Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates

Summary

Human Cytomegalovirus (HCMV) infection of neonates represents an important cause of mental retardation, yet the molecular events leading to virus-induced pathogenesis are still poorly understood. To investigate the dynamics of brain infection, we adapted whole-animal in vivo imaging to perform time-course analysis of neonates infected with a luciferase-recombinant virus.

Abstract

Human Cytomegalovirus (HCMV or HHV-5) is a life-threatening pathogen in immune-compromised individuals. Upon congenital or neonatal infection, the virus can infect and replicate in the developing brain, which may induce severe neurological damage, including deafness and mental retardation. Despite the potential severity of the symptoms, the therapeutic options are limited by the unavailability of a vaccine and the absence of a specific antiviral therapy. Furthermore, a precise description of the molecular events occurring during infection of the central nervous system (CNS) is still lacking since observations mostly derive from the autopsy of infected children. Several animal models, such as rhesus macaque CMV, have been developed and provided important insights into CMV pathogenesis in the CNS. However, despite its evolutionary proximity with humans, this model was limited by the intracranial inoculation procedure used to infect the animals and consistently induce CNS infection. Furthermore, ethical considerations have promoted the development of alternative models, among which neonatal infection of newborn mice with mouse cytomegalovirus (MCMV) has recently led to significant advances. For instance, it was reported that intraperitoneal injection of MCMV to Balb/c neonates leads to infection of neurons and glial cells in specific areas of the brain. These findings suggested that experimental inoculation of mice might recapitulate the deficits induced by HCMV infection in children. Nevertheless, a dynamic analysis of MCMV infection of neonates is difficult to perform because classical methodology requires the sacrifice of a significant number of animals at different time points to analyze the viral burden and/or immune-related parameters. To circumvent this bottleneck and to enable future investigations of rare mutant animals, we applied in vivo imaging technology to perform a time-course analysis of the viral dissemination in the brain upon peripheral injection of a recombinant MCMV expressing luciferase to C57Bl/6 neonates.

Introduction

Human Cytomegalovirus (HCMV/HHV-5) is a member of the β-herpesvirus family. HCMV is a highly prevalent, opportunistic pathogen which is usually acquired during early life as an asymptomatic infection ¹. Like all herpesviruses, HCMV persists throughout the entire life of the host whose immune system tightly controls viral replication. Episodes of viral reactivation mostly occur in immunocompromised individuals such as transplant patients receiving drugs to prevent graft rejection ². In adults, HCMV has also been linked to glioblastomas ³. In addition, HCMV is a prominent pathogen for newborns with immature immunity ^4-6....

Protocol

1. Preparation of Viral Suspension

1. Obtain the Smith strain of MCMV expressing Luciferase (MCMV-Luc ¹⁵) from Ulrich Koszninowski's laboratory. In this recombinant, the Luciferase gene is inserted in the ie2 locus of the MCMV genome.
2. To amplify MCMV-Luc, infect a murine bone marrow stromal cell line (M2-10B4, ATCC #CRL-1972) with MCMV at different multiplicity of infection (MOI, 0.001 to 1) ¹⁶. For this, add virus to cultured cells in serum free medium at 37 °C. After one hour, remove the supernatant and replace it with DMEM supplemented with 10% fetal calf serum (FCS). Five days later, harvest the culture medium ....

Results

A representative experiment is illustrated in Figure 1. Upon intraperitoneal injection of 50 PFUs of MCMV-Luc (Panel A shows a similar injection performed with Methylene blue to visualize the subcutaneous route of the needle), neonates were anesthetized and received simultaneously 0.3 mg of the luciferase substrate (Luciferin, Caliper). Fifteen minutes later, animals were placed ventral side up in the acquisition chamber of the IVIS 50 (In Vivo Imaging System, Caliper) and the light emitted by the whole .......

Discussion

Using in vivo imaging technology to monitor MCMV-Luc dissemination in mice neonates, we were able to observe viral spread to the brain of mutant animals, as opposed to wild-type. Further dissection of the animal and ex vivo imaging of the brain confirmed the presence of luminescent virus in the central nervous system. In addition, we also performed immunohistochemistry (not shown) on brain thin sections with an antibody specific for MCMV E1 early protein and observed that indeed, MCMV is present in the .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagent/Material
DMEM	Fisher Scientific	W3523A
Methylen blue	Sigma Aldrich	319112
Insulin needles	VWR	613-4897
Ketamine	CentraVet	Ket 201
Xylazine/Vetranal	Sigma Aldrich	46995
DPBS	DUTSCHER	P0436500
Luciferin	Caliper	760504
gentamycin	Sigma Aldrich	G1272
penicillin/streptomycin	Gibco	15070
carboxymethylcellulose	Sigma Aldrich	C4888
formaldehyde	Sigma Aldrich	F8775
crystal violet	Sigma Aldrich	C3886
Equipment
IVIS 50	Caliper/Perkin Elmer