{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,10]],"date-time":"2026-04-10T02:08:38Z","timestamp":1775786918101,"version":"3.50.1"},"reference-count":46,"publisher":"American Mosquito Control Association","issue":"1","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2022,3]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n        <jats:p>Eastern equine encephalitis virus (EEEV) is a highly pathogenic alphavirus that causes periodic outbreaks in the eastern USA. Mosquito abatement programs are faced with various challenges with surveillance and control of EEEV and other mosquito-borne illnesses. Environmental sampling of mosquito populations can be technically complex. Here we report the identification of biomarkers, development and validation of a colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of EEEV. Positive samples are easily visualized by a color change from pink to yellow. The assay was validated using EEEV from viral culture, experimentally spiked mosquito pools, and previously tested mosquito pools. The RT-LAMP assay detected viral titers down to approximately 10% of what would be present in a single infectious mosquito, based upon EEEV viral titers determined by previous competency studies. The RT-LAMP assay efficiently detected EEEV in combined aliquots from previously homogenized pools of mosquitoes, allowing up to 250 individual mosquitoes to be tested in a single reaction. No false positive results were obtained from RNA prepared from negative mosquito pools acquired from known and potential EEEV vectors. The colorimetric RT-LAMP assay is highly accurate, technically simple, and does not require sophisticated equipment, making it a cost-effective alternative to real time reverse transcriptase-polymerase chain reaction (RT-PCR) for vector surveillance.<\/jats:p>","DOI":"10.2987\/21-7047","type":"journal-article","created":{"date-parts":[[2022,3,11]],"date-time":"2022-03-11T22:38:37Z","timestamp":1647038317000},"page":"7-18","source":"Crossref","is-referenced-by-count":3,"title":["Development and Validation of a Colorimetric Reverse Transcriptase Loop-Mediated Isothermal Amplification Assay for Detection of Eastern Equine Encephalitis Virus in Mosquitoes"],"prefix":"10.2987","volume":"38","author":[{"given":"Mikayla D.","family":"Maddison","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Zhiru","family":"Li","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kristi M.","family":"Miley","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Catherine B.","family":"Poole","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Clotilde K. S.","family":"Carlow","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Thomas R.","family":"Unnasch","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1569","reference":[{"key":"i1943-6270-38-1-7-Alhassan1","series-title":"PloS One","first-page":"e108927","article-title":"A simple isothermal DNA amplification method to screen black flies for Onchocerca volvulus infection","volume":"9","author":"Alhassan","year":"2014","unstructured":"Alhassan\n              A,\n            \n            \n              Makepeace\n              BL,\n            \n            \n              LaCourse\n              EJ,\n            \n            \n              Osei-Atweneboana\n              MY,\n            \n            \n              Carlow\n              CKS.\n            \n          \n          2014.\n\t\t\t\t\tA simple isothermal DNA amplification method to screen black flies for Onchocerca 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seasonal patterns of host utilization by Culiseta melanura (Diptera: Culicidae) in Florida","volume":"54","author":"Blosser","year":"2017","unstructured":"Blosser\n              EM,\n            \n            \n              Lord\n              CC,\n            \n            \n              Stenn\n              T,\n            \n            \n              Acevedo\n              C,\n            \n            \n              Hassan\n              HK,\n            \n            \n              Reeves\n              LE,\n            \n            \n              Unnasch\n              TR,\n            \n            \n              Burkett-Cadena\n              ND.\n            \n          \n          2017.\n\t\t\t\t\tEnvironmental drivers of seasonal patterns of host utilization by Culiseta melanura (Diptera: Culicidae) in Florida.\n\t\t\t\t\tJ Med Entomol54:\n\t\t\t\t\t1365\u2013\n\t\t\t\t\t1374."},{"key":"i1943-6270-38-1-7-BurkettCadena1","series-title":"J Med Entomol","first-page":"1074","article-title":"Ecology of Culiseta melanura and other mosquitoes (Diptera: Culicidae) from Walton County, FL, during winter period 2013-2014","volume":"52","author":"Burkett-Cadena","year":"2015","unstructured":"Burkett-Cadena\n              ND,\n            \n            \n              Bingham\n              AM,\n            \n            \n              Hunt\n              B,\n            \n            \n              Morse\n              G,\n            \n            \n              Unnasch\n              TR.