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. 2009 Sep 30;392(2):153-61.
doi: 10.1016/j.virol.2009.05.035. Epub 2009 Aug 3.

Investigation of the impact of the common animal facility contaminant murine norovirus on experimental murine cytomegalovirus infection

Affiliations

Investigation of the impact of the common animal facility contaminant murine norovirus on experimental murine cytomegalovirus infection

Carmen M Doom et al. Virology. .

Abstract

Murine norovirus (MNV) is a recently discovered pathogen that has become a common contaminant of specific pathogen-free mouse colonies. MNV-1 induces a robust interferon-beta response and causes histopathology in some mouse strains, suggesting that it may impact other mouse models of infection. Despite many concerns about MNV-1 contamination, there is little information about its impact on immune responses to other infections. This study addresses whether MNV-1 infection has an effect on a model of murine cytomegalovirus (MCMV) infection. Exposure to MNV-1 resulted in a decreased CD8 T cell response to immunodominant MCMV epitopes in both BALB/c and C57BL/6 mice. However, MNV-1 did not impact MCMV titers in either mouse strain, nor did it stimulate reactivation of latent MCMV. These data suggest that while MNV-1 has a mild impact on the immune response to MCMV, it is not likely to affect most experimental outcomes in immunocompetent mice in the MCMV model.

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Figures

Figure 1
Figure 1
MNV infection does not alter the magnitude of MCMV infection. A) BALB/c (i.–iii.) or C57BL/6 (i.–iv.) mice were infected p.o. with 3x107 PFU MNV-1 CW3 on day -2, day -1, or day 0. On day 0, mice were infected i.p. with 5x105 PFU MCMV MW97.01. Four days later (d 4) mice were sacrificed. The salivary glands and spleen were harvested into 1 mL DMEM-complete and the other organs were snap frozen in liquid nitrogen. MCMV titers in different organs were tested by standard plaque assay using 10% tissue homogenates. Each dot represents an individual mouse; the short, solid line represents the mean. The limit of detection is 101 PFU and is indicated by the dotted line. n=5 or 12 for each group. Roman numerals (i.–iv.) represent similar experiments performed on different days. MNV was given on day 0 in BALB/c i. and C57BL/6 i. MNV was given on day -1 in BALB/c ii. and C57BL/6 ii. and iii. MNV was given on day -2 in BALB/c iii. and C57BL/6 iv. All mice were MNV seronegative. All p-values comparing organ virus titers with and without MNV infection were > 0.100, unless otherwise noted. In each case where there was a significant difference between organ virus titers, there is an example provided from a similar experiment where no significant difference was found.
Figure 2
Figure 2
MNV infection does not alter the kinetics of MCMV infection. C57BL/6 or BALB/c mice were infected p.o. with 3x107 PFU MNV-1 CW3 on day -2. On day 0, mice were infected i.p. with 5x105 PFU MCMV MW97.01. A) 3, 7, and 14 days later or B) 21 days later, mice were sacrificed. C) C57BL/6 mice were infected p.o. with 108 PFU MNV-1 CR6 (passage 2) on day -2. On day 0, mice were infected i.p. with 5x105 PFU MCMV MW97.01. Twenty-one days later, mice were sacrificed. The salivary glands and spleen were harvested into 1 mL DMEM-complete and the other organs were snap frozen in liquid nitrogen. MCMV titers in different organs were tested by standard plaque assay using 10% tissue homogenates. Each dot represents an individual mouse; the short, solid line represents the mean. The limit of detection is 101 PFU and is indicated by the dotted line. n=5 for each group. B) i–ii. represent identical experiments performed on different days. All mice were MNV seronegative, except in B) 12/15 MNV-infected mice were MNV-seropositive and C) 5/5 MNV-infected mice were MNV-seropositive. All p-values comparing organ virus titers with and without MNV infection were > 0.200, unless otherwise noted.
Figure 3
Figure 3
MNV infection impacts the immunodominant CD8 T cell response to MCMV. BALB/c mice were infected p.o. with 3x107 PFU MNV-1 CW3 on day -2. On day 0, mice were infected i.p. with 5x105 PFU MCMV MW97.01. A week later (d 7), the spleens were harvested and splenocytes were incubated with MCMV peptides (10−6 M) for 7 hours in the presence of brefeldin A. They were then stained for surface CD8-α, fixed, permeabilized, and stained for intracellular IFN-γ. The percentage of IFN-γ+ CD8-α+ T cells were measured by flow cytometry on an LSR II and analyzed by FloJo software. Error bars indicate SEM. n=6 for each group. All mice were MNV-1 seronegative. All p-values comparing an epitope-specific response with and without MNV infection were > 0.100, unless otherwise noted.
Figure 4
Figure 4
MCMV MW97.01 does not reactivate in response to MNV infection. A) BALB/c or B) C57BL/6 mice were infected i.p. with 5x105 PFU MCMV MW97.01 on day ≥ 30. On day 0, mice were infected p.o. with 3x107 PFU MNV-1 CW3. Either 1 (left panels) or 2 (right panels) weeks later, mice were sacrificed. C) BALB/c (left) or C57BL/6 mice (right) were infected i.p. with 5x105 PFU MCMV-K181 on day ≥ 30. On day 0, mice were infected p.o. with 3x107 PFU MNV-1 CW3. Two weeks later, mice were sacrificed. The salivary glands, spleen, and lung were harvested into 1 mL DMEM-complete. MCMV titers in these organs were tested by standard plaque assay using 10% tissue homogenates. Each dot represents an individual mouse; the short, solid line represents the mean. The limit of detection is 101 PFU and is indicated by the dotted line. n=5 for each group. All mice were MNV-1 seronegative. All p-values comparing organ virus titers with and without MNV infection were > 0.400, unless otherwise noted.

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