A Focus-Forming Assay for Quantifying Virus Titers in the Organs of an Infected Mouse

Abstract

Source: Brien, J. D. et al., Isolation and Quantification of Zika Virus from Multiple Organs in a Mouse. J. Vis. Exp. (2019)

The video demonstrates a procedure for quantifying the Zika virus in multiple mouse organs via a focus-forming assay. The process involves infecting mammalian cell monolayers with virus-infected tissue homogenates, followed by the use of methyl cellulose to prevent viral spread. Subsequently, immunostaining is employed to enable precise quantification and measurement of organ-specific infectivity.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

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.

1. Infect mice using the chosen dose and route, based on the phenotype required. For this protocol, infect 8-10 week-old male and female type I interferon receptor-deficient (Ifnar1^-/-) C57BL/6 mice.
   1. Anesthetize Ifnar1^-/-

Materials

List of materials used in this article

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