Mouse Hepatitis Virus Infection Research Articles

Coronavirus envelope protein activates TMED10-mediated unconventional secretion of inflammatory factors

The precise cellular mechanisms underlying heightened proinflammatory cytokine production during coronavirus infection remain incompletely understood. Here we identify the envelope (E) protein in severe coronaviruses (SARS-CoV-2, SARS, or MERS) as a potent inducer of interleukin-1 release, intensifying lung inflammation through the activation of TMED10-mediated unconventional protein secretion (UcPS). In contrast, the E protein of mild coronaviruses (229E, HKU1, or OC43) demonstrates a less pronounced effect. The E protein of severe coronaviruses contains an SS/DS motif, which is not present in milder strains and facilitates interaction with TMED10. This interaction enhances TMED10-oligomerization, facilitating UcPS cargo translocation into the ER-Golgi intermediate compartment (ERGIC)—a pivotal step in interleukin-1 UcPS. Progesterone analogues were identified as compounds inhibiting E-enhanced release of proinflammatory factors and lung inflammation in a Mouse Hepatitis Virus (MHV) infection model. These findings elucidate a molecular mechanism driving coronavirus-induced hyperinflammation, proposing the E-TMED10 interaction as a potential therapeutic target to counteract the adverse effects of coronavirus-induced inflammation.

The KDM6A-KMT2D-p300 axis regulates susceptibility to diverse coronaviruses by mediating viral receptor expression.

Identification of host determinants of coronavirus infection informs mechanisms of pathogenesis and may provide novel therapeutic targets. Here, we demonstrate that the histone demethylase KDM6A promotes infection of diverse coronaviruses, including SARS-CoV, SARS-CoV-2, MERS-CoV and mouse hepatitis virus (MHV) in a demethylase activity-independent manner. Mechanistic studies reveal that KDM6A promotes viral entry by regulating expression of multiple coronavirus receptors, including ACE2, DPP4 and Ceacam1. Importantly, the TPR domain of KDM6A is required for recruitment of the histone methyltransferase KMT2D and histone deacetylase p300. Together this KDM6A-KMT2D-p300 complex localizes to the proximal and distal enhancers of ACE2 and regulates receptor expression. Notably, small molecule inhibition of p300 catalytic activity abrogates ACE2 and DPP4 expression and confers resistance to all major SARS-CoV-2 variants and MERS-CoV in primary human airway and intestinal epithelial cells. These data highlight the role for KDM6A-KMT2D-p300 complex activities in conferring diverse coronaviruses susceptibility and reveal a potential pan-coronavirus therapeutic target to combat current and emerging coronaviruses. One Sentence Summary: The KDM6A/KMT2D/EP300 axis promotes expression of multiple viral receptors and represents a potential drug target for diverse coronaviruses.

Strictinin, a Major Ingredient in Yunnan Kucha Tea Possessing Inhibitory Activity on the Infection of Mouse Hepatitis Virus to Mouse L Cells.

Theacrine and strictinin of Yunnan Kucha tea prepared from a mutant variety of wild Pu'er tea plants were two major ingredients responsible for the anti-influenza activity. As the COVID-19 outbreak is still lurking, developing safe and cost-effective therapeutics is an urgent need. This study aimed to evaluate the effects of these tea compounds on the infection of mouse hepatitis virus (MHV), a β-coronavirus serving as a surrogate for SARS-CoV. Treatment with strictinin (100 μM), but not theacrine, completely eliminated MHV infection, as indicated by a pronounced reduction in plaque formation, nucleocapsid protein expression, and progeny production of MHV. Subsequently, a time-of-drug addition protocol, including pre-, co-, or post-treatment, was exploited to further evaluate the possible mechanism of antiviral activity mediated by strictinin, and remdesivir, a potential drug for the treatment of SARS-CoV-2, was used as a positive control against MHV infection. The results showed that all three treatments of remdesivir (20 μM) completely blocked MHV infection. In contrast, no significant effect on MHV infection was observed when cells were pre-treated with strictinin (100 μM) prior to infection, while significant inhibition of MHV infection was observed when strictinin was introduced upon viral adsorption (co-treatment) and after viral entry (post-treatment). Of note, as compared with the co-treatment group, the inhibitory effect of strictinin was more striking in the post-treatment group. These results indicate that strictinin suppresses MHV infection by multiple mechanisms; it possibly interferes with viral entry and also critical step(s) of viral infection. Evidently, strictinin significantly inhibited MHV infection and might be a suitable ingredient for protection against coronavirus infection.

