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Arbovirus Detection in Insect Vectors by Rapid, High-Throughput Pyrosequencing

BACKGROUND: Despite the global threat caused by arthropod-borne viruses, there is not an efficient method for screening vector populations to detect novel viral sequences. Current viral detection and surveillance methods based on culture can be costly and time consuming and are predicated on prior k...

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Autores principales: Bishop-Lilly, Kimberly A., Turell, Michael J., Willner, Kristin M., Butani, Amy, Nolan, Nichole M. E., Lentz, Shannon M., Akmal, Arya, Mateczun, Al, Brahmbhatt, Trupti N., Sozhamannan, Shanmuga, Whitehouse, Chris A., Read, Timothy D.
Formato: Texto
Lenguaje:English
Publicado: Public Library of Science 2010
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2976685/
https://www.ncbi.nlm.nih.gov/pubmed/21085471
http://dx.doi.org/10.1371/journal.pntd.0000878
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author Bishop-Lilly, Kimberly A.
Turell, Michael J.
Willner, Kristin M.
Butani, Amy
Nolan, Nichole M. E.
Lentz, Shannon M.
Akmal, Arya
Mateczun, Al
Brahmbhatt, Trupti N.
Sozhamannan, Shanmuga
Whitehouse, Chris A.
Read, Timothy D.
author_facet Bishop-Lilly, Kimberly A.
Turell, Michael J.
Willner, Kristin M.
Butani, Amy
Nolan, Nichole M. E.
Lentz, Shannon M.
Akmal, Arya
Mateczun, Al
Brahmbhatt, Trupti N.
Sozhamannan, Shanmuga
Whitehouse, Chris A.
Read, Timothy D.
author_sort Bishop-Lilly, Kimberly A.
collection PubMed
description BACKGROUND: Despite the global threat caused by arthropod-borne viruses, there is not an efficient method for screening vector populations to detect novel viral sequences. Current viral detection and surveillance methods based on culture can be costly and time consuming and are predicated on prior knowledge of the etiologic agent, as they rely on specific oligonucleotide primers or antibodies. Therefore, these techniques may be unsuitable for situations when the causative agent of an outbreak is unknown. METHODOLOGY/PRINCIPAL FINDINGS: In this study we explored the use of high-throughput pyrosequencing for surveillance of arthropod-borne RNA viruses. Dengue virus, a member of the positive strand RNA Flavivirus family that is transmitted by several members of the Aedes genus of mosquitoes, was used as a model. Aedes aegypti mosquitoes experimentally infected with dengue virus type 1 (DENV-1) were pooled with noninfected mosquitoes to simulate samples derived from ongoing arbovirus surveillance programs. Using random-primed methods, total RNA was reverse-transcribed and resulting cDNA subjected to 454 pyrosequencing. CONCLUSIONS/SIGNIFICANCE: In two types of samples, one with 5 adult mosquitoes infected with DENV-1- and the other with 1 DENV-1 infected mosquito and 4 noninfected mosquitoes, we identified DENV-1 DNA sequences. DENV-1 sequences were not detected in an uninfected control pool of 5 adult mosquitoes. We calculated the proportion of the Ae. aegypti metagenome contributed by each infecting Dengue virus genome (p(IP)), which ranged from 2.75×10(−8) to 1.08×10(−7). DENV-1 RNA was sufficiently concentrated in the mosquito that its detection was feasible using current high-throughput sequencing instrumentation. We also identified some of the components of the mosquito microflora on the basis of the sequence of expressed RNA. This included members of the bacterial genera Pirellula and Asaia, various fungi, and a potentially uncharacterized mycovirus.
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spelling pubmed-29766852010-11-17 Arbovirus Detection in Insect Vectors by Rapid, High-Throughput Pyrosequencing Bishop-Lilly, Kimberly A. Turell, Michael J. Willner, Kristin M. Butani, Amy Nolan, Nichole M. E. Lentz, Shannon M. Akmal, Arya Mateczun, Al Brahmbhatt, Trupti N. Sozhamannan, Shanmuga Whitehouse, Chris A. Read, Timothy D. PLoS Negl Trop Dis Research Article BACKGROUND: Despite the global threat caused by arthropod-borne viruses, there is not an efficient method for screening vector populations to detect novel viral sequences. Current viral detection and surveillance methods based on culture can be costly and time consuming and are predicated on prior knowledge of the etiologic agent, as they rely on specific oligonucleotide primers or antibodies. Therefore, these techniques may be unsuitable for situations when the causative agent of an outbreak is unknown. METHODOLOGY/PRINCIPAL FINDINGS: In this study we explored the use of high-throughput pyrosequencing for surveillance of arthropod-borne RNA viruses. Dengue virus, a member of the positive strand RNA Flavivirus family that is transmitted by several members of the Aedes genus of mosquitoes, was used as a model. Aedes aegypti mosquitoes experimentally infected with dengue virus type 1 (DENV-1) were pooled with noninfected mosquitoes to simulate samples derived from ongoing arbovirus surveillance programs. Using random-primed methods, total RNA was reverse-transcribed and resulting cDNA subjected to 454 pyrosequencing. CONCLUSIONS/SIGNIFICANCE: In two types of samples, one with 5 adult mosquitoes infected with DENV-1- and the other with 1 DENV-1 infected mosquito and 4 noninfected mosquitoes, we identified DENV-1 DNA sequences. DENV-1 sequences were not detected in an uninfected control pool of 5 adult mosquitoes. We calculated the proportion of the Ae. aegypti metagenome contributed by each infecting Dengue virus genome (p(IP)), which ranged from 2.75×10(−8) to 1.08×10(−7). DENV-1 RNA was sufficiently concentrated in the mosquito that its detection was feasible using current high-throughput sequencing instrumentation. We also identified some of the components of the mosquito microflora on the basis of the sequence of expressed RNA. This included members of the bacterial genera Pirellula and Asaia, various fungi, and a potentially uncharacterized mycovirus. Public Library of Science 2010-11-09 /pmc/articles/PMC2976685/ /pubmed/21085471 http://dx.doi.org/10.1371/journal.pntd.0000878 Text en This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. https://creativecommons.org/publicdomain/zero/1.0/ This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration, which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.
spellingShingle Research Article
Bishop-Lilly, Kimberly A.
Turell, Michael J.
Willner, Kristin M.
Butani, Amy
Nolan, Nichole M. E.
Lentz, Shannon M.
Akmal, Arya
Mateczun, Al
Brahmbhatt, Trupti N.
Sozhamannan, Shanmuga
Whitehouse, Chris A.
Read, Timothy D.
Arbovirus Detection in Insect Vectors by Rapid, High-Throughput Pyrosequencing
title Arbovirus Detection in Insect Vectors by Rapid, High-Throughput Pyrosequencing
title_full Arbovirus Detection in Insect Vectors by Rapid, High-Throughput Pyrosequencing
title_fullStr Arbovirus Detection in Insect Vectors by Rapid, High-Throughput Pyrosequencing
title_full_unstemmed Arbovirus Detection in Insect Vectors by Rapid, High-Throughput Pyrosequencing
title_short Arbovirus Detection in Insect Vectors by Rapid, High-Throughput Pyrosequencing
title_sort arbovirus detection in insect vectors by rapid, high-throughput pyrosequencing
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2976685/
https://www.ncbi.nlm.nih.gov/pubmed/21085471
http://dx.doi.org/10.1371/journal.pntd.0000878
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