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 minutes to ensure complete knock-down.</li>
	<li>Tap anesthetized mosquitoes onto a pre-chilled pan placed on a bed of dry ice. Separate individual female mosquitoes using fine forceps and place in plastic portion cups. Cover with lid and place cups on wet ice. </li>
	<li>Identify mosquitoes to species using descriptive taxonomic keys based on external mosquito morphology with the aid of a stereo dissecting microscope (10-60X zoom) mounted on an electronic chill table. </li>
	<li>Combine identified mosquito species into pools of &le;50 individuals in 2 mL snap-cap vials containing a copper BB. Vials are labeled with a unique identifier. </li>
	<li>Following identification of all mosquitoes, vials are placed in labeled bags and held in a Styrofoam cooler containing dry ice.</li>
	<li>Store mosquito pools in a -80&deg; C freezer until virus testing. </li>
</ol>
2. Biosafety considerations to isolate virus from mosquitoes 
<ol>
	<li>The following procedures are performed in a biosafety level-3 (BSL-3) laboratory by staff who are trained to work at this level of containment1. Laboratory facilities, equipment, procedures and practices are subject to institutional, state, and federal oversight and inspection. Seek the appropriate training and approval before attempting the following protocols.</li>
	<li>Wear the appropriate personal protective equipment (PPE) for each procedure such as disposable gowns, gloves, face shields, and respirators.</li>
	<li>Disinfect work surfaces with 70% alcohol before and after completing procedures.</li>
	<li>Cell cultures and potentially infectious material are handled in a Class II biosafety cabinet (BSC).</li>
	<li>Mosquito pools are homogenized in a mixer-mill that is placed inside a BSC.</li>
	<li>Mosquito homogenates are centrifuged in an aerosol-tight microcentrifuge. If this procedure cannot be performed within a BSC, the operator should wear a respirator when operating and retrieving samples from the centrifuge.</li>
	<li>Pipets and pipet tips handling material in the BSC (biosafety cabinet) are soaked in 10% bleach prior to autoclaving. All laboratory waste is decontaminated by autoclaving prior to disposal.</li>
</ol>
3. Preparation of Vero cells 
<ol>
	<li>Decant culture media from one large tissue culture flask (175 cm2) of confluent Vero cells (Clone E6) into waste beaker containing bleach.</li>
	<li>Wash Vero cells by adding a 5 mL aliquot of PBS and swirl over layer of cells and along sides of flask making sure to thoroughly cover entire monolayer.</li>
	<li>Decant PBS into waste beaker.</li>
	<li>Add 2.5 mL of Trypsin-EDTA and swirl over layer of cells making sure to thoroughly cover entire monolayer.</li>
	<li>Incubate flask in incubator for 5 minutes at 37&deg;C, 5% CO2.</li>
	<li>Gently shake and swirl flask to remove cells from bottom surface. The cells should slide off easily; incubate an additional 1-5 minutes if cells do not easily slide off.</li>
	<li>Add 5 mL of Minimal Essential Media (MEM), 5% fetal bovine serum (FBS), using a serological pipet and pipet up-and-down 10-20 times to break up the clumps of cells.</li>
	<li>Add an additional 15 mL MEM, 5% FBS for a total volume of 22.5 mL and mix well.</li>
	<li>Place 40 small tissue culture flasks (25 cm2) upright in two racks (20 flasks/rack).</li>
	<li>Place the racks containing flasks inside the biosafety cabinet and remove flask caps, placing caps directly in front of flasks.</li>
	<li>Dispense 4 mL of MEM, 5% FBS into each flask using a serological pipet.</li>
	<li>Dispense 0.5 mL suspended cells into each flask (&tilde;1:6 splitting ratio)Replace flask caps.</li>
	<li>Add 50 mL of MEM, 5% FBS back to the original large tissue culture flask containing the remaining cell suspension (&tilde;2.5 mLs) and return flask to the incubator. The same large tissue culture flask may be re-used up to 5 passages and then a new flask should be set-up to replace it.</li>
	<li>Place small tissue culture flasks in stacks on tray. Swirl the tray to make sure that media uniformly covers the bottom of the flaks.</li>
	<li>Place small flasks in the incubator at 37&deg; C, 5% CO2 and grow overnight until 80-90% confluent.</li>
</ol>
4. Preparation of mosquito pools 
<ol>
	<li>Keep mosquito pools cold during testing procedures. Use pre-chilled freezer racks and mixer-mill cassettes, ice-cold reagents, and a refrigerated centrifuge when processing mosquito pools. </li>
	<li>Place tubes containing mosquitoes into a pre-chilled freezer rack and add 1- 1.5 mL PBS-G to each tube of mosquitoes.</li>
