JOURNAL OF VIROLOGY, July 2009, p. 7357–7360 0022-538X/09/$08.00⫹0 doi:10.1128/JVI.00623-09 Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 83, No. 14
Murine Norovirus Infection Has No Significant Effect on Adaptive Immunity to Vaccinia Virus or Influenza A Virus䌤 Scott E. Hensley,1 Amelia K. Pinto,2 Heather D. Hickman,1 Robin J. Kastenmayer,3 Jack R. Bennink,1 Herbert W. Virgin,2 and Jonathan W. Yewdell1* Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 208921; Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri 631102; and Comparative Medicine Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 208923 Received 25 March 2009/Accepted 23 April 2009
Murine norovirus (MNV) is endemic in many research mouse colonies. Although MNV infections are typically asymptomatic in immunocompetent mice, the effects of MNV infection on subsequent experimental viral infections are poorly documented. Here, we infected C57BL/6 mice with MNV and then with either vaccinia virus or influenza A virus. MNV infection had no effect on CD8ⴙ T-cell or antibody responses to secondary viruses or to secondary virus-induced morbidity or mortality. While our findings suggest that MNV has little influence on host immunity in immunocompetent mice, we would urge caution regarding the potential effects of MNV on immune responses to viruses and other pathogens, which must be determined on a system-by-system basis. infection on adaptive immune responses in wt mice to influenza A virus (IAV) and vaccinia virus (VV). We infected C57BL/6 mice perorally with a high dose (3 ⫻ 107 PFU/mouse) of a plaque-purified MNV stock derived from MNV-CR6p2 (13). The capacity of this plaque-purified virus to persist in wt mice has been confirmed by quantitative PCR analysis and a plaque assay (D. Strong, L. Thackray, and H. Virgin, unpublished observation). We confirmed that the mice were infected by measuring anti-MNV antibodies (Abs) by using an enzyme-linked immunosorbent assay (ELISA) (data not shown). For all experiments, mice were infected with MNV at Washington University and shipped 4 to 5 days later to NIAID for further study. To contain MNV, infected mice were housed in microisolator cages in a quarantine room. In some experiments, control mice were housed in the same room as MNV-infected mice. Sera collected from control mice did not contain anti-MNV Abs as determined by ELISA (data not shown), confirming that transmission of MNV between mice housed in microisolator cages can be prevented by proper cage changing and aseptic handling of samples from infected mice. Upon intraperitoneal (i.p.) infection with either VV or IAV, mice mount robust CD8⫹ T-cell responses that peak, respectively, on day 6 or 7. Anti-VV and anti-IAV CD8⫹ T-cell responses in C57BL/6 mice conform to a well-established immunodominance hierarchy (3, 10). To determine to what extent MNV infection alters the magnitude and/or immunodominance hierarchy of CD8⫹ T-cell responses, we infected C57BL/6 mice i.p. with either VV or IAV 19 days following MNV infection. As controls, naïve mice (MNV negative) were infected with either virus. Lymphocytes were isolated from mice 6 days postinfection with VV and 7 days postinfection with IAV. The fraction of antigen-specific CD8⫹ T cells present in spleen and peritoneal exudate cells (PEC) was determined by intracellular IFN-␥ staining after stimulation with synthetic peptides. MNV infection had little effect on the magnitude of splenic or PEC CD8⫹ T cells responding to VV (Fig.
