We gratefully acknowledge the important contributions of Drs. John Anderson, Andrew Main and Shirley Tirrell to this study. This work was supported in part by grants from the Centers for Disease Control and Prevention (U50/CCU116806-01-1) and the US Department of Agriculture (58-6615-1-218, CONH00768, and CONH00773).

1. Mosquito sorting and identification 
<ol>
	<li>The following procedures are performed on an open laboratory bench in a dedicated biosafety level-2 (BSL-2) laboratory by staff who are trained to work with live mosquitoes. A "cold-chain" is maintained throughout the procedure.</li>
	<li>Anesthetize live adult mosquitoes by placing mosquito collection bag in a -20&deg; freezer for 5 minutes. Transfer collection bag to an insulated container containing dry ice. Close lid and expose mosquitoes to carbon-dioxide for 1-3...

<ol>
	<li>Chosewood, L. C., Wilson, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biosafety in Microbiological and Biomedical Laboratories. , (2007).</li><li>Anderson, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+West+Nile+virus+from+mosquitoes,+crows,+and+a+Cooper's+hawk+in+Connecticut.">Isolation of West Nile virus from mosquitoes, crows, and a Cooper's hawk in Connecticut.</a> Science. 286, 2331-2333 (1999).</li><li>Armstrong, P. M., Andreadis, T. G., Anderson, J. F., Main, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolations+of+Potosi+virus+from+mosquitoes+(Diptera:+Culicidae)+collected+in+Connecticut.">Isolations of Potosi virus from mosquitoes (Diptera: Culicidae) collected in Connecticut.</a> J Med Entomol. 42, 875-881 (2005).</li><li>Armstrong, P. M., Andreadis, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+genetic+variant+of+La+Crosse+virus+(Bunyaviridae)+isolated+from+New+England.">A new genetic variant of La Crosse virus (Bunyaviridae) isolated from New England.</a> Am J Trop Med Hyg. 75, 491-496 (2006).</li><li>Andreadis, T. G., Anderson, J. F., Vossbrinck, C. R., Main, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+of+West+Nile+virus+in+Connecticut,+USA:+a+five+year+analysis+of+mosquito+data+1999-2003.">Epidemiology of West Nile virus in Connecticut, USA: a five year analysis of mosquito data 1999-2003.</a> Vector Borne Zoonotic Dis. 4, 360-378 (2004).</li><li>Andreadis, T. G., Anderson, J. F., Tirrell-Peck, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+isolations+of+eastern+equine+encephalitis+and+highlands+J+viruses+from+mosquitoes+(Diptera:+Culicidae)+during+a+1996+epizootic+in+southeastern+Connecticut.">Multiple isolations of eastern equine encephalitis and highlands J viruses from mosquitoes (Diptera: Culicidae) during a 1996 epizootic in southeastern Connecticut.</a> J Med Entomol. 35, 296-302 (1998).</li><li>Armstrong, P. M., Andreadis, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eastern+equine+encephalitis+virus+in+mosquitoes+and+their+role+as+bridge+vectors.">Eastern equine encephalitis virus in mosquitoes and their role as bridge vectors.</a> Emerg Infect Dis. 16, 1869-1874 (2010).</li><li>Andreadis, T. G., Anderson, J. F., Armstrong, P. M., Main, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolations+of+Jamestown+Canyon+virus+(Bunyaviridae:+Orthobunyavirus)+from+field-collected+mosquitoes+(Diptera:+Culicidae)+in+Connecticut,+USA:+a+ten-year+analysis,+1997-2006.">Isolations of Jamestown Canyon virus (Bunyaviridae: Orthobunyavirus) from field-collected mosquitoes (Diptera: Culicidae) in Connecticut, USA: a ten-year analysis, 1997-2006.</a> Vector Borne Zoonotic Dis. 8, 175-188 (2008).</li><li>Beaty, B. J., Calisher, C. H., Shope, R., Lennette, E. H., Lennette, D. A., Lennette, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+11.">Chapter 11.</a> Diagnostic Procedures for Viral, Rickettsial, and Chlamydial Infections. , 189-212 (1995).</li><li>Karabatsos, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> International Catalogue of Arboviruses Including Certain Other Viruses of Vertebrates. , (1985).</li><li>Lanciotti, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+detection+of+west+nile+virus+from+human+clinical+specimens,+field-collected+mosquitoes,+and+avian+samples+by+a+TaqMan+reverse+transcriptase-PCR+assay.">Rapid detection of west nile virus from human clinical specimens, field-collected mosquitoes, and avian samples by a TaqMan reverse transcriptase-PCR assay.</a> J Clin Microbiol. 38, 4066-4071 (2000).</li><li>Lambert, A. J., Martin, D. A., Lanciotti, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+North+American+eastern+and+western+equine+encephalitis+viruses+by+nucleic+acid+amplification+assays.">Detection of North American eastern and western equine encephalitis viruses by nucleic acid amplification assays.</a> J Clin Microbiol. 41, 379-385 (2003).</li></ol>

No conflicts of interest declared.