	<li>Retrieve mixer-mill cassettes from the freezer. Place 24 tubes into each cassette and then secure in the mixer-mill.</li>
	<li>Homogenize the samples for 4 minutes at 25 cycles/second. The mixer mill shakes the tubes at high velocity and a metal BB inside each tube disrupts the mosquito tissue. </li>
	<li>Centrifuge tubes for 6 minutes at 7,000 rpm. </li>
	<li>Place tubes back into freezer racks until inoculated into Vero cells.</li>
</ol>
5. Inoculation of Vero cells with mosquito pool homogenates
<ol>
	<li>Check at least one tissue culture flask from each series under inverted microscope to assure adequate confluency and cell health; expect about 80-90% coverage. </li>
	<li>Line up 20 small tissue culture flasks in a rack and label flasks with corresponding accession numbers from each mosquito pool.</li>
	<li>Loosen caps and decant most of the media from tissue culture flasks into waste beaker. Leave enough media in the flask to completely coat cell monolayer.</li>
	<li>Aliquot 100&mu;L of supernatant from each processed mosquito pool into the corresponding tissue culture flask. </li>
	<li>Replace and tighten culture flask cap.</li>
	<li>Lay culture flasks flat on shaker for 5 minutes (room temperature) at 35-40 rpm.</li>
	<li>Return 20 tissue culture flasks to upright position in a rack and place in the BSC.</li>
	<li>Uncapping 3 tissue culture flasks at one time, dispense 4 mL growth media into each flask using a serological pipette; replace caps.</li>
	<li>Be sure that the pipette tip does not contact the culture flask neck or lip. Change pipette tips as necessary if contact is made with the flask.</li>
	<li>Incubate tissue culture flasks at 37&deg;C, 5% CO2.</li>
</ol>
6. Screening Vero cells for virus growth
<ol>
	<li>Screen inoculated tissue culture flasks for signs of viral infection, cytopathic effect (CPE), from 3-7 days post-inoculation. </li>
	<li>Visually inspect cell cultures for CPE and/or contamination by tilting the flasks and examining the media that pools at the bottom of the flask. The media will appear cloudy as the cells deteriorate during CPE or due to growth of yeast, fungus, or bacterial contamination.</li>
	<li>Re-examine cell cultures that exhibit cloudy media under an inverted microscope. Viruses will cause cells to become deformed and will break loose from the culture flasks. Cell cultures with visible contaminants, e.g. yeast, other bacteria, fungus or heavy mosquito debris, are re-tested by passing the original mosquito pool through a 0.22&mu;M syringe filter.</li>
	<li>Aseptically dispense 1-2 mLs of media from virus-positive cell cultures into labeled cryotubes using a serological pipette.</li>
	<li>Virus cultures are stored at -80&deg; C until identified using the appropriate diagnostic assays. </li>
</ol>
7. Preparation of reagents
<ol>
	<li>MEM, 5% FBS. Dissolve 37.6 g of powdered MEM in a final volume of 4 L of dd H20. Dispense solution into 500 mL aliquots in media bottles and autoclave. When solution has cooled, aseptically add 26 mL heat-inactivated fetal bovine serum, 5.2 mL L- Glutamine, 5.2 mL anti-biotic/mycotic, and 10.4 mL 7.5% sodium bicarbonate to each bottle. Store at 4&deg; C.</li>
	<li>PBS-G. Dissolve 16 g NaCl, 0.4 g KCl, 2.3 g Na2HPO4, 0.4 g KH2PO4, and 4 mL of 0.5% Phenol Red into 1000 mL dd H20. Adjust pH to 7.1-7.4 with 2 N NaOH (if necessary). In a separate beaker, heat 600 mL of H20 to a boil. Remove from hot-plate, add 10 g gelatin, and dissolve. Add dissolved gelatin solution to the PBS solution and adjust final volume to 2 L with dd H2O. Dispense 100 mL of solution into bottles and autoclave. When solution has cooled, aseptically add 42.8 mL heat-inactivated rabbit serum and 1.4 mL anti-biotic/mycotic. Store at 4&deg; C.</li>
	<li>PBS cell wash. Add 50 mL 10X Dulbecco&#x2019;s PBS to 400 mL dd H2O. Adjust pH to 7.1 with 2 N NaOH. Adjust final volume to 500 mL with dd H2O. Filter solution with 0.2uM vacuum filter unit. Dispense into 5 mL aliquots in 8 mL screw-cap tubes. Store at 4&deg; C.</li>
</ol>
8. Representative Results
Nine different of viruses from 4 taxonomic families were recovered from mosquitoes collected during continuous statewide surveillance in Connecticut from 1997-2010 (table 1). Five of these viruses are known to cause human disease, the most important pathogens being West Nile virus and eastern equine encephalitis virus. New discoveries include: the first isolation of WNV from mosquitoes collected in North America in 19992, the first isolation of Potosi virus in northeastern US in 20003, and the first isolation of La Crosse virus in New England in 20054.

