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

Summary

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.

Abstract

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.

Introduction

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 Asia^2,3,4,5, and epidemics within Central and South America (reviewed in reference⁶). However, it was not until recently that ZIKV was thought to cause severe ....

Protocol

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.

Discussion

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.......

Materials

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