SARS-CoV-2 mitochondriopathy in COVID-19 pneumonia exacerbates hypoxemia

RationaleSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19 pneumonia. We hypothesize that SARS-CoV-2 causes alveolar injury and hypoxemia by damaging mitochondria in airway epithelial cells (AEC) and pulmonary artery smooth muscle cells (PASMC), triggering apoptosis and bioenergetic impairment, and impairing hypoxic pulmonary vasoconstriction (HPV), respectively. ObjectivesWe examined the effects of: A) human betacoronaviruses, SARS-CoV-2 and HCoV-OC43, and individual SARS-CoV-2 proteins on apoptosis, mitochondrial fission, and bioenergetics in AEC; and B) SARS-CoV-2 proteins and mouse hepatitis virus (MHV-1) infection on HPV. MethodsWe used transcriptomic data to identify temporal changes in mitochondrial-relevant gene ontology (GO) pathways post-SARS-CoV-2 infection. We also transduced AECs with SARS-CoV-2 proteins (M, Nsp7 or Nsp9) and determined effects on mitochondrial permeability transition pore (mPTP) activity, relative membrane potential, apoptosis, mitochondrial fission, and oxygen consumption rates (OCR). In human PASMC, we assessed the effects of SARS-CoV-2 proteins on hypoxic increases in cytosolic calcium, an HPV proxy. In MHV-1 pneumonia, we assessed HPV via cardiac catheterization and apoptosis using the TUNEL assay. ResultsSARS-CoV-2 regulated mitochondrial apoptosis, mitochondrial membrane permeabilization and electron transport chain (ETC) GO pathways within 2 hours of infection. SARS-CoV-2 downregulated ETC Complex I and ATP synthase genes, and upregulated apoptosis-inducing genes. SARS-CoV-2 and HCoV-OC43 upregulated and activated dynamin-related protein 1 (Drp1) and increased mitochondrial fission. SARS-CoV-2 and transduced SARS-CoV-2 proteins increased apoptosis inducing factor (AIF) expression and activated caspase 7, resulting in apoptosis. Coronaviruses also reduced OCR, decreased ETC Complex I activity and lowered ATP levels in AEC. M protein transduction also increased mPTP opening. In human PASMC, M and Nsp9 proteins inhibited HPV. In MHV-1 pneumonia, infected AEC displayed apoptosis and HPV was suppressed. BAY K8644, a calcium channel agonist, increased HPV and improved SpO2. ConclusionsCoronaviruses, including SARS-CoV-2, cause AEC apoptosis, mitochondrial fission, and bioenergetic impairment. SARS-CoV-2 also suppresses HPV by targeting mitochondria. This mitochondriopathy is replicated by transduction with SARS-CoV-2 proteins, indicating a mechanistic role for viral-host mitochondrial protein interactions. Mitochondriopathy is a conserved feature of coronaviral pneumonia that may exacerbate hypoxemia and constitutes a therapeutic target.