<table border="1">
<tbody>
 <tr>
 <td>Virus</td>
 <td>Family</td>
 <td>No. isolates</td>
 <td>No. locations</td>
 <td>No. years detected</td>
 <td>Human disease</td>
 <td>Refs.</td>
 </tr>
 <tr>
 <td>West Nile virus</td>
 <td>Flaviviridae</td>
 <td>1,002</td>
 <td>94</td>
 <td>12</td>
 <td>Moderate to severe, fever, encephalitis</td>
 <td>2,5</td>
 </tr>
 <tr>
 <td>Eastern equine encephalitis virus</td>
 <td>Togaviridae</td>
 <td>292</td>
 <td>41</td>
 <td>10</td>
 <td>Severe, encephalitis</td>
 <td>6,7</td>
 </tr>
 <tr>
 <td>Highlands J virus</td>
 <td>Togaviridae</td>
 <td>178</td>
 <td>39</td>
 <td>11</td>
 <td>Not known</td>
 <td>6</td>
 </tr>
 <tr>
 <td>Jamestown Canyon virus</td>
 <td>Bunyaviridae</td>
 <td>305</td>
 <td>74</td>
 <td>14</td>
 <td>Mild to moderate, fever, meningitis</td>
 <td>8</td>
 </tr>
<tr>
 <td>Potosi virus</td>
 <td>Bunyaviridae</td>
 <td>259</td>
 <td>76</td>
 <td>4</td>
 <td>Not known</td>
 <td>3</td>
 </tr>
 <tr>
 <td>Cache Valley virus</td>
 <td>Bunyaviridae</td>
 <td>114</td>
 <td>51</td>
 <td>8</td>
 <td>Mild to severe, fever, neurological illness in two documented cases</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Trivittatus virus</td>
 <td>Bunyaviridae</td>
 <td>83</td>
 <td>21</td>
 <td>11</td>
 <td>Not known</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>La Crosse virus</td>
 <td>Bunyaviridae</td>
 <td>1</td>
 <td>1</td>
 <td>1</td>
 <td>Moderate to severe, encephalitis</td>
 <td>4</td>
 </tr>
 <tr>
 <td>Flanders virus</td>
 <td>Rhabdoviridae</td>
 <td>4</td>
 <td>4</td>
 <td>2</td>
 <td>Not known</td>
 <td>&nbsp;</td>
 </tr>
</tbody>
</table>
Table 1. Viruses detected in field-collected mosquitoes by Vero cell culture assay. Mosquitoes were collected during statewide surveillance in Connecticut from 1997-2010.

