qPCR and Microarray

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