Characterizing and Quantifying Arbovirus Transmission by <i>Aedes aegypti</i> Using Forced Salivation and Analysis of Bloodmeals

Arbovirus transmission studies are dependent on the ability to estimate the titer of virus transmitted from infectious mosquitoes to a host. There are several methods for estimating virus titer in mosquito saliva, including (1) using forced salivation (FS) whereby the infectious mosquito’s proboscis...

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Main Authors: Megan R. Miller, Madeleine R. Sorensen, Erin D. Markle, Taylor C. Clarkson, Ashley L. Knight, Michelle J. Savran, Brian D. Foy
Format: Article
Language:English
Published: MDPI AG 2021-03-01
Series:Insects
Subjects:
Online Access:https://www.mdpi.com/2075-4450/12/4/304
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author Megan R. Miller
Madeleine R. Sorensen
Erin D. Markle
Taylor C. Clarkson
Ashley L. Knight
Michelle J. Savran
Brian D. Foy
author_facet Megan R. Miller
Madeleine R. Sorensen
Erin D. Markle
Taylor C. Clarkson
Ashley L. Knight
Michelle J. Savran
Brian D. Foy
author_sort Megan R. Miller
collection DOAJ
description Arbovirus transmission studies are dependent on the ability to estimate the titer of virus transmitted from infectious mosquitoes to a host. There are several methods for estimating virus titer in mosquito saliva, including (1) using forced salivation (FS) whereby the infectious mosquito’s proboscis is forced into a capillary tube containing media to collect and test their saliva for virus, and (2) by quantifying virus expectorated into host tissues or into the blood contained in an artificial feeder immediately after blood feeding. We studied FS and bloodmeals to estimate and compare titers of Zika virus and chikungunya virus transmitted by the mosquito vector <i>Aedes aegypti</i>. Infectious virus and viral genomes of both viruses were detected more often from individual mosquitoes using immersion oil for the FS media compared to fetal bovine serum (FBS) plus glycerol, but the FS media had no influence on virus quantification from positive samples. FS virus titers were equivalent when comparing individuals or groups of mosquitoes that never received a blood meal compared to those that were blood fed immediately prior, showing that blood feeding does not influence FS. This suggested that performing FS on mosquitoes after blood feeding might be an efficient way to estimate virus transmitted during blood feeding. However, detecting virus from the blood remaining in an artificial feeder post-blood feeding was mostly unsuccessful relative to quantifying virus from FS of the post-blood fed mosquitoes. In contrast, immunocompromised mice always became infected after being fed on by Zika-infected mosquitoes, even when no infectious virus was detected in their saliva by FS post-blood feed. Due to this discrepancy, we tested the ingested bloodmeals of individual mosquitoes that fed on artificial blood feeders for virus, and compared these to virus in their saliva harvested from FS and to virus in their bodies. These experiments revealed ~50–100 times higher virus titers in the dissected bloodmeals compared to those detected in the same mosquitoes’ saliva, demonstrating how mosquitoes re-ingest much of their saliva during artificial blood feeding, and highlighting a large increase in virus transmission during <i>Aedes aegypti</i> blood feeding. Both FS and the dissected bloodmeals of artificially blood-fed mosquitoes showed that the quantity of viral RNA expectorated by mosquitoes was 2–5 logs more than the quantity of infectious virus. The results from this study add critical information to understanding and quantifying the transmission of <i>Aedes aegypti</i> arboviruses.
