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Timothy J Nice - One of the best experts on this subject based on the ideXlab platform.
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CD300lf Conditional Knockout Mouse Reveals Strain-Specific Cellular Tropism of Murine Norovirus.
Journal of virology, 2021Co-Authors: Vincent R. Graziano, Timothy J Nice, Megan T. Baldridge, Mia Madel Alfajaro, Cameron O. Schmitz, Renata B. Filler, Madison S. Strine, Jin Wei, Leon L. Hsieh, Sanghyun LeeAbstract:Noroviruses are a leading cause of gastrointestinal infection in humans and mice. Understanding human Norovirus (HuNoV) cell tropism has important implications for our understanding of viral pathogenesis. Murine Norovirus (MNoV) is extensively used as a surrogate model for HuNoV. We previously identified CD300lf as the receptor for MNoV. Here, we generated a Cd300lf conditional knockout (CD300lfF/F ) mouse to elucidate the cell tropism of persistent and nonpersistent strains of Murine Norovirus. Using this mouse model, we demonstrated that CD300lf expression on intestinal epithelial cells (IECs), and on tuft cells in particular, is essential for transmission of the persistent MNoV strain CR6 (MNoVCR6) in vivo In contrast, the nonpersistent MNoV strain CW3 (MNoVCW3) does not require CD300lf expression on IECs for infection. However, deletion of CD300lf in myelomonocytic cells (LysM Cre+) partially reduces CW3 viral load in lymphoid and intestinal tissues. Disruption of CD300lf expression on B cells (CD19 Cre), neutrophils (Mrp8 Cre), and dendritic cells (CD11c Cre) did not affect MNoVCW3 viral RNA levels. Finally, we show that the transcription factor STAT1, which is critical for the innate immune response, partially restricts the cell tropism of MNoVCW3 to LysM+ cells. Taken together, these data demonstrate that CD300lf expression on tuft cells is essential for MNoVCR6; that myelomonocytic cells are a major, but not exclusive, target cell of MNoVCW3; and that STAT1 signaling restricts the cellular tropism of MNoVCW3 This study provides the first genetic system for studying the cell type-specific role of CD300lf in Norovirus pathogenesis.IMPORTANCE Human Noroviruses (HuNoVs) are a leading cause of gastroenteritis resulting in up to 200,000 deaths each year. The receptor and cell tropism of HuNoV in immunocompetent humans are unclear. We use Murine Norovirus (MNoV) as a model for HuNoV. We recently identified CD300lf as the sole physiologic receptor for MNoV. Here, we leverage this finding to generate a Cd300lf conditional knockout mouse to decipher the contributions of specific cell types to MNoV infection. We demonstrate that persistent MNoVCR6 requires CD300lf expression on tuft cells. In contrast, multiple CD300lf+ cell types, dominated by myelomonocytic cells, are sufficient for nonpersistent MNoVCW3 infection. CD300lf expression on epithelial cells, B cells, neutrophils, and dendritic cells is not critical for MNoVCW3 infection. Mortality associated with the MNoVCW3 strain in Stat1-/- mice does not require CD300lf expression on LysM+ cells, highlighting that both CD300lf receptor expression and innate immunity regulate MNoV cell tropism in vivo.
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cd300lf conditional knockout mouse reveals strain specific cellular tropism for Murine Norovirus
Journal of Virology, 2020Co-Authors: Vincent R. Graziano, Timothy J Nice, Megan T. Baldridge, Mia Madel Alfajaro, Cameron O. Schmitz, Renata B. Filler, Madison S. Strine, Jin Wei, Leon L. Hsieh, Sanghyun LeeAbstract:Noroviruses are a leading cause of gastrointestinal infection in humans and mice. Understanding human Norovirus (HuNoV) cell tropism has important implications for our understanding of viral pathogenesis. Murine Norovirus (MNoV) is extensively used as a surrogate model for HuNoV. We previously identified CD300lf as the receptor for MNoV. Here, we generated a Cd300lf conditional knockout (CD300lfF/F ) mouse to elucidate the cell tropism of persistent and non-persistent strains of Murine Norovirus. Using this mouse model, we demonstrate that CD300lf expression on intestinal epithelial cells (IECs), and on tuft cells in particular, is essential for transmission of the persistent MNoV strain CR6 (MNoVCR6) in vivo In contrast, the non-persistent MNoV strain CW3 (MNoVCW3) does not require CD300lf expression on IECs for infection. However, deletion of CD300lf in myelomonocytic cells (LysM Cre+) partially reduces CW3 viral load in lymphoid and intestinal tissues. Disruption of CD300lf expression on B cells (CD19 Cre), neutrophils (Mrp8 Cre), and dendritic cells (CD11c Cre) did not affect MNoVCW3 viral RNA levels. Finally, we show that the transcription factor STAT1, which is critical for the innate immune response, partially restricts the cell tropism of MNoVCW3 to LysM+ cells. Taken together, these data demonstrate that CD300lf expression on tuft cells is essential for MNoVCR6, that myelomonocytic cells are a major, but not exclusive, target cell of MNoVCW3, and that STAT1 signaling restricts the cellular tropism of MNoVCW3 This provides the first genetic system to study the cell type-specific role of CD300lf in Norovirus pathogenesis.IMPORTANCE Human Noroviruses (HuNoVs) are a leading cause of gastroenteritis resulting in up to 200,000 deaths each year. The receptor and cell tropism of HuNoV in immunocompetent humans are unclear. We use Murine Norovirus (MNoV) as a model for HuNoV. We recently identified CD300lf as the sole physiologic receptor for MNoV. Here, we leverage this finding to generate a Cd300lf conditional knockout mouse to decipher the contributions of specific cell types to MNoV infection. We demonstrate that persistent MNoVCR6 requires CD300lf expression on tuft cells. In contrast, multiple CD300lf+ cell types, dominated by myelomonocytic cells, are sufficient for non-persistent MNoVCW3 infection. CD300lf expression on epithelial cells, B cells, neutrophils, and dendritic cells is not critical for MNoVCW3 infection. Mortality associated with MNoVCW3 strain in Stat1-/- mice does not require CD300lf expression on LysM+ cells, highlighting that both CD300lf receptor expression and innate immunity regulate MNoV cell tropism in vivo.
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CD300lf conditional knockout mouse reveals strain-specific cellular tropism for Murine Norovirus
2020Co-Authors: Vincent R. Graziano, Timothy J Nice, Megan T. Baldridge, Mia Madel Alfajaro, Cameron O. Schmitz, Renata B. Filler, Madison S. Strine, Jin Wei, Leon L. Hsieh, Sanghyun LeeAbstract:Noroviruses are a leading cause of gastrointestinal infection in humans and mice. Understanding human Norovirus (HuNoV) cell tropism has important implications for our understanding of viral pathogenesis. Murine Norovirus (MNoV) is extensively used as a surrogate model for HuNoV. We previously identified CD300lf as the receptor for MNoV. Here, we generated a Cd300lf conditional knockout (CD300lfF/F) mouse to elucidate the cell tropism of persistent and non-persistent strains of Murine Norovirus. Using this mouse model, we demonstrate that CD300lf expression on intestinal epithelial cells (IECs), and on tuft cells in particular, is essential for transmission of the persistent MNoV strain CR6 (MNoVCR6) in vivo. In contrast, the nonpersistent MNoV strain CW3 (MNoVCW3) does not require CD300lf expression on IECs for infection. However, deletion of CD300lf in myelomonocytic cells (LysM Cre+) partially reduces CW3 viral load in lymphoid and intestinal tissues. Disruption of CD300lf expression on B cells (CD19 Cre), neutrophils (Mrp8 Cre), and dendritic cells (CD11c Cre) did not affect CW3 viral RNA levels. Finally, we show that the transcription factor STAT1, which is critical for the innate immune response, partially restricts the cell tropism of MNoVCW3 to LysM+ cells. Taken together, these data demonstrate that CD300lf expression on tuft cells is essential for MNoVCR6, that myelomonocytic cells are a major, but not exclusive, target cell of MNoVCW3, and that STAT1 signaling restricts the cellular tropism of MNoVCW3. This provides the first genetic system to study the cell type-specific role of CD300lf in Norovirus pathogenesis.
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hoil1 is essential for the induction of type i and iii interferons by mda5 and regulates persistent Murine Norovirus infection
Journal of Virology, 2018Co-Authors: Jerome James Miner, Arwa Qaqish, Azad D Darbandi, Victoria L Hartley, Timothy J Nice, Stefan T. Peterson, Donna A Macduff, Megan T. Baldridge, Kazuhiro IwaiAbstract:The linear ubiquitin chain assembly complex (LUBAC), composed of heme-oxidized IRP2 ubiquitin ligase 1 (HOIL1), HOIL1-interacting protein (HOIP), and SHANK-associated RH domain-interacting protein (SHARPIN), is a crucial regulator of multiple immune signaling pathways. In humans, HOIL1 or HOIP deficiency is associated with an immune disorder involving autoinflammation, immunodeficiency, and inflammatory bowel disease (IBD)-like symptoms. During viral infection, LUBAC is reported to inhibit the induction of interferon (IFN) by the cytosolic RNA sensor retinoic acid-inducible gene I (RIG-I). Surprisingly, we found that HOIL1 is essential for the induction of both type I and type III IFNs, as well as the phosphorylation of IFN regulatory factor 3 (IRF3), during Murine Norovirus (MNoV) infection in cultured dendritic cells. The RIG-I-like receptor, melanoma differentiation-associated protein 5 (MDA5), is also required for IFN induction and IRF3 phosphorylation during MNoV infection. Furthermore, HOIL1 and MDA5 were required for IFN induction after Theiler’s Murine encephalomyelitis virus infection and poly(I·C) transfection, but not Sendai virus or vesicular stomatitis virus infection, indicating that HOIL1 and LUBAC are required selectively for MDA5 signaling. Moreover, Hoil1−/− mice exhibited defective control of acute and persistent Murine Norovirus infection and defective regulation of MNoV persistence by the microbiome as also observed previously for mice deficient in interferon lambda (IFN-λ) receptor, signal transducer and activator of transcription factor 1 (STAT1), and IRF3. These data indicate that LUBAC plays a critical role in IFN induction to control RNA viruses sensed by MDA5. IMPORTANCE Human Noroviruses are a leading cause of gastroenteritis throughout the world but are challenging to study in vivo and in vitro. Murine Norovirus (MNoV) provides a tractable genetic and small-animal model to study Norovirus biology and immune responses. Interferons are critical mediators of antiviral immunity, but excessive expression can dysregulate the immune system. IFN-λ plays an important role at mucosal surfaces, including the gastrointestinal tract, and both IFN-λ and commensal enteric bacteria are important modulators of persistent MNoV infection. LUBAC, of which HOIL1 is a component, is reported to inhibit type I IFN induction after RIG-I stimulation. We show, in contrast, that HOIL1 is critical for type I and III IFN induction during infection with MNoV, a virus that preferentially activates MDA5. Moreover, HOIL1 regulates MNoV infection in vivo. These data reveal distinct functions for LUBAC in these closely related signaling pathways and in modulation of IFN expression.
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The Role of Interferon in Persistent Viral Infection: Insights from Murine Norovirus
Trends in Microbiology, 2018Co-Authors: Timothy J Nice, Bridget A. Robinson, Jacob A. Van WinkleAbstract:Persistent viral infections result from evasion or avoidance of sterilizing immunity, extend the timeframe of virus transmission, and can trigger disease. Prior studies in mouse models of persistent infection have suggested that ineffective adaptive immune responses are necessary for persistent viral infection. However, recent work in the Murine Norovirus (MNV) model of persistent infection demonstrates that innate immunity can control both early and persistent viral replication independently of adaptive immune effector functions. Interferons (IFNs) are central to the innate control of persistent MNV, apart from a role in modulating adaptive immunity. Furthermore, subtypes of IFN play distinct tissue-specific roles in innate control of persistent MNV infection. Type I IFN (IFN-α/β) controls systemic replication, and type III IFN (IFN-λ) controls MNV persistence in the intestinal epithelium. In this article, we review recent findings in the MNV model, highlighting the role of IFNs and innate immunity in clearing persistent viral infection, and discussing the broader implications of these findings for control of persistent human infections.
Mieke Uyttendaele - One of the best experts on this subject based on the ideXlab platform.
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Inactivation of Murine Norovirus 1 and Bacteroides fragilis Phage B40-8 by Mesophilic and Thermophilic Anaerobic Digestion of Pig Slurry
Applied and Environmental Microbiology, 2010Co-Authors: Leen Baert, Bart De Gusseme, Nico Boon, Willy Verstraete, Johan Debevere, Mieke UyttendaeleAbstract:Mesophilic (37°C) and thermophilic (52°C) anaerobic digestion of pig slurry induced at least a 4-log decrease in Murine Norovirus 1, used as a surrogate virus for porcine Norovirus, after 13 and 7 days, respectively. Bacteroides fragilis phage B40-8, employed as a universal viral model, was lowered by 2.5 log after 7 days. The viral titer declined due to temperature and matrix effects.
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Inactivation of Murine Norovirus 1 and Bacteroides fragilis Phage B40-8 by Mesophilic and Thermophilic Anaerobic Digestion of Pig Slurry
Applied and environmental microbiology, 2010Co-Authors: Leen Baert, Bart De Gusseme, Nico Boon, Willy Verstraete, Johan Debevere, Mieke UyttendaeleAbstract:Mesophilic (37 degrees C) and thermophilic (52 degrees C) anaerobic digestion of pig slurry induced at least a 4-log decrease in Murine Norovirus 1, used as a surrogate virus for porcine Norovirus, after 13 and 7 days, respectively. Bacteroides fragilis phage B40-8, employed as a universal viral model, was lowered by 2.5 log after 7 days. The viral titer declined due to temperature and matrix effects.
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detection of Murine Norovirus 1 by using plaque assay transfection assay and real time reverse transcription pcr before and after heat exposure
Applied and Environmental Microbiology, 2008Co-Authors: Leen Baert, Christiane E. Wobus, Larissa B. Thackray, Johan Debevere, Els Van Coillie, Mieke UyttendaeleAbstract:The correlation between the detection of Murine Norovirus 1 RNA by real-time reverse transcription-PCR and the infectivity by plaque assay before and after heat exposure (80°C) was examined. No correlation was found in the current study. Moreover, heat inactivation had a much stronger detrimental effect on virus infectivity than on the integrity of the viral genome.
Christiane E. Wobus - One of the best experts on this subject based on the ideXlab platform.
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Newly isolated mAbs broaden the neutralizing epitope in Murine Norovirus
Journal of General Virology, 2014Co-Authors: Abimbola O Kolawole, Christine M Rippinger, Chunsheng Xia, Monica Gamez, Ryan E. Yucha, Thomas J. Smith, Christiane E. WobusAbstract:Here, we report the isolation and functional characterization of mAbs against two Murine Norovirus (MNV) strains, MNV-1 and WU20, which were isolated following oral infection of mice. The mAbs were screened for reactivity against the respective homologous and heterologous MNV strain by ELISA. Selected mAbs were of IgA, IgG1, IgG2a or IgG2b isotype and showed a range of Western blot reactivities from non-binding to strong binding, suggesting recognition of conformational and linear epitopes. Some of the anti-MNV-1 antibodies neutralized both MNV-1 and WU20 infections in culture and in mice, but none of the anti-WU20 mAbs neutralized either virus. The non-neutralizing anti-MNV-1 IgG2b antibody 5C4.10 was mapped to the S domain of the MNV-1 capsid, whilst the epitopes of the neutralizing anti-MNV-1 IgA antibodies 2D3.7 and 4F9.4 were mapped to the P domain. Generation of neutralization escape viruses showed that two mutations (V339I and D348E) in the C′D′ loop of the MNV-1 P domain mediated escape from mAb 2D3.7 and 4F9.4 neutralization. These findings broaden the known neutralizing epitopes of MNV to the main surface-exposed loops of the P domain. In addition, the current panel of antibodies provides valuable reagents for studying Norovirus biology and development of diagnostic tools.
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multiple effects of dendritic cell depletion on Murine Norovirus infection
Journal of General Virology, 2013Co-Authors: Mariam B Gonzalezhernandez, Michael D Elftman, Cheryl Perkins, Kenneth S Henderson, Nobuhiko Kamada, Gabriel Nunez, Christiane E. WobusAbstract:Dendritic cells (DCs) are permissive to Murine Norovirus (MNV) infection in vitro and in vivo. However, their roles during infection in vivo are not well defined. To determine the role of DCs during infection, conventional DCs were depleted from CD11c-DTR mice and infected with a persistent MNV strain. Viral titres in the intestine and secondary lymphoid organs were determined at early time points during infection, and anti-MNV antibody responses were analysed later during infection. Depletion of conventional DCs resulted in increased viral loads in intestinal tissues, impaired generation of antibody responses, and a failure of MNV to efficiently infect lymphoid tissues. These data suggest that DCs play multiple roles in MNV pathogenesis, in both innate immunity and the efficient generation of adaptive immune responses against MNV, as well as by promoting the dissemination of MNV to secondary lymphoid tissues. This is the first study to probe the roles of DCs in controlling and/or facilitating a Norovirus infection in vivo and provides the basis for further studies aimed at defining mechanisms by which DCs control MNV replication and promote viral dissemination.
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Murine Norovirus-1 entry into permissive macrophages and dendritic cells is pH-independent.
Virus Research, 2009Co-Authors: Jeffrey W. Perry, Stefan Taube, Christiane E. WobusAbstract:Murine Norovirus (MNV) is a recently discovered mouse pathogen. Unlike the fastidious human Noroviruses that cause the overwhelming majority of non-bacterial gastroenteritis worldwide, MNV readily infects cells in culture. Its replication in primary Murine macrophages and dendritic cells and their derived cell lines allows the study of Norovirus cell entry for the first time. In this study we determined the role of pH during MNV-1 infection since the low pH environment of endosomes often triggers uncoating of viruses. We demonstrated that MNV-1 viral titers by plaque assay and expression of the non-structural protein VPg by immunofluorescence were not affected by pH in cultured and primary macrophages and dendritic cells in the presence of two known endosome acidification inhibitors, bafilomycin A1 and chloroquine. These data indicate that MNV-1 enters permissive cells in a pH-independent manner.
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antibody is critical for the clearance of Murine Norovirus infection
Journal of Virology, 2008Co-Authors: Karen A Chachu, David W Strong, Anna D Lobue, Ralph S Baric, Christiane E. Wobus, Herbert W VirginAbstract:Human Noroviruses cause more than 90% of epidemic nonbacterial gastroenteritis. However, the role of B cells and antibody in the immune response to Noroviruses is unclear. Previous studies have demonstrated that human Norovirus specific antibody levels increase upon infection, but they may not be protective against infection. In this report, we used Murine Norovirus (MNV), an enteric Norovirus, as a model to determine the importance of Norovirus specific B cells and immune antibody in clearance of Norovirus infection. We show here that mice genetically deficient in B cells failed to clear primary MNV infection as effectively as wild-type mice. In addition, adoptively transferred immune splenocytes derived from B-cell-deficient mice or antibody production-deficient mice were unable to efficiently clear persistent MNV infection in RAG1 / mice. Further, adoptive transfer of either polyclonal anti-MNV serum or neutralizing anti-MNV monoclonal antibodies was sufficient to reduce the level of MNV infection both systemically and in the intestine. Together, these data demonstrate that antibody plays an important role in the clearance of MNV and that immunoglobulin G anti-Norovirus antibody can play an important role in clearing mucosal infection.
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detection of Murine Norovirus 1 by using plaque assay transfection assay and real time reverse transcription pcr before and after heat exposure
Applied and Environmental Microbiology, 2008Co-Authors: Leen Baert, Christiane E. Wobus, Larissa B. Thackray, Johan Debevere, Els Van Coillie, Mieke UyttendaeleAbstract:The correlation between the detection of Murine Norovirus 1 RNA by real-time reverse transcription-PCR and the infectivity by plaque assay before and after heat exposure (80°C) was examined. No correlation was found in the current study. Moreover, heat inactivation had a much stronger detrimental effect on virus infectivity than on the integrity of the viral genome.
Mi Young Lim - One of the best experts on this subject based on the ideXlab platform.
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Effect of Temperature, pH, and NaCl on the Inactivation Kinetics of Murine Norovirus
Journal of Food Protection, 2012Co-Authors: Kyeongjin Seo, Jung Eun Lee, Mi Young LimAbstract:We investigated the resistance of Murine Norovirus (MNV) and coliphage MS2, a culturable human Norovirus surrogate, to temperature, salt, and pH. Virus inactivation was measured by plaque, real-tim...
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disinfection kinetics of Murine Norovirus using chlorine and chlorine dioxide
Water Research, 2010Co-Authors: Mi Young Lim, Ju-mi KimAbstract:We determined the disinfection efficiency of chlorine and chlorine dioxide (ClO(2)) using Murine Norovirus (MNV) and coliphage MS2 as surrogates for human Norovirus. Experiments were performed in oxidant demand-free buffer (pH 7.2) at 5 degrees C and 20 degrees C. The extent of virus inactivation by a disinfectant was quantified using three different analytical methods: plaque, short template real-time TaqMan reverse transcriptase-polymerase chain reaction (RT-PCR), and long template RT-PCR assays. Rapid inactivation of MNV by both chlorine and chlorine dioxide was observed by the plaque assay. According to the efficiency factor Hom model, Ct values of 0.314mg/Lmin and 0.247mg/Lmin were required for a 4-log reduction of MNV at 5 degrees C by chlorine and chlorine dioxide, respectively. Lower Ct values were required at 20 degrees C. Both long template and short template RT-PCR assays significantly underestimated the virus inactivation compared to the plaque assay. Our study demonstrates that adequate treatment of water with either chlorine or ClO(2) is likely to effectively control the waterborne transmission of human Norovirus.
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Characterization of ozone disinfection of Murine Norovirus.
Applied and Environmental Microbiology, 2009Co-Authors: Mi Young Lim, Ju-mi Kim, Jung Eun LeeAbstract:Despite the importance of human Noroviruses (NoVs) in public health, little information concerning the effectiveness of ozone against NoVs is available. We determined the efficacy of ozone disinfection using Murine Norovirus (MNV) as a surrogate of human NoV. MNV in ozone demand-free buffer was exposed to a predetermined dose of ozone at two different pHs and temperatures. The virus remaining in the solution was analyzed by plaque assay, real-time TaqMan reverse transcriptase PCR (RT-PCR) (short template), and long-template conventional RT-PCR. Under all conditions, more than 99% of the MNV was inactivated by ozone at 1 mg/liter within 2 min. Both RT-PCR assays significantly underestimated the inactivation of MNV, compared with that measured by plaque assay. Our results indicate that NoV may be more resistant to ozone than has been previously reported. Nevertheless, proper ozone disinfection practices can be used to easily control its transmission in water.
Sanghyun Lee - One of the best experts on this subject based on the ideXlab platform.
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CD300lf Conditional Knockout Mouse Reveals Strain-Specific Cellular Tropism of Murine Norovirus.
Journal of virology, 2021Co-Authors: Vincent R. Graziano, Timothy J Nice, Megan T. Baldridge, Mia Madel Alfajaro, Cameron O. Schmitz, Renata B. Filler, Madison S. Strine, Jin Wei, Leon L. Hsieh, Sanghyun LeeAbstract:Noroviruses are a leading cause of gastrointestinal infection in humans and mice. Understanding human Norovirus (HuNoV) cell tropism has important implications for our understanding of viral pathogenesis. Murine Norovirus (MNoV) is extensively used as a surrogate model for HuNoV. We previously identified CD300lf as the receptor for MNoV. Here, we generated a Cd300lf conditional knockout (CD300lfF/F ) mouse to elucidate the cell tropism of persistent and nonpersistent strains of Murine Norovirus. Using this mouse model, we demonstrated that CD300lf expression on intestinal epithelial cells (IECs), and on tuft cells in particular, is essential for transmission of the persistent MNoV strain CR6 (MNoVCR6) in vivo In contrast, the nonpersistent MNoV strain CW3 (MNoVCW3) does not require CD300lf expression on IECs for infection. However, deletion of CD300lf in myelomonocytic cells (LysM Cre+) partially reduces CW3 viral load in lymphoid and intestinal tissues. Disruption of CD300lf expression on B cells (CD19 Cre), neutrophils (Mrp8 Cre), and dendritic cells (CD11c Cre) did not affect MNoVCW3 viral RNA levels. Finally, we show that the transcription factor STAT1, which is critical for the innate immune response, partially restricts the cell tropism of MNoVCW3 to LysM+ cells. Taken together, these data demonstrate that CD300lf expression on tuft cells is essential for MNoVCR6; that myelomonocytic cells are a major, but not exclusive, target cell of MNoVCW3; and that STAT1 signaling restricts the cellular tropism of MNoVCW3 This study provides the first genetic system for studying the cell type-specific role of CD300lf in Norovirus pathogenesis.IMPORTANCE Human Noroviruses (HuNoVs) are a leading cause of gastroenteritis resulting in up to 200,000 deaths each year. The receptor and cell tropism of HuNoV in immunocompetent humans are unclear. We use Murine Norovirus (MNoV) as a model for HuNoV. We recently identified CD300lf as the sole physiologic receptor for MNoV. Here, we leverage this finding to generate a Cd300lf conditional knockout mouse to decipher the contributions of specific cell types to MNoV infection. We demonstrate that persistent MNoVCR6 requires CD300lf expression on tuft cells. In contrast, multiple CD300lf+ cell types, dominated by myelomonocytic cells, are sufficient for nonpersistent MNoVCW3 infection. CD300lf expression on epithelial cells, B cells, neutrophils, and dendritic cells is not critical for MNoVCW3 infection. Mortality associated with the MNoVCW3 strain in Stat1-/- mice does not require CD300lf expression on LysM+ cells, highlighting that both CD300lf receptor expression and innate immunity regulate MNoV cell tropism in vivo.
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cd300lf conditional knockout mouse reveals strain specific cellular tropism for Murine Norovirus
Journal of Virology, 2020Co-Authors: Vincent R. Graziano, Timothy J Nice, Megan T. Baldridge, Mia Madel Alfajaro, Cameron O. Schmitz, Renata B. Filler, Madison S. Strine, Jin Wei, Leon L. Hsieh, Sanghyun LeeAbstract:Noroviruses are a leading cause of gastrointestinal infection in humans and mice. Understanding human Norovirus (HuNoV) cell tropism has important implications for our understanding of viral pathogenesis. Murine Norovirus (MNoV) is extensively used as a surrogate model for HuNoV. We previously identified CD300lf as the receptor for MNoV. Here, we generated a Cd300lf conditional knockout (CD300lfF/F ) mouse to elucidate the cell tropism of persistent and non-persistent strains of Murine Norovirus. Using this mouse model, we demonstrate that CD300lf expression on intestinal epithelial cells (IECs), and on tuft cells in particular, is essential for transmission of the persistent MNoV strain CR6 (MNoVCR6) in vivo In contrast, the non-persistent MNoV strain CW3 (MNoVCW3) does not require CD300lf expression on IECs for infection. However, deletion of CD300lf in myelomonocytic cells (LysM Cre+) partially reduces CW3 viral load in lymphoid and intestinal tissues. Disruption of CD300lf expression on B cells (CD19 Cre), neutrophils (Mrp8 Cre), and dendritic cells (CD11c Cre) did not affect MNoVCW3 viral RNA levels. Finally, we show that the transcription factor STAT1, which is critical for the innate immune response, partially restricts the cell tropism of MNoVCW3 to LysM+ cells. Taken together, these data demonstrate that CD300lf expression on tuft cells is essential for MNoVCR6, that myelomonocytic cells are a major, but not exclusive, target cell of MNoVCW3, and that STAT1 signaling restricts the cellular tropism of MNoVCW3 This provides the first genetic system to study the cell type-specific role of CD300lf in Norovirus pathogenesis.IMPORTANCE Human Noroviruses (HuNoVs) are a leading cause of gastroenteritis resulting in up to 200,000 deaths each year. The receptor and cell tropism of HuNoV in immunocompetent humans are unclear. We use Murine Norovirus (MNoV) as a model for HuNoV. We recently identified CD300lf as the sole physiologic receptor for MNoV. Here, we leverage this finding to generate a Cd300lf conditional knockout mouse to decipher the contributions of specific cell types to MNoV infection. We demonstrate that persistent MNoVCR6 requires CD300lf expression on tuft cells. In contrast, multiple CD300lf+ cell types, dominated by myelomonocytic cells, are sufficient for non-persistent MNoVCW3 infection. CD300lf expression on epithelial cells, B cells, neutrophils, and dendritic cells is not critical for MNoVCW3 infection. Mortality associated with MNoVCW3 strain in Stat1-/- mice does not require CD300lf expression on LysM+ cells, highlighting that both CD300lf receptor expression and innate immunity regulate MNoV cell tropism in vivo.
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CD300lf conditional knockout mouse reveals strain-specific cellular tropism for Murine Norovirus
2020Co-Authors: Vincent R. Graziano, Timothy J Nice, Megan T. Baldridge, Mia Madel Alfajaro, Cameron O. Schmitz, Renata B. Filler, Madison S. Strine, Jin Wei, Leon L. Hsieh, Sanghyun LeeAbstract:Noroviruses are a leading cause of gastrointestinal infection in humans and mice. Understanding human Norovirus (HuNoV) cell tropism has important implications for our understanding of viral pathogenesis. Murine Norovirus (MNoV) is extensively used as a surrogate model for HuNoV. We previously identified CD300lf as the receptor for MNoV. Here, we generated a Cd300lf conditional knockout (CD300lfF/F) mouse to elucidate the cell tropism of persistent and non-persistent strains of Murine Norovirus. Using this mouse model, we demonstrate that CD300lf expression on intestinal epithelial cells (IECs), and on tuft cells in particular, is essential for transmission of the persistent MNoV strain CR6 (MNoVCR6) in vivo. In contrast, the nonpersistent MNoV strain CW3 (MNoVCW3) does not require CD300lf expression on IECs for infection. However, deletion of CD300lf in myelomonocytic cells (LysM Cre+) partially reduces CW3 viral load in lymphoid and intestinal tissues. Disruption of CD300lf expression on B cells (CD19 Cre), neutrophils (Mrp8 Cre), and dendritic cells (CD11c Cre) did not affect CW3 viral RNA levels. Finally, we show that the transcription factor STAT1, which is critical for the innate immune response, partially restricts the cell tropism of MNoVCW3 to LysM+ cells. Taken together, these data demonstrate that CD300lf expression on tuft cells is essential for MNoVCR6, that myelomonocytic cells are a major, but not exclusive, target cell of MNoVCW3, and that STAT1 signaling restricts the cellular tropism of MNoVCW3. This provides the first genetic system to study the cell type-specific role of CD300lf in Norovirus pathogenesis.