Human norovirus (NoV) infections cause greater than 90% of nonbacterial gastroenteritis cases (4, 5) and are an important public health concern. Murine noroviruses (MNV) were recently identified (7) as highly pathogenic agents in immunocompromised mice, and serological studies indicate that over 20% of mice in research colonies are exposed to MNV (6). As with NoV, MNV is spread through the fecal-oral route. While NoV rapidly causes gastrointestinal symptoms and fever in healthy individuals, MNV is typically asymptomatic in immunocompetent mice. MNV isolates are both genetically and biologically diverse (13). In wild-type (wt) mice, some strains of MNV are rapidly cleared, while others persist (13). Controlling MNV infections requires elements of both innate and adaptive immunity. Mice with defects in interferon (IFN) signaling pathways demonstrate increased MNV lethality (7, 9). CD4⫹ and CD8⫹ T cells and B cells are all needed for complete MNV clearance (1, 2). Natural exposure of immunocompromised mice to MNV leads to inflammation of the liver, lungs, and peritoneal and pleural cavities (14). It is well established that infection with natural mouse viruses can greatly impact immune responses to infections with other viruses. The prevalence of MNV in research mouse colonies might therefore lead to irreproducible and variable results that significantly impact research efforts. Indeed, MNV was recently reported to alter disease progression in a mouse model of bacterium-induced inflammatory bowel disease (8). Concern over the potential effects of MNV on viral immunology research prompted a dedicated workshop at the 2008 Keystone Viral Immunity meeting (http://www.keystonesymposia .org). In the present study, we examined the effect of MNV
* Corresponding author. Mailing address: Building 33, Room 2E13, 33 North Drive, Bethesda, MD 20892. Phone: (301) 402-4602. Fax: (301) 402-4802. E-mail:
[email protected]. 䌤 Published ahead of print on 29 April 2009. 7357
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FIG. 1. MNV exposure does not alter CD8⫹ T-cell responses to VV or IAV. MNV-infected and naïve C57BL/6 mice were infected i.p. with ⬃1 ⫻ 106 PFU of VV (A and B) or ⬃1 ⫻ 107 50% tissue culture infective dose units of IAV (C and D), and specific CD8⫹ T cells were determined by intracellular IFN-␥ staining after restimulating lymphocytes with peptides. Lymphocytes isolated from the spleen (A and C) and peritoneal cavity (B and D) were tested. MNV infections were completed 19 days prior to VV or IAV infections. Means and SEM are shown in panels A and C. A two-way analysis of variance and Bonferroni statistical analysis were completed for these experiments. Cells were pooled for peritoneal lavage samples as shown in panels B and D. Four to five mice/group were used for each experiment; data are representative of two independent experiments.
1A and B) or IAV (Fig. 1C and D) infection. Regardless of MNV exposure history, splenic and PEC responses were dominated by B8R- and A8R-specific CD8⫹ T cells following VV infection (Fig. 1A and B) and by PA-specific and NP-specific CD8⫹ T cells following IAV infection (Fig. 1C and D). To examine the effect of MNV infection on antiviral Ab
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FIG. 2. MNV exposure does not alter Ab responses to VV or IAV. MNV-infected and naïve C57BL/6 mice were infected i.n. with ⬃1 ⫻ 103 PFU of VV (A and C) or ⬃50 50% tissue culture infective dose units of IAV (B and D), and virus-specific Abs were determined by ELISA (A, C, and D) or hemagglutination inhibition (B). The ELISA results shown in panel A measured the total IgG, while the ELISA results shown in panels C and D measured the individual isotype indicated. MNV infections were completed 19 days prior to VV or IAV infections. Means and standard errors of the means are shown in panels A, C, and D. Means are shown as lines in panel B. A two-way analysis of variance and Bonferroni statistical analysis were completed for experiments shown in panels A, C, and D, and t tests were completed for the experiment shown in panel B. Four to five mice/group were used for each experiment. O.D., optical density; HAI, hemagglutination inhibition.
responses, MNV-infected and control C57BL/6 mice were infected intranasally (i.n.) with a sublethal dose of either VV or IAV. Three weeks later, levels of anti-VV and anti-IAV Abs were determined by ELISA and hemagglutination inhibition
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FIG. 3. MNV does not increase morbidity following subsequent i.n. infection with VV or IAV. MNV-infected and naïve C57BL/6 mice were infected i.n. with a sublethal dose of VV (⬃1 ⫻ 103 PFU) (A) or IAV (⬃50 50% tissue culture infective dose units) (B), and weight loss was recorded for 16 days postinfection. MNV infections were completed 19 days prior to VV or IAV infections. A two-way analysis of variance and Bonferroni statistical analysis were completed. Four to five mice/group were used for each experiment.
assays, respectively. MNV infection did not significantly modify the magnitude of Ab responses to VV (Fig. 2A) or IAV (Fig. 2B). Next, we determined the effect of MNV infection on heavy chain class switching of anti-VV or anti-IAV Ab responses. Anti-VV and anti-IAV Ab responses exhibited similar heavy chain profiles dominated by immunoglobulin G2b (IgG2b) Abs regardless of MNV status (Fig. 2C and D). Thus, the CD8⫹ T-cell and Ab response to both VV and IAV appears to be essentially unaffected by chronic MNV infection. Since IgG anti-VV or anti-IAV Ab responses are entirely dependent on CD4⫹ T-cell help (11, 12), we can also infer that MNV also does not significantly affect CD4⫹ T-cell responses to VV or IAV. T-cell and Ab responses, together with innate immune mechanisms, collaborate to control viral replication and limit pathogenesis. To examine the effect of chronic MNV infection on VV-induced or IAV-induced pathogenesis, we infected C57BL/6 mice i.n. with a lethal or sublethal dose of VV or IAV and monitored body weight over a 16-day period. MNVCR6p2 infection had no significant effect on morbidity or mortality from either virus (Fig. 3 and 4). Since MNV isolates are highly diverse, we decided to examine the effects of a second strain of MNV (MNV-CW3) which is fully cleared in immunocompetent mice. Mice that cleared MNV-CW3 (19 days post-MNV infection) were infected i.n. with VV or IAV. Once again, this strain of MNV had no effect on VV-induced or IAV-induced morbidity or mortality (Fig. 3 and 4). Future
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FIG. 4. MNV does not increase mortality following subsequent i.n. infection with VV or IAV. MNV-infected and naïve C57BL/6 mice were infected i.n. with VV (⬃1 ⫻ 104 PFU) (A) or IAV (⬃500 50% tissue culture infective dose units) (B), and survival was monitored for 16 days postinfection. MNV infections were completed 19 days prior to VV or IAV infections. Eight to 10 mice/group were used for each experiment.
studies should address the extent to which other MNV strains affect the generation of adaptive immune responses to secondary viral infections. Taken together, these data demonstrate that MNV infection has no significant effects on the measured immune response to VV or IAV. Our results cannot, however, be simply extrapolated to other viruses or microorganisms. Rather, the effect of MNV infection on host immunity in mouse model disease systems needs to be established on a system-by-system basis. Without this knowledge, the possible confounding effects of MNV infection will continue to undermine the confidence in results obtained using mice in colonies in which MNV infections are endemic. We thank the staff of the Comparative Medical Branch of the National Institute of Allergy and Infectious Diseases. This work was supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases and grants AI054483 and AI065982. REFERENCES 1. Chachu, K. A., A. D. LoBue, D. W. Strong, R. S. Baric, and H. W. Virgin. 2008. Immune mechanisms responsible for vaccination against and clearance of mucosal and lymphatic norovirus infection. PLoS Pathog. 4:e1000236. 2. Chachu, K. A., D. W. Strong, A. D. LoBue, C. E. Wobus, R. S. Baric, and H. W. Virgin. 2008. Antibody is critical for the clearance of murine norovirus infection. J. Virol. 82:6610–6617. 3. Chen, W., J. R. Bennink, P. A. Morton, and J. W. Yewdell. 2002. Mice deficient in perforin, CD4⫹ T cells, or CD28-mediated signaling maintain the typical immunodominance hierarchies of CD8⫹ T-cell responses to influenza virus. J. Virol. 76:10332–10337. 4. Estes, M. K., B. V. Prasad, and R. L. Atmar. 2006. Noroviruses everywhere: has something changed? Curr. Opin. Infect. Dis. 19:467–474.
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