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spelling doaj.art-15921f95d0fa41498cb54c7ebb7cd7d32023-11-21T13:22:46ZengMDPI AGInsects2075-44502021-03-0112430410.3390/insects12040304Characterizing and Quantifying Arbovirus Transmission by <i>Aedes aegypti</i> Using Forced Salivation and Analysis of BloodmealsMegan R. Miller0Madeleine R. Sorensen1Erin D. Markle2Taylor C. Clarkson3Ashley L. Knight4Michelle J. Savran5Brian D. Foy6Center for Vector-Borne Infectious Diseases, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523-1685, USACenter for Vector-Borne Infectious Diseases, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523-1685, USACenter for Vector-Borne Infectious Diseases, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523-1685, USACenter for Vector-Borne Infectious Diseases, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523-1685, USACenter for Vector-Borne Infectious Diseases, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523-1685, USACenter for Vector-Borne Infectious Diseases, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523-1685, USACenter for Vector-Borne Infectious Diseases, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523-1685, USAArbovirus transmission studies are dependent on the ability to estimate the titer of virus transmitted from infectious mosquitoes to a host. There are several methods for estimating virus titer in mosquito saliva, including (1) using forced salivation (FS) whereby the infectious mosquito’s proboscis is forced into a capillary tube containing media to collect and test their saliva for virus, and (2) by quantifying virus expectorated into host tissues or into the blood contained in an artificial feeder immediately after blood feeding. We studied FS and bloodmeals to estimate and compare titers of Zika virus and chikungunya virus transmitted by the mosquito vector <i>Aedes aegypti</i>. Infectious virus and viral genomes of both viruses were detected more often from individual mosquitoes using immersion oil for the FS media compared to fetal bovine serum (FBS) plus glycerol, but the FS media had no influence on virus quantification from positive samples. FS virus titers were equivalent when comparing individuals or groups of mosquitoes that never received a blood meal compared to those that were blood fed immediately prior, showing that blood feeding does not influence FS. This suggested that performing FS on mosquitoes after blood feeding might be an efficient way to estimate virus transmitted during blood feeding. However, detecting virus from the blood remaining in an artificial feeder post-blood feeding was mostly unsuccessful relative to quantifying virus from FS of the post-blood fed mosquitoes. In contrast, immunocompromised mice always became infected after being fed on by Zika-infected mosquitoes, even when no infectious virus was detected in their saliva by FS post-blood feed. Due to this discrepancy, we tested the ingested bloodmeals of individual mosquitoes that fed on artificial blood feeders for virus, and compared these to virus in their saliva harvested from FS and to virus in their bodies. These experiments revealed ~50–100 times higher virus titers in the dissected bloodmeals compared to those detected in the same mosquitoes’ saliva, demonstrating how mosquitoes re-ingest much of their saliva during artificial blood feeding, and highlighting a large increase in virus transmission during <i>Aedes aegypti</i> blood feeding. Both FS and the dissected bloodmeals of artificially blood-fed mosquitoes showed that the quantity of viral RNA expectorated by mosquitoes was 2–5 logs more than the quantity of infectious virus. The results from this study add critical information to understanding and quantifying the transmission of <i>Aedes aegypti</i> arboviruses.https://www.mdpi.com/2075-4450/12/4/304arbovirusestransmission<i>Aedes aegypti</i>saliva
spellingShingle Megan R. Miller
Madeleine R. Sorensen
Erin D. Markle
Taylor C. Clarkson
Ashley L. Knight
Michelle J. Savran
Brian D. Foy
Characterizing and Quantifying Arbovirus Transmission by <i>Aedes aegypti</i> Using Forced Salivation and Analysis of Bloodmeals
Insects
arboviruses
transmission
<i>Aedes aegypti</i>
saliva
title Characterizing and Quantifying Arbovirus Transmission by <i>Aedes aegypti</i> Using Forced Salivation and Analysis of Bloodmeals
title_full Characterizing and Quantifying Arbovirus Transmission by <i>Aedes aegypti</i> Using Forced Salivation and Analysis of Bloodmeals
title_fullStr Characterizing and Quantifying Arbovirus Transmission by <i>Aedes aegypti</i> Using Forced Salivation and Analysis of Bloodmeals
title_full_unstemmed Characterizing and Quantifying Arbovirus Transmission by <i>Aedes aegypti</i> Using Forced Salivation and Analysis of Bloodmeals
title_short Characterizing and Quantifying Arbovirus Transmission by <i>Aedes aegypti</i> Using Forced Salivation and Analysis of Bloodmeals
title_sort characterizing and quantifying arbovirus transmission by i aedes aegypti i using forced salivation and analysis of bloodmeals
topic arboviruses
transmission
<i>Aedes aegypti</i>
saliva
url https://www.mdpi.com/2075-4450/12/4/304
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