<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 ...

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Immunology and Infection

18.3K vistas.  The Connecticut Agricultural Experiment Station. Se describe un método para procesar y la pantalla de campo recogidos mosquitos para una diversidad de virus mediante el ensayo de cultivo de células Vero. Mediante el empleo de esta técnica, hemos detectado nueve virus distintos de 4 familias taxonómicas de mosquitos recogidos en Connecticut.

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

Live Cell Imaging of Alphaherpes Virus Anterograde Transport and Spread

Advances in live cell fluorescence microscopy techniques, as well as the construction of recombinant viral strains that express fluorescent fusion proteins have enabled real-time visualization of transport and spread of alphaherpes virus infection of neurons. The utility of novel fluorescent fusion proteins to viral membrane, tegument, and capsids, in conjunction with live cell imaging, identified viral particle assemblies undergoing transport within axons. Similar tools have been successfully employed for analyses of cell-cell spread of viral particles to quantify the number and diversity of virions transmitted between cells. Importantly, the techniques of live cell imaging of anterograde transport and spread produce a wealth of information including particle transport velocities, distributions of particles, and temporal analyses of protein localization. Alongside classical viral genetic techniques, these methodologies have provided critical insights into important mechanistic questions. In this article we describe in detail the imaging methods that were developed to answer basic questions of alphaherpes virus transport and spread.

Live cell imaging of alphaherpes virus infections enables analysis of the dynamic events of directed transport and intercellular spread. Here, we present methodologies that utilize recombinant viral strains expressing fluorescent fusion proteins to facilitate visualization of viral assemblies during infection of primary neurons.

Advances in live cell fluorescence microscopy  techniques, as well as the construction of recombinant viral strains that  express fluorescent fusion ...

Organoids as Model for Infectious Diseases: Culture of Human and Murine Stomach Organoids and Microinjection of Helicobacter Pylori

Recently infection biologists have employed stem cell derived cultures to answer the need for new and better models to study host-pathogen interactions. Three cellular sources have been used: Embryonic stem cells (ESC), induced pluripotent stem cells (iPSC) or adult stem cells. Here, culture of mouse and human gastric organoids derived from adult stem cells is described and used for infection with the gastric pathogen Helicobacter pylori. Human gastric glands are isolated from resection material, seeded in a basement matrix and embedded in medium containing growth factors epidermal growth factor (EGF), R-spondin, Noggin, Wnt, fibroblast growth factor (FGF) 10, gastrin and transforming growth factor (TGF) beta inhibitor. In these conditions, gastric glands grow into 3-dimensional organoids containing 4 lineages of the stomach. The organoids expand indefinitely and can be frozen and thawed similarly as cell lines. For infection studies, bacteria are microinjected into the lumen of the organoids. Infected organoids are processed for imaging. The described methods can be adapted to other organoids and infections with other bacteria, viruses or parasites. This allows the study of infection-induced changes in primary cells.

Stem cell derived cultures harbor tremendous potential to model infectious diseases. Here, the culture of mouse and human gastric organoids derived from adult stem cells is described. The organoids are microinjected with the gastric pathogen Helicobacter pylori.

Recently infection biologists have employed stem cell derived cultures to answer the need for new and better models to study host-pathogen interactions. ...

Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons

Zika Virus (ZIKV) is an emerging pathogen that is linked to fetal developmental abnormalities such as microcephaly, eye defects, and impaired growth. ZIKV is an RNA virus of the Flaviviridae family. ZIKV is mainly transmitted by mosquitoes, but can also be spread by maternal to fetal vertical transmission as well as sexual contact. To date, there are no reliable treatment or vaccine options available to protect those infected by the virus. The development of a reproducible, effective Zika virus infectious cell culture system is critical for studying the molecular mechanisms of ZIKV replication as well as drug and vaccine development. In this regard, a protocol describing a mammalian cell-based in vitro Zika virus culture system for viral production and growth analysis is reported here. Details on the formation of plaques by Zika virus on a cell monolayer and plaque assay for measuring viral titer are presented. Viral genome replication kinetics and double-stranded RNA genome replicatory intermediates are determined. This culture platform was utilized to screen against a library of a small set of cytokines resulting in the identification of interferon-&#945; (IFN-&#945;), IFN-&#946; and IFN-&#947; as potent inhibitors of Zika viral growth. In summary, an in vitro infectious Zika viral culture system and various virological assays are demonstrated in this study, which has the potential to greatly benefit the research community in elucidating further the mechanisms of viral pathogenesis and the evolution of viral virulence. Antiviral IFN-alpha can further be evaluated as a prophylactic, post-exposure prophylactic, and treatment option for Zika virus infections in high-risk populations, including infected pregnant women.

Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons

Zika Virus (ZIKV), an emerging pathogen, is linked to fetal developmental abnormalities and microcephaly. The establishment of an effective infectious cell culture system is crucial for studies of ZIKV replication as well as vaccine and drug development. In this study, various virological assays pertaining to ZIKV are illustrated and discussed.

Zika Virus (ZIKV) is an emerging pathogen that is linked to fetal developmental abnormalities such as microcephaly, eye defects, and impaired growth. ZIKV ...

Rescue and Characterization of Recombinant Virus from a New World Zika Virus Infectious Clone

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral evolution. Here we describe the construction of an infectious clone for Zika virus (ZIKV), which is currently causing an explosive outbreak in the Americas. To prevent toxicity to bacteria that is commonly observed with flavivirus-derived plasmids, we generated a two-plasmid system which separates the genome at the NS1 gene and is more stable than full-length constructs that could not be successfully recovered without mutations. After digestion and ligation to join the two fragments, full-length viral RNA can be generated by in vitro transcription with T7 RNA polymerase. Following electroporation of transcribed RNA into cells, virus was recovered that exhibited similar in vitro growth kinetics and in vivo virulence and infection phenotypes in mice and mosquitoes, respectively.

This protocol describes the recovery of infectious Zika virus from a two-plasmid infectious cDNA clone.

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral ...

Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for the rapid bedside inactivation of the Ebola virus during blood sampling for safe nucleic acid (NA) tests by adding a commercial lysis/binding buffer directly into the vacuum blood collection tubes. Using this bedside inactivation approach, we have developed a safe, rapid, and simplified bedside NA extraction method for the subsequent detection of a virus in lysis/binding buffer-inactivated whole blood. The NA extraction is directly performed in the blood collection tubes and requires no equipment or electricity. 
After the blood is collected into the lysis/binding buffer, the contents are mixed by flipping the tube by hand, and the mixture is incubated for 20 min at room temperature. Magnetic glass particles (MGPs) are added to the tube, and the contents are mixed by flipping the collection tube by hand. The MGPs are then collected on the side of the blood collection tube using a magnetic holder or a magnet and a rubber band. The MGPs are washed three times, and after the addition of elution buffer directly into the collection tube, the NAs are ready for NA tests, such as qPCR or isothermal loop amplification (LAMP), without the removal of the MGPs from the reaction. The NA extraction method is not dependent on any laboratory facilities and can easily be used anywhere (e.g., in field hospitals and hospital isolation wards). When this NA extraction method is combined with LAMP and a portable instrument, a diagnosis can be obtained within 40 min of the blood collection.

Here, we present a protocol for the rapid virus nucleic acid extraction from the virus-inactivated whole blood. The extraction is performed directly in the blood collection tubes and requires no equipment or electricity. The method is not dependent on laboratory facilities and can be used anywhere (e.g., in field hospitals).

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, mortality, and immunosuppression in infected birds. In this study, we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with IBDV, and the quantification of viral replication. The addition of chicken CD40 ligand significantly increased cell proliferation fourfold over six days of culture and significantly enhanced cell viability. Two strains of IBDV, a cell-culture adapted strain, D78, and a very virulent strain, UK661, replicated well in the ex vivo cell cultures. This model will be of use in determining how cells respond to IBDV infection and will permit a reduction in the number of infected birds used in IBDV pathogenesis studies. The model can also be expanded to include other viruses and could be applied to different species of birds.

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Here we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with infectious bursal disease virus, and the quantification of viral replication.

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, ...