Vero cell culture assay serves as an effective method to screen field-collected mosquitoes for a diversity of viruses. This contrasts to molecular methods that specifically target one or a few viruses of interest. Moreover, we have found that Vero cell culture assay is equally if not more sensitive than RT-PCR7 and provides us with virus isolates that are stored in our reference collection for future studies. Nevertheless, cell culture systems may not be appropriate in many instances. This approach incurs a...

<table border="1">
	<tbody>
	 <tr>
 	<th>Name of reagent/ equipment</th>
 	<th>Company</th>
 	<th>Catalog Number</th>
 	</tr>
 	<tr>
 	<td>Fetal bovine serum</td>
 	<td>Gibco &ndash; Invitrogen</td>
 	<td>16140</td>
 	</tr>
 	<tr>
 <td>Anti-biotic/mycotic</td>
 <td>Gibco &ndash; Invitrogen</td>
 <td>15240</td>
 </tr>
 <tr>
 <td>Sodium bicarbonate NaHCO3, 7.5% Soln.</td>
 <td>Gibco &ndash; Invitrogen</td>
 <td>25080</td>
 </tr>
 <tr>
 <td>L-Glutamine 200mM 100x</td>
 <td>Gibco-Invitrogen</td>
 <td>25030</td>
 </tr>
 <tr>
 <td>Powdered minimal essential medium (MEM)</td>
 <td>Gibco &ndash; Invitrogen</td>
 <td>11700</td>
 </tr>
 <tr>
 <td>10X Dulbecco&rsquo;s PBS</td>
 <td>Gibco &ndash; Invitrogen</td>
 <td>14080</td>
 </tr>
 <tr>
 <td>Trypsin-EDTA</td>
 <td>Gibco &ndash; Invitrogen</td>
 <td>15400</td>
 </tr>
 <tr>
 <td>Rabbit serum</td>
 <td>Gibco &ndash; Invitrogen</td>
 <td>16120</td>
 </tr>
 <tr>
 <td>Vero Cells (Clone E6)</td>
 <td>ATCC</td>
 <td>CRL-1586</td>
 </tr>
 <tr>
 <td>Small tissue culture flasks, vented caps, 25 cm2</td>
 <td>Falcon - Becton Dickinson</td>
 <td>353112</td>
 </tr>
 <tr>
 <td>Large tissue culture flasks, vented caps, 175 cm2</td>
 <td>Falcon &ndash; Becton Dickinson</td>
 <td>353108</td>
 </tr>
 <tr>
 <td>Copper-coated BB&rsquo;s, 4.5 mm</td>
 <td>Crosman</td>
 <td>0767</td>
 </tr>
 <tr>
 <td>Mixer Mill</td>
 <td>Retsch</td>
 <td>MM300</td>
 </tr>
 <tr>
 <td>Isopack freezer racks</td>
 <td>Eppendorf</td>
 <td>022510240</td>
 </tr>
 </tbody>
</table>

detection of infectious virus from field-collected mosquitoes by vero cell culture assay

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many of these viruses have intensified in their endemic ranges and expanded to new territories, necessitating effective surveillance and control programs to respond to these threats. One strategy to monitor virus activity involves collecting large numbers of mosquitoes from endemic sites and testing them for viral infection. In this article, we describe how to handle, process, and screen field-collected mosquitoes for infectious virus by Vero cell culture assay. Mosquitoes are sorted by trap location and species, and grouped into pools containing &le;50 individuals. Pooled specimens are homogenized in buffered saline using a mixer-mill and the aqueous phase is inoculated onto confluent Vero cell cultures (Clone E6). Cell cultures are monitored for cytopathic effect from days 3-7 post-inoculation and any viruses grown in cell culture are identified by the appropriate diagnostic assays. By utilizing this approach, we have isolated 9 different viruses from mosquitoes collected in Connecticut, USA, and among these, 5 are known to cause human disease. Three of these viruses (West Nile virus, Potosi virus, and La Crosse virus) represent new records for North America or the New England region since 1999. The ability to detect a wide diversity of viruses is critical to monitoring both established and newly emerging viruses in the mosquito population.

Watch this Scientific Journal Video about Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay at JoVE.com

Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay

We describe a method to process and screen field-collected mosquitoes for a diversity of viruses by Vero cell culture assay. By employing this technique, we have detected 9 different viruses from 4 taxonomic families in mosquitoes collected in Connecticut.

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many ...

detection-infectious-virus-from-field-collected-mosquitoes-vero-cell

Research

JoVE Journal

Immunology and Infection

18.3K 조회수.  The Connecticut Agricultural Experiment Station. 우리는 처리 방법을 설명하고 화면의 필드 수집 베로 세포 배양 분석에 의해 바이러스의 다양성을 위해 모기. 이 기술을 채용함으로써, 우리는 코네티컷에서 수집한 모기 4 taxonomic 가정에서 9 가지 바이러스를 감지했습니다.