\n            \n          \n          2015.\n\t\t\t\t\tEcology of Culiseta melanura and other mosquitoes (Diptera: Culicidae) from Walton County, FL, during winter period 2013-2014.\n\t\t\t\t\tJ Med Entomol52:\n\t\t\t\t\t1074\u2013\n\t\t\t\t\t1082."},{"key":"i1943-6270-38-1-7-Calvert1","series-title":"PLoS One","first-page":"e0185340","article-title":"Rapid colorimetric detection of Zika virus from serum and urine specimens by reverse transcription loop-mediated isothermal amplification (RT-LAMP)","volume":"12","author":"Calvert","year":"2017","unstructured":"Calvert\n              AE,\n            \n            \n              Biggerstaff\n              BJ,\n            \n            \n              Tanner\n              NA,\n            \n            \n              Lauterbach\n              M,\n            \n            \n              Lanciotti\n              RS.\n            \n          \n          2017.\n\t\t\t\t\tRapid colorimetric detection of Zika virus from serum and urine specimens by reverse transcription loop-mediated isothermal amplification (RT-LAMP).\n\t\t\t\t\tPLoS One12:\n\t\t\t\t\te0185340."},{"key":"i1943-6270-38-1-7-daSilva1","series-title":"Scientific Reports","first-page":"1","article-title":"Development and validation of reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of ZIKV in mosquito samples from Brazil","volume":"9","author":"da Silva","year":"2019","unstructured":"da Silva\n              SJR,\n            \n            \n              Paiva\n              MHS,\n            \n            \n              Guedes\n              DRD,\n            \n            \n              Krokovsky\n              L,\n            \n            \n              de Melo\n              FL,\n            \n            \n              da Silva\n              MAL,\n            \n            \n              da Silva\n              A,\n            \n            \n              Ayres\n              CFJ,\n            \n            \n              Pena\n              LJ.\n            \n          \n          2019.\n\t\t\t\t\tDevelopment and validation of reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of ZIKV in mosquito samples from Brazil.\n\t\t\t\t\tScientific Reports9:\n\t\t\t\t\t1\u2013\n\t\t\t\t\t12."},{"key":"i1943-6270-38-1-7-daSilva2","series-title":"Viruses","first-page":"19","article-title":"Loop-mediated isothermal amplification (LAMP) for the diagnosis of Zika virus: A review","volume":"12","author":"da Silva","year":"2020","unstructured":"da Silva\n              SJR,\n            \n            \n              Pardee\n              K,\n            \n            \n              Pena\n              L.\n            \n          \n          2020.\n\t\t\t\t\tLoop-mediated isothermal amplification (LAMP) for the diagnosis of Zika virus: A review.\n\t\t\t\t\tViruses12:\n\t\t\t\t\t19."},{"key":"i1943-6270-38-1-7-GoncalvesDdSHookerDJDongYBaranNKyrylosPIturbeOrmaetxeISimmonsCPONeillSL1","series-title":"Parasites & Vectors","first-page":"404","article-title":"Detecting wMel Wolbachia in field-collected Aedes aegypti mosquitoes using loop-mediated isothermal amplification (LAMP)","volume":"12","author":"Gon\u00e7alves","year":"2019","unstructured":"Gon\u00e7alves\n              DdS,\n            \n            \n              Hooker\n              DJ,\n            \n            \n              Dong\n              Y,\n            \n            \n              Baran\n              N,\n            \n            \n              Kyrylos\n              P,\n            \n            \n              Iturbe-Ormaetxe\n              I,\n            \n            \n              Simmons\n              CP,\n            \n            \n              O'Neill\n              SL.\n            \n          \n          2019.\n\t\t\t\t\tDetecting wMel Wolbachia in field-collected Aedes aegypti mosquitoes using loop-mediated isothermal amplification (LAMP).\n\t\t\t\t\tParasites & Vectors12:\n\t\t\t\t\t404."},{"key":"i1943-6270-38-1-7-HeberleinLarson1","series-title":"Am J Trop Med Hyg","first-page":"1266","article-title":"Complex epidemiological dynamics of eastern equine encephalitis virus in Florida","volume":"100","author":"Heberlein-Larson","year":"2019","unstructured":"Heberlein-Larson\n              LA,\n            \n            \n              Tan\n              Y,\n            \n            \n              Stark\n              LM,\n            \n            \n              Cannons\n              AC,\n            \n            \n              Shilts\n              MH,\n            \n            \n              Unnasch\n              TR,\n            \n            \n              Das\n              SR.\n            \n          \n          2019.\n\t\t\t\t\tComplex epidemiological dynamics of eastern equine encephalitis virus in Florida.\n\t\t\t\t\tAm J Trop Med Hyg100:\n\t\t\t\t\t1266\u2013\n\t\t\t\t\t1274."},{"key":"i1943-6270-38-1-7-Honnold1","series-title":"Virol J","first-page":"152","article-title":"Eastern equine encephalitis virus in mice I: clinical course and outcome are dependent on route of 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transcription-loop-mediated isothermal amplification","volume":"8","author":"Lamb","year":"2018","unstructured":"Lamb\n              LE,\n            \n            \n              Bartolone\n              SN,\n            \n            \n              Tree\n              MO,\n            \n            \n              Conway\n              MJ,\n            \n            \n              Rossignol\n              J,\n            \n            \n              Smith\n              CP,\n            \n            \n              Chancellor\n              MB.\n            \n          \n          2018.\n\t\t\t\t\tRapid detection of Zika virus in urine samples and infected mosquitos by reverse transcription-loop-mediated isothermal amplification.\n\t\t\t\t\tScientific Reports8:\n\t\t\t\t\t3803\u2013\n\t\t\t\t\t3803."},{"key":"i1943-6270-38-1-7-Lambert1","series-title":"J Clin Microbiol","first-page":"379","article-title":"Detection of North American eastern and western equine encephalitis viruses 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change","volume":"45","author":"Ludwig","year":"2019","unstructured":"Ludwig\n              A,\n            \n            \n              Zheng\n              H,\n            \n            \n              Vrbova\n              L,\n            \n            \n              Drebot\n              MA,\n            \n            \n              Iranpour\n              M,\n            \n            \n              Lindsay\n              LR.\n            \n          \n          2019.\n\t\t\t\t\tIncreased risk of endemic mosquito-borne diseases in Canada due to climate change.\n\t\t\t\t\tCanada Commun Disease report = Releve des maladies transmissibles au Canada45:\n\t\t\t\t\t91\u2013\n\t\t\t\t\t97."},{"key":"i1943-6270-38-1-7-Molaei1","series-title":"PLoS One","first-page":"e0136743","article-title":"Vector-host interactions of Culiseta melanura in a focus of eastern Equine encephalitis virus activity in southeastern Virginia","volume":"10","author":"Molaei","year":"2015","unstructured":"Molaei\n              G,\n            \n            \n              Armstrong\n              PM,\n            \n            \n              Abadam\n              CF,\n            \n            \n              Akaratovic\n              KI,\n            \n            \n              Kiser\n              JP,\n            \n            \n              Andreadis\n              TG.\n            \n          \n          2015.\n\t\t\t\t\tVector-host interactions of Culiseta melanura in a focus of eastern Equine encephalitis virus activity in southeastern Virginia.\n\t\t\t\t\tPLoS One10:\n\t\t\t\t\te0136743."},{"key":"i1943-6270-38-1-7-Oliver1","series-title":"Parasit Vectors","first-page":"362","article-title":"Twenty years of surveillance for eastern equine encephalitis virus in mosquitoes in New York State from 1993 to 2012","volume":"11","author":"Oliver","year":"2018","unstructured":"Oliver\n              J,\n           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     SR,\n            \n            \n              Dash\n              PK,\n            \n            \n              Tripathi\n              NK,\n            \n            \n              Lakshmi\n              V,\n            \n            \n              Mamidi\n              N,\n            \n            \n              Shrivastva\n              A,\n            \n            \n              Gupta\n              N,\n            \n            \n              Saxena\n              P,\n            \n            \n              Babu\n              JP,\n            \n            \n              Rao\n              PV,\n            \n            \n              Morita\n              K.\n            \n          \n          2007.\n\t\t\t\t\tRapid and real-time detection of Chikungunya virus by reverse transcription loop-mediated isothermal amplification assay.\n\t\t\t\t\tJ Clin Microbiol45:\n\t\t\t\t\t351\u2013\n\t\t\t\t\t357."},{"key":"i1943-6270-38-1-7-Parida2","series-title":"J Clin Microbiol","first-page":"4172","article-title":"Development and evaluation of reverse transcription-loop-mediated isothermal amplification assay for rapid and real-time detection of Japanese encephalitis virus","volume":"44","author":"Parida","year":"2006","unstructured":"Parida\n              MM,\n            \n            \n              Santhosh\n              SR,\n            \n            \n              Dash\n              PK,\n            \n            \n              Tripathi\n              NK,\n            \n            \n              Saxena\n              P,\n            \n            \n              Ambuj\n              S,\n            \n            \n              Sahni\n              AK,\n            \n            \n              Lakshmana Rao\n              PV\n            ,\n\t\t\t\t\t\tMoritaK.\n          2006.\n\t\t\t\t\tDevelopment and evaluation of reverse transcription-loop-mediated isothermal amplification assay for rapid and real-time detection of Japanese 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