Identification of Mouse and Human Antibody Repertoires by Next-Generation Sequencing

The immense adaptability of antigen recognition by antibodies is the basis of the acquired immune system. Despite our understanding of the molecular mechanisms underlying the production of the vast repertoire of antibodies by the acquired immune systems, it has not yet been possible to arrive at a global view of a complete antibody repertoire. In particular, B cell repertoires have been regarded as a black box because of their astronomical number of antibody clones. However, next-generation sequencing technologies are enabling breakthroughs to increase our understanding of the B cell repertoire. In this report, we describe a simple and efficient method to visualize and analyze whole individual mouse and human antibody repertoires. From the immune organs, representatively from spleen in mice and peripheral blood mononuclear cells in humans, total RNA was prepared, reverse transcribed, and amplified using the 5&#39;-RACE method. Using a universal forward primer and antisense primers for the antibody class-specific constant domains, antibody mRNAs were uniformly amplified in proportions reflecting their frequencies in the antibody populations. The amplicons were sequenced by next-generation sequencing (NGS), yielding more than 105 antibody sequences per immunological sample. We describe the protocols for antibody sequence analyses including V(D)J-gene-segment annotation, a bird&#39;s-eye view of the antibody repertoire, and our computational methods.

Here, we describe protocols for the analysis and visualization of the structure and constitution of whole antibody repertoires. This involves the acquisition of vast sequences of antibody RNA using next-generation sequencing.

The immense adaptability of antigen recognition by antibodies is the basis of the acquired immune system. Despite our understanding of the molecular ...

Human Serum Anti-aquaporin-4 Immunoglobulin G Detection by Cell-based Assay

Anti-aquaporin-4 (AQP4) immunoglobulin G (IgG) is the core diagnostic biomarker for neuromyelitis optica spectrum disorders (NMOSD). The cell-based assay (CBA) is a widely used method to detect anti-AQP4 IgG in human serum with high sensitivity and specificity. Briefly, serum anti-AQP4 IgG is captured by AQP4-transfected cell that is fixed on the biochip then detected by a fluorescein-labelled secondary antibody. Fluorescence microscopy is utilized to visualize the fluorescence, and the intensity of fluorescence is evaluated by at least two experienced clinicians. A final diagnosis of NMOSD can be made based on the combination of anti-AQP4 IgG detection results, clinical manifestations, and neuroradiological findings. According to previous studies, CBA is more sensitive and specific than other anti-AQP4 IgG detection methods, and it can be applied to both clinical diagnosis and studies of NMOSD. The method has limitations; for example, an international scale to evaluate serum anti-AQP4 IgG titers is still lacking. Here, a detailed protocol for human serum anti-AQP4 IgG detection using CBA is described.

Cell-based assay is a widely used method to detect serum anti-aquaporin-4 immunoglobulin G. This method could be applied to clinical diagnosis and scientific researches of neuromyelitis optical spectrum disorders.

Anti-aquaporin-4 (AQP4) immunoglobulin G (IgG) is the core diagnostic biomarker for neuromyelitis optica spectrum disorders (NMOSD). The cell-based assay ...

Purification of High Yield Extracellular Vesicle Preparations Away from Virus

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection setting. The presented method is meant to isolate EVs away from virions (i.e., HIV-1), allowing for a higher efficiency and yield compared to conventional ultracentrifugation methods. Our protocol contains three steps: EV precipitation, density gradient separation, and particle capture. Downstream assays (i.e., Western blot, and PCR) can be run directly following particle capture. This method is advantageous over other isolation methods (i.e., ultracentrifugation) as it allows for the use of minimal starting volumes. Furthermore, it is more user friendly than alternative EV isolation methods requiring multiple ultracentrifugation steps. However, the presented method is limited in its scope of functional EV assays as it is difficult to elute intact EVs from our particles. Furthermore, this method is tailored towards a strictly research-based setting and would not be commercially viable.

This protocol isolates extracellular vesicles (EVs) away from virions with high efficiency and yield by incorporating EV precipitation, density gradient ultracentrifugation, and particle capture to allow for a streamlined workflow and a reduction of starting volume requirements, resulting in reproducible preparations for use in all EV research.

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection ...

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine the rate of infection, disseminated infection, and potential transmission of the virus in the mosquito population in question. These procedures are widely utilized with various modifications in vector competence evaluations globally. They are important in determining the potential role that a given mosquito (i.e., species, population, individual) may play in the transmission of a given agent.

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

The presented protocol can determine the vector competence of Aedes aegypti mosquito populations for a given virus, such as Zika, in a containment setting.

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine ...