Dr. Pinto is funded by a seed grant from the Saint Louis University School of Medicine and startup funds from Saint Louis University School of Medicine. Dr. Brien is funded by a K22AI104794 early investigator award from the NIH NIAID as well a seed grant from the Saint Louis University School. For all funded individuals the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

All procedures of the present study are in accordance with the guidelines set by the St. Louis University Animal Care and Use Committee. SLU is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC).
1. Organ Isolation
NOTE: The virus is not stable at room temperature (RT) so the number of animals harvested at one time must be planned carefully to preserve viral titers.
<ol>
	<li><stro...

<ol>
	<li>Lazear, H. M., Diamond, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zika+Virus:+New+Clinical+Syndromes+and+Its+Emergence+in+the+Western+Hemisphere.">Zika Virus: New Clinical Syndromes and Its Emergence in the Western Hemisphere.</a> Journal of Virology. 90 (10), 4864-4875 (2016).</li><li>Simpson, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zika+Virus+Infection+in+Man.">Zika Virus Infection in Man.</a> Transactions of the Royal Society of Tropical Medicine and Hygiene. 58, 335-338 (1964).</li><li>Fagbami, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiological+investigations+on+arbovirus+infections+at+Igbo-Ora,+Nigeria.">Epidemiological investigations on arbovirus infections at Igbo-Ora, Nigeria.</a> Tropical and Geographical Medicine. 29 (2), 187-191 (1977).</li><li>McCrae, A. W., Kirya, B. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zika+virus:+history+of+a+newly+emerging+arbovirus.">Zika virus: history of a newly emerging arbovirus.</a> Lancet Infect Dis. 16 (7), e119-e126 (2016).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zika+virus+outbreaks+in+the+Americas.">Zika virus outbreaks in the Americas.</a> The Weekly Epidemiological Record. 90 (45), 609-610 (2015).</li><li>Brien, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Immunological basis of age-related vulnerability to viral infection. , (2007).</li><li>Brien, J. D., Uhrlaub, J. L., Nikolich-Zugich, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=West+Nile+virus-specific+CD4+T+cells+exhibit+direct+antiviral+cytokine+secretion+and+cytotoxicity+and+are+sufficient+for+antiviral+protection.">West Nile virus-specific CD4 T cells exhibit direct antiviral cytokine secretion and cytotoxicity and are sufficient for antiviral protection.</a> Journal of Immunology. 181 (12), 8568-8575 (2008).</li><li>Brien, J. D., Uhrlaub, J. L., Hirsch, A., Wiley, C. A., Nikolich-Zugich, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Key+role+of+T+cell+defects+in+age-related+vulnerability+to+West+Nile+virus.">Key role of T cell defects in age-related vulnerability to West Nile virus.</a> Journal of Experimental Medicine. 206 (12), 2735-2745 (2009).</li><li>Brien, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genotype-specific+neutralization+and+protection+by+antibodies+against+dengue+virus+type+3.">Genotype-specific neutralization and protection by antibodies against dengue virus type 3.</a> Journal of Virology. 84 (20), 10630-10643 (2010).</li><li>Shrestha, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+therapeutic+antibodies+that+neutralize+homologous+and+heterologous+genotypes+of+dengue+virus+type+1.">The development of therapeutic antibodies that neutralize homologous and heterologous genotypes of dengue virus type 1.</a> PLoS Pathogens. 6 (4), e1000823 (2010).</li><li>Brien, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interferon+regulatory+factor-1+(IRF-1)+shapes+both+innate+and+CD8(+)+T+cell+immune+responses+against+West+Nile+virus+infection.">Interferon regulatory factor-1 (IRF-1) shapes both innate and CD8(+) T cell immune responses against West Nile virus infection.</a> PLoS Pathogens. 7 (9), e1002230 (2011).</li><li>Pinto, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+temporal+role+of+type+I+interferon+signaling+in+CD8++T+cell+maturation+during+acute+West+Nile+virus+infection.">A temporal role of type I interferon signaling in CD8+ T cell maturation during acute West Nile virus infection.</a> PLoS Pathogens. 7 (12), e1002407 (2011).</li><li>Brien, J. D., Lazear, H. M., Diamond, M. 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D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protection+by+immunoglobulin+dual-affinity+retargeting+antibodies+against+dengue+virus.">Protection by immunoglobulin dual-affinity retargeting antibodies against dengue virus.</a> Journal of Virology. 87 (13), 7747-7753 (2013).</li><li>Messaoudi, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chikungunya+virus+infection+results+in+higher+and+persistent+viral+replication+in+aged+rhesus+macaques+due+to+defects+in+anti-viral+immunity.">Chikungunya virus infection results in higher and persistent viral replication in aged rhesus macaques due to defects in anti-viral immunity.</a> PLoS Neglected Tropical Diseases. 7 (7), e2343 (2013).</li><li>Pinto, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+hydrogen+peroxide-inactivated+virus+vaccine+elicits+humoral+and+cellular+immunity+and+protects+against+lethal+West+Nile+virus+infection+in+aged+mice.">A hydrogen peroxide-inactivated virus vaccine elicits humoral and cellular immunity and protects against lethal West Nile virus infection in aged mice.</a> Journal of Virology. 87 (4), 1926-1936 (2013).</li><li>Sukupolvi-Petty, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+analysis+of+antibodies+against+dengue+virus+type+4+reveals+strain-dependent+epitope+exposure+that+impacts+neutralization+and+protection.">Functional analysis of antibodies against dengue virus type 4 reveals strain-dependent epitope exposure that impacts neutralization and protection.</a> Journal of Virology. 87 (16), 8826-8842 (2013).</li><li>Pinto, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deficient+IFN+signaling+by+myeloid+cells+leads+to+MAVS-dependent+virus-induced+sepsis.">Deficient IFN signaling by myeloid cells leads to MAVS-dependent virus-induced sepsis.</a> PLoS Pathogens. 10 (4), e1004086 (2014).</li><li>Pinto, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+New+Therapeutics+Using+a+More+Immunocompetent+Mouse+Model+of+Antibody-Enhanced+Dengue+Virus+Infection.">Defining New Therapeutics Using a More Immunocompetent Mouse Model of Antibody-Enhanced Dengue Virus Infection.</a> MBio. 6 (5), (2015).</li><li>Pinto, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+and+Murine+IFIT1+Proteins+Do+Not+Restrict+Infection+of+Negative-Sense+RNA+Viruses+of+the+Orthomyxoviridae,+Bunyaviridae,+and+Filoviridae+Families.">Human and Murine IFIT1 Proteins Do Not Restrict Infection of Negative-Sense RNA Viruses of the Orthomyxoviridae, Bunyaviridae, and Filoviridae Families.</a> Journal of Virology. 89 (18), 9465-9476 (2015).</li><li>Hassert, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD4+T+cells+mediate+protection+against+Zika+associated+severe+disease+in+a+mouse+model+of+infection.">CD4+T cells mediate protection against Zika associated severe disease in a mouse model of infection.</a> PLoS Pathog. 14 (9), e1007237 (2018).</li><li>Pinto, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deficient+IFN+signaling+by+myeloid+cells+leads+to+MAVS-dependent+virus-induced+sepsis.">Deficient IFN signaling by myeloid cells leads to MAVS-dependent virus-induced sepsis.</a> PLoS Pathog. 10 (4), e1004086 (2014).</li><li>Brien, J. D., Lazear, H. M., Diamond, M. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+lectin+pathway+of+complement+activation+contributes+to+protection+from+West+Nile+virus+infection.">The lectin pathway of complement activation contributes to protection from West Nile virus infection.</a> Virology. 412 (1), 101-109 (2011).</li><li>Lazear, H. M., Pinto, A. K., Vogt, M. R., Gale, M., Diamond, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beta+interferon+controls+West+Nile+virus+infection+and+pathogenesis+in+mice.">Beta interferon controls West Nile virus infection and pathogenesis in mice.</a> Journal of Virology. 85 (14), 7186-7194 (2011).</li></ol>

ZIKV infection can cause a neurological disease therefore the current animal models to study pathogenesis, immune responses and protective efficacy of vaccines and antivirals need to focus on viral control within the CNS. One of the challenges in focusing on CNS disease is that it often comes at the expense of studying peripheral infection. The organ isolation methods proposed here focuses on the need to rapidly evaluate ZIKV infection in both the periphery and the CNS in order to assess CNS mediated ZIKV associated dise...

Zika virus (ZIKV) is an arbovirus that belongs to the flaviviridae family, which includes important neuroinvasive human pathogens such as Powassan virus (POWV), Japanese encephalitis virus (JEV), and West Nile virus (WNV)1. Following its isolation and identification, there have been periodic reports of human ZIKV infections in Africa and Asia2,3,4,5, and epidemics within Central and South America (reviewed in reference6). However, it was not until recently that ZIKV was thought to cause severe disease7. Now there are thousands of cases of neurological disease and birth defects linked to ZIKV infections. The rapid emergence of ZIKV has prompted many questions relating to: why there is an increase in disease severity, what is the immunological response to ZIKV infection and are there viral and/or immune mediated pathologies linked to the increase in neurological manifestations and birth defects. There is now a rush to understand the central nervous system (CNS) related disease associated with ZIKV as well as the need to rapidly test the efficacy of the antivirals and vaccines against ZIKV. It is against this backdrop that we have developed methods for the rapid analysis of ZIKV titers in both the periphery and CNS using a ZIKV-specific focus forming assays (FFA).
Small animal models are important for understanding disease progression and for the early evaluation of vaccines, therapeutics, and anti-virals. We have established small animal models for the study of arbovirus disease by using various mouse strains to model human infection and protection against viral pathogens8,9,10,11,12,13,14,15,16,17,18,19,20,21,22. Using this prior experience, we began to modify techniques used for the assessment of WNV and Dengue virus, a related flavivirus for the assessment of ZIKV titer in both peripheral organs as well as the CNS21,23,24. The advantages of these methods over other assays are: 1) that they combine the ability to harvest both peripheral and CNS organs for the analysis; 2) the methods are adaptable for flow cytometry, for measurements of innate and adaptive immune responses, along with viral titers on the same animal in the same organ; 3) the harvest technique is adaptable for histological analysis; 4) the ZIKV FFA is a rapid high throughput method for viral titer analysis; and 5) these methods can be applied to the assessment of viral titers in the organs of animals infected with most pathogens25.

<table>
	<tbody>
		<tr>
			<td>1-bromo-3-chloropropane (BCP)</td>
			<td>MRC gene</td>
			<td>BP151</td>
			<td></td>
		</tr>
		<tr>
			<td>10cc syringe</td>
			<td>Thermo Fisher Scientific</td>
			<td>BD 309642</td>
			<td></td>
		</tr>
		<tr>
			<td>18G needle</td>
			<td>Thermo Fisher Scientific</td>
			<td>22-557-145</td>
			<td></td>
		</tr>
		<tr>
			<td>1cc TB syringe</td>
			<td>Thermo Fisher Scientific</td>
			<td>14-823-16H</td>
			<td></td>
		</tr>
		<tr>
			<td>20cc syringe</td>
			<td>Thermo Fisher Scientific</td>
			<td>05-561-66</td>
			<td></td>
		</tr>
		<tr>
			<td>24 tube beadmill</td>
			<td>Thermo Fisher Scientific</td>
			<td>15 340 163</td>
			<td></td>
		</tr>
		<tr>
			<td>3.2 mm stainless steel beads</td>
			<td>Thermo Fisher Scientific</td>
			<td>NC9084634</td>
			<td></td>
		</tr>
		<tr>
			<td>37C Tissue Culture incubator</td>
			<td>Nuair</td>
			<td>5800</td>
			<td></td>
		</tr>
		<tr>
			<td>4G2 antibody</td>
			<td>in house</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96 well flat bottom plates</td>
			<td>Midsci</td>
			<td>TP92696</td>
			<td></td>
		</tr>
		<tr>
			<td>96well round bottom plates</td>
			<td>Midsci</td>
			<td>TP92697</td>
			<td></td>
		</tr>
		<tr>
			<td>Basix 1.5ml eppendorf tubes</td>
			<td>Thermo Fisher Scientific</td>
			<td>02-682-002</td>
			<td></td>
		</tr>
		<tr>
			<td>Concentrated Germicidal Bleach</td>
			<td>Staples</td>
			<td>30966CT</td>
			<td></td>
		</tr>
		<tr>
			<td>CTL S6 Analyzer</td>
			<td>CTL</td>
			<td>CTL S6 Universal Analyzer</td>
			<td></td>
		</tr>
		<tr>
			<td>curved cutting scissors</td>
			<td>Fine Science Tools</td>
			<td>14061-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle&#8217;s Medium - high glucose With 4500 mg/L glucose</td>
			<td>MilliporeSigma</td>
			<td>D5671</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol (molecular biology-grade)</td>
			<td>MilliporeSigma</td>
			<td>e7023</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>MilliporeSigma</td>
			<td>F0926-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fine Science Tools</td>
			<td>11036-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Glacial acetic acid</td>
			<td>MilliporeSigma</td>
			<td>537020</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse HRP-labeled antibody</td>
			<td>MilliporeSigma</td>
			<td>8924</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES 1 M</td>
			<td>MilliporeSigma</td>
			<td>H3537-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol (molecular biology-grade)</td>
			<td>MilliporeSigma</td>
			<td>I9516</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine/Xylazine cocktail</td>
			<td>Comparative Medicine</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>MilliporeSigma</td>
			<td>g7513</td>
			<td></td>
		</tr>
		<tr>
			<td>Magmax RNA purification kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM1830</td>
			<td></td>
		</tr>
		<tr>
			<td>Methylcellulose</td>
			<td>MilliporeSigma</td>
			<td>M0512</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Ependorf</td>
			<td>5424R</td>
			<td></td>
		</tr>
		<tr>
			<td>MiniCollect 0.5ml EDTA tubes</td>
			<td>Bio-one</td>
			<td>450480</td>
			<td></td>
		</tr>
		<tr>
			<td>o-ring tubes</td>
			<td>Thermo Fisher Scientific</td>
			<td>21-403-195</td>
			<td></td>
		</tr>
		<tr>
			<td>one step q RT-PCR mix</td>
			<td>Thermo Fisher Scientific</td>
			<td>4392938</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Thermo Fisher Scientific</td>
			<td>EMS- 15713-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>MilliporeSigma</td>
			<td>d8537-500ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Proline multichannel pipettes</td>
			<td>Sartorius</td>
			<td>72230/72240</td>
			<td></td>
		</tr>
		<tr>
			<td>Proline single channel pipettes</td>
			<td>Sartorius</td>
			<td>728230</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAse free water</td>
			<td>Thermo Fisher Scientific</td>
			<td>10-977-023</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAzol BD</td>
			<td>MRC gene</td>
			<td>RB192</td>
			<td></td>
		</tr>
		<tr>
			<td>Rocking Platform</td>
			<td>Thermo Fisher Scientific</td>
			<td>11-676-333</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Fisher</td>
			<td>MT10040CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>MilliporeSigma</td>
			<td>s7900</td>
			<td></td>
		</tr>
		<tr>
			<td>spoon/spatula</td>
			<td>Fine Science Tools</td>
			<td>10090-17</td>
			<td></td>
		</tr>
		<tr>
			<td>straight cutting scissors</td>
			<td>Fine Science Tools</td>
			<td>14060-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>MilliporeSigma</td>
			<td>t8787</td>
			<td></td>
		</tr>
		<tr>
			<td>True Blue Substrate</td>
			<td>VWR</td>
			<td>95059-168</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>MilliporeSigma</td>
			<td>T3924-100ML</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and quantification of zika virus from multiple organs in a mouse

The methods being presented demonstrate laboratory procedures for the isolation of organs from Zika virus infected animals and the quantification of viral load. The purpose of the procedure is to quantify viral titers in peripheral and CNS areas of the mouse at different time points post infection or under different experimental conditions to identify virologic and immunological factors that regulate Zika virus infection. The organ isolation procedures demonstrated allow for both focus forming assay quantification and quantitative PCR assessment of viral titers. The rapid organ isolation techniques are designed for the preservation of virus titer. Viral titer quantification by focus forming assay allows for the rapid throughput assessment of Zika virus. The benefit of the focus forming assay is the assessment of infectious virus, the limitation of this assay is the potential for organ toxicity reducing the limit of detection. Viral titer assessment is combined with quantitative PCR, and using a recombinant RNA copy control viral genome copy number within the organ is assessed with low limit of detection. Overall these techniques provide an accurate rapid high throughput method for the analysis of Zika viral titers in the periphery and CNS of Zika virus infected animals and can be applied to the assessment of viral titers in the organs of animals infected with most pathogens, including Dengue virus.

To evaluate ZIKV titers using the protocol described above Ifnar1-/- mice were infected with ZIKV (PRVABC59) via subcutaneous (SC) injection to the footpad. Here, the administration of 1 x 105 FFU of ZIKV to 8-12 week old Ifnar1-/- mice SC is not lethal but the virus can replicate in both the periphery and CNS. This dose and route are used to study host pathogen immune responses and pathogenicity. Administration of 1 x 105 FFU ...

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Isolation and Quantification of Zika Virus from Multiple Organs in a Mouse

The goal of the protocol is to demonstrate the techniques used to investigate viral disease by isolating and quantifying Zika virus, from multiple organs in a mouse following infection.

The methods being presented demonstrate laboratory procedures for the isolation of organs from Zika virus infected animals and the quantification of viral ...

In this video we describe a set of procedures designed to interrogate the impact of a viral infection and the resulting immunological response using a Murine model of Zika infection. This protocol is significant because it allows us to develop a mechanistic understanding of infection and immunity by identifying the extent to which a virus has spread throughout the body, transversing physical barriers and accessing tissues. These techniques allow a detailed understanding of viral pathogenesis and provides the ability to dissect the mechanism of protection provided by vaccines or therapeutics.Here we demonstrate this method using Zika virus infection of mice, but this method can be applied to a range of viral infections including other flaviviruses like Dengue virus and alphaviruses like Chikungunya virus. Harvesting the organs of the central nervous system can be challenging, so it's important for a person to plan a small experiment, and be prepared to practice the techniques beforehand. This protocol allows for the rapid dissection of viral spread within small animal models with the ability to visualize, capture, and store experimental data.It is important to visually demonstrate this method to allow the experimenter to understand how to harvest the central nervous system organs. It's also important to see the level of cell confluency and the intensity of the viral foci. To begin, use forceps to remove the pelt of the infected mouse.Decapitate the head of the mouse with scissors, followed by the removal of the arms and legs. To harvest the brain, use serrated La Grange scissors to cut the skull through the foramen magnum. Peel off the skull with forceps, and scoop out the brain with a spatula.Using strong blunted scissors, remove the bone surrounding the spine, then cut across the pelvic bone to expose the vertebral foramen at the lumbar level. Then, remove as much muscle as possible surrounding the spinal column. Carefully, place the beveled tip of the needle inside the vertebral foramen, avoiding excessive pressure to prevent the needle trespassing the vertebral body.Hold strongly to exert pressure on the vertebral body and the needle, and press the syringe plunger to expel the cord into the petri dish. Immediately transfer the spinal cord in the labeled tube, and place in the dry ice bath for further homogenization. For homogenization, add one milliliter of DMEM and homogenize the sample in a BeadBeater.After ensuring organs are homogenized, centrifuge and aliquot samples on ice and store in the minus 80 degrees Celsius until needed. On the day of the assay, remove the homogenized samples from the minus 80 degrees Celsius freezer, and allow them to thaw. With the samples on ice, dilute Zika virus tenfold into the first well of the dilution plate and use a multichannel pipette to do tenfold serial dilutions down the plate, changing tips each time.By adding 20 microliters of sample into 180 microliters of growth media, change pipette tips between each dilution. Prepare the focus forming plate by removing the media from the prepared flat bottom 96-well plate covering virocells. To prevent the monolayer from drying out, immediately add 100 microliters of the virus dilution to each well in the viroplate.Use the same set of tips to add from the lowest to the highest concentration. Rock plate side to side two to four times and incubate at 37 degrees Celsius in 5%carbon dioxide for one to two hours. Warm up 2%methylcellulose to room temperature.Then, dilute the 2%methylcellulose solution in growth media at a ratio of approximately two to one. After incubation, add 125 microliters of the methylcellulose growth media to each well of the 96-well plates. Incubate again at 37 degrees Celsius in 5%carbon dioxide for 32 to 40 hours.To fix the virocells, in a biosafety cabinet add 50 microliters of 5%paraformaldehyde over the top of the methylcellulose layer in each well of the 96-well plates. Incubate for 60 minutes at room temperature. Alternatively, cover the plates with Parafilm and place them at four degrees Celsius overnight.After that, use a pipette to remove overlay and media off cells into a disposable container inside the biosafety cabinet. Wash gently with 150 microliters of PBS per well. Remove PBS from the plates and remove the plate from the biosafety cabinet.Repeat the PBS wash twice, then add 150 microliters per well one times FFA wash buffer and leave at room temperature for five to 10 minutes to permeabilize the fixed cells. Next, use primary Zika antibody to detect infection. Prepare the primary antibody 4G2 at a concentration of one microgram per milliliter in FFA staining buffer.Flick FFA wash buffer from the plates, and add 50 microliters of the primary antibody in FFA staining buffer to each well. Then seal the plates with Parafilm, or plastic film, and incubate overnight at four degrees Celsius. Prepare the secondary go anti-mouse HRP labeled antibody at a concentration of one to 5, 000 in FFA staining buffer.After removing the antibody solution, wash cells three times with 150 microliters FFA wash buffer per well. Remove the wash buffer by flicking into the sink each time. Stain the cells with the secondary antibody in FFA staining buffer at 50 microliters per well and incubate one to two hours at room temperature.After that, wash cells three times with FFA wash buffer, again, removing the wash buffer by flicking into the sink each time. Add 50 microliters of the TrueBlue Substrate into each well. Wait two to 15 minutes until spots are fully defined with minimal background.After the spots are visible, wash gently with water using a hand to shield the monolayer from the force of the water running. Tap dry on paper towels. Shortly after, use a dissecting scope to count manually the spots in each well, or use an automated spot counter.In the ImmunoCapture software, select a dilution with easily distinguished foci between 20 to 200 per well for each sample and calculate titer in focus-forming units per millimeter using the average of duplicate wells. In this experiment, viral burden was shown in the peripheral and central nervous system tissues after Ifnar mice were given Zika virus subcutaneously. The limit of detection was between 100 to 500 FFU per gram, based upon organ.When performing the focus-forming assay, technical mistakes can result in suboptimal results. Organ toxicity, driven by high concentration of liver, altered the sensitivity of the assay. When the viral titer in the organ was lower than the toxicity, the FFA could not accurately record the viral titers.Kidney cells illustrated toxicity with high organ concentration but was overcome by the viral titer at low organ concentrations. Vigorous pipetting or washing can remove the monolayer, leading to lost data inaccurate reporting of titer results. Fibers or hairs that are present in lab bench absorbent paper, can contaminate individual wells and cause significant errors if using an automated counting program.Cell density is another issue, which can dramatically impact the success of a focus-forming assay. Cells at approximately 60%confluency at the start of the assay, compared to cells plated at 90%confluency, showed dramatic difference impacting the focus-forming assay. Planning the size and scope of the experiment so that organ harvest is completed efficiently, is the most important part of the procedure, followed by starting the focus-forming assay with high quality cells.This procedure has been used to quantify virus for multiple viral families. This protocol can also be adapted to screen for therapeutic compounds and quantify neutralizing antibody titers. These techniques are used to quantitate live virus and appropriate safety steps should be taken according to the guidelines laid out in the BMBL.

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Research

JoVE Journal

Immunology and Infection

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