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Immunology and Infection

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

Published: July 6th, 2013

DOI:

10.3791/50409

1ImmunoRhumatologie Moléculaire, INSERM UMR_S 1109, Centre de Recherche d'Immunologie et d'Hématologie, Université de Strasbourg

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.

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.

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....

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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. Afte.......

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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 .......

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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 .......

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We thank Lee Tuddenham (IBMC, Strasbourg) for amplifying and titrating MCMV-Luc and Thomas Baumert (INSERM U748, Strasbourg) for permission to use the animal facility of the Institute of Virology. Financial support from INSERM, Université de Strasbourg and Agence Nationale de la Recherche (ANR-08-MIEN-005-01) is acknowledged. The initial participation of Sonia Beroud and Laetitia Lelieur during their Master project is also acknowledged.

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Name Company Catalog Number Comments
Reagent/Material
DMEM Fisher Scientific W3523A
Methylene 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

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