Neuroimmunomodulatory balance between pro and anti‐inflammatory cytokines regulate m‐CoV induced alteration of gap junction protein Cx43

Mouse hepatitis virus (MHV), a murine β‐CoV, is an experimental model used to understand the viral‐induced acute neuroinflammation and chronic progressive demyelination, which are the characteristic hallmarks of the human neurological disease Multiple Sclerosis. MHV induced neuroinflammation provides an excellent cause‐effective platform to understand how the virus can initiate neuroinflammation by causing direct neuroglial cell dystrophy, leading to chronic progressive myelin damage with or without concurrent axonal loss. Previous studies have postulated that a significant nexus between host proteins is also involved in regulating host response to viral replication. One such protein is a transmembrane protein connexin 43 (Cx43) belonging to the gap junction family. Cx43 is one of the most abundant gap junction proteins in astrocytes responsible for metabolic coupling with other CNS cells to maintain homeostasis. MHV infection in mice results in a significant decrease in the expression of Cx43 during the acute stage (day 5‐6) of MHV infection when viral titer reaches its peak and the brain presents with acute encephalitis characterized by perivascular cuffing and microglial nodule formation. Surprisingly, Cx43 returns to normal levels as infectious viral particles go below the detection limit by day 10 p.i. which marks the transition of innate immune responses to adaptive immunity. Interestingly, this reduced expression of Cx43 at Day 5 accords with increased pro‐inflammatory cytokines, oxidative stress, and activation of UPR pathway proteins. This study investigated the transcriptional regulation of TNF‐α, Il‐6, IL‐10, IL‐1β and TGF‐β in the alteration of Cx43 expression and trafficking during day 5/6 and day 10 post MHV infection. TNF‐α, IL‐10, IL‐6,IL‐1β are the major inflammatory cytokines upregulated during the acute stage of MHV infection (5‐6 p.i.). Additionally, TNF‐α and IL‐1β are known to be negative regulators of Cx43 expression and could be attributed to the decrease in Cx43 observed in our studies. Furthermore, our studies also revealed that in MHV infection, apart from the very high expression of IL‐10 another anti‐inflammatory cytokine TGF‐β expression increases when proinflammatory milieu of cytokines shifts towards the anti‐inflammatory condition in the inflamed brain. As TGF‐β is a positive regulator of Cx43, it may be responsible for the restoration of Cx43 expression and its trafficking to the cell surface resulting in functional gap junctional communication. This re‐establishment of intercellular communication maybe essential to bring back the homeostasis in the inflamed brain. The balance between pro‐inflammatory cytokines and the anti‐inflammatory mediators may regulate the host response to counteract the viral infection by altering the gap junctional communication. This study thus presents crucial evidence to support the nexus between immune inflammatory mediators and host regulatory responders in MHV induced neuroinflammation.

Activation of TCA cycle restrains virus-metabolic hijacking and viral replication in mouse hepatitis virus-infected cells

BackgroundOne of coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused coronavirus disease 2019 (COVID-19) pandemic and threatened worldwide. However, therapy for COVID-19 has rarely been proven to possess specific efficacy. As the virus relies on host metabolism for its survival, several studies have reported metabolic intervention by SARS-CoV-2.ResultsWe investigated the coronavirus-metabolic hijacking using mouse hepatitis virus (MHV) as a surrogate for SARS-CoV-2. Based on the altered host metabolism by MHV infection, an increase of glycolysis with low mitochondrial metabolism, we tried to investigate possible therapeutic molecules which increase the TCA cycle. Endogenous metabolites and metabolic regulators were introduced to restrain viral replication by metabolic intervention. We observed that cells deprived of cellular energy nutrition with low glycolysis strongly suppress viral replication. Furthermore, viral replication was also significantly suppressed by electron transport chain inhibitors which exhaust cellular energy. Apart from glycolysis and ETC, pyruvate supplement suppressed viral replication by the TCA cycle induction. As the non-glucose metabolite, fatty acids supplement decreased viral replication via the TCA cycle. Additionally, as a highly possible therapeutic metabolite, nicotinamide riboside (NR) supplement, which activates the TCA cycle by supplying NAD+, substantially suppressed viral replication.ConclusionsThis study suggests that metabolite-mediated TCA cycle activation suppresses replication of coronavirus and suggests that NR might play a role as a novel therapeutic metabolite for coronavirus.

Basic reproduction numbers of three strains of mouse hepatitis viruses in mice.

Mouse hepatitis virus (MHV) is a murine coronavirus and one of the most important pathogens in laboratory mice. Although various strains of MHV have been isolated, they are generally excreted in the feces and transmitted oronasally via aerosols and contaminated bedding. In this study, we attempted to determine the basic reproduction numbers (R0) of three strains of MHV to improve our understanding of MHV infections in mice. Five‐week‐old female C57BL/6J mice were inoculated intranasally with either the Y, NuU, or JHM variant strain of MHV and housed with two naïve mice. After 4 weeks, the presence or absence of anti‐MHV antibody in the mice was determined by ELISA. We also examined the distribution of MHV in the organs of Y, NuU, or JHM variant‐infected mice. Our data suggest that the transmissibility of MHV is correlated with viral growth in the gastrointestinal tract of infected mice. To the best of our knowledge, this is the first report to address the basic reproduction numbers among pathogens in laboratory animals.

CD40L protects against mouse hepatitis virus-induced neuroinflammatory demyelination

Neurotropic mouse hepatitis virus (MHV-A59/RSA59) infection in mice induces acute neuroinflammation due to direct neural cell dystrophy, which proceeds with demyelination with or without axonal loss, the pathological hallmarks of human neurological disease, Multiple sclerosis (MS). Recent studies in the RSA59-induced neuroinflammation model of MS showed a protective role of CNS-infiltrating CD4+ T cells compared to their pathogenic role in the autoimmune model. The current study further investigated the molecular nexus between CD4+ T cell-expressed CD40Ligand and microglia/macrophage-expressed CD40 using CD40L-/- mice. Results demonstrate CD40L expression in the CNS is modulated upon RSA59 infection. We show evidence that CD40L-/- mice are more susceptible to RSA59 induced disease due to reduced microglia/macrophage activation and significantly dampened effector CD4+ T recruitment to the CNS on day 10 p.i. Additionally, CD40L-/- mice exhibited severe demyelination mediated by phagocytic microglia/macrophages, axonal loss, and persistent poliomyelitis during chronic infection, indicating CD40-CD40L as host-protective against RSA59-induced demyelination. This suggests a novel target in designing prophylaxis for virus-induced demyelination and axonal degeneration, in contrast to immunosuppression which holds only for autoimmune mechanisms of inflammatory demyelination.

Phosphatidylserine receptors enhance SARS-CoV-2 infection.

Phosphatidylserine (PS) receptors enhance infection of many enveloped viruses through virion-associated PS binding that is termed apoptotic mimicry. Here we show that this broadly shared uptake mechanism is utilized by SARS-CoV-2 in cells that express low surface levels of ACE2. Expression of members of the TIM (TIM-1 and TIM-4) and TAM (AXL) families of PS receptors enhance SARS-CoV-2 binding to cells, facilitate internalization of fluorescently-labeled virions and increase ACE2-dependent infection of SARS-CoV-2; however, PS receptors alone did not mediate infection. We were unable to detect direct interactions of the PS receptor AXL with purified SARS-CoV-2 spike, contrary to a previous report. Instead, our studies indicate that the PS receptors interact with PS on the surface of SARS-CoV-2 virions. In support of this, we demonstrate that: 1) significant quantities of PS are located on the outer leaflet of SARS-CoV-2 virions, 2) PS liposomes, but not phosphatidylcholine liposomes, reduced entry of VSV/Spike pseudovirions and 3) an established mutant of TIM-1 which does not bind to PS is unable to facilitate entry of SARS-CoV-2. As AXL is an abundant PS receptor on a number of airway lines, we evaluated small molecule inhibitors of AXL signaling such as bemcentinib for their ability to inhibit SARS-CoV-2 infection. Bemcentinib robustly inhibited virus infection of Vero E6 cells as well as multiple human lung cell lines that expressed AXL. This inhibition correlated well with inhibitors that block endosomal acidification and cathepsin activity, consistent with AXL-mediated uptake of SARS-CoV-2 into the endosomal compartment. We extended our observations to the related betacoronavirus mouse hepatitis virus (MHV), showing that inhibition or ablation of AXL reduces MHV infection of murine cells. In total, our findings provide evidence that PS receptors facilitate infection of the pandemic coronavirus SARS-CoV-2 and suggest that inhibition of the PS receptor AXL has therapeutic potential against SARS-CoV-2.

Phase separation drives RNA virus-induced activation of the NLRP6 inflammasome

NLRP6 is important in host defense by inducing functional outcomes including inflammasome activation and interferon production. Here, we show that NLRP6 undergoes liquid-liquid phase separation (LLPS) upon interaction with double-stranded RNA (dsRNA) invitro and in cells, and an intrinsically disordered poly-lysine sequence (K350-354) of NLRP6 is important for multivalent interactions, phase separation, and inflammasome activation. Nlrp6-deficient or Nlrp6K350-354A mutant mice show reduced inflammasome activation upon mouse hepatitis virus or rotavirus infection, and in steady state stimulated by intestinal microbiota, implicating NLRP6 LLPS in anti-microbial immunity. Recruitment of ASC via helical assembly solidifies NLRP6 condensates, and ASC further recruits and activates caspase-1. Lipoteichoic acid, a known NLRP6 ligand, also promotes NLRP6 LLPS, and DHX15, a helicase in NLRP6-induced interferon signaling, co-forms condensates with NLRP6 and dsRNA. Thus, LLPS of NLRP6 is a common response to ligand stimulation, which serves to direct NLRP6 to distinct functional outcomes depending on the cellular context.

CD40-CD40L interaction is critical in mounting host immunity against m-CoV Mouse Hepatitis Virus induced neuroinflammatory demyelination

Abstract Chronic progressive neuroinflammatory disease multiple sclerosis (MS) is characterized by loss of neuronal functions resulting from demyelination with or without axonal degeneration. Infiltration of T lymphocytes and activation of microglia and their interplay are the major pathophysiological events leading to neurodegeneration in MS. Our studies in Mouse Hepatitis Virus (MHV) induced neuroinflammatory model demonstrated a protective role of CNS infiltrating CD4+ T cells. In the absence of CD4+ T cells, microglial activation fails to resolve, and mice are more susceptible to acute poliomyelitis and chronic demyelination with axonal bulbar vacuolation. Our studies also revealed that CD40L (expressed on CD4+ T cells) upregulates upon MHV infection but is downregulated in CD4−/− mice CNS. This led to a further delineation of the CD4-microglia nexus at the molecular level using CD40L−/− mice. Results showed that the absence of CD40L renders mice highly susceptible to MHV infection due to reduced microglia/macrophage activation and significantly dampened effector CD4+ T recruitment to the CNS at the acute-adaptive bridging phase (day 7–10 p.i.) of inflammation. Moreover, CD40L−/− mice exhibited severe demyelination, axonal loss, and persistent poliomyelitis at the chronic phase of infection, highlighting the protective role of CD40-CD40L in MHV induced neuroinflammatory demyelination. Together, these studies highlight that migration of peripheral T cells and their interaction with microglia via CD40-CD40L is essential to eliminate the virus and provide long-term neuroprotection. These findings can lead to designing potential therapeutic interventions against MS, targeting CD40-CD40L interaction.

Application of Tensor Decomposition to Gene Expression of Infection of Mouse Hepatitis Virus Can Identify Critical Human Genes and Efffective Drugs for SARS-CoV-2 Infection.

To better understand the genes with altered expression caused by infection with the novel coronavirus strain SARS-CoV-2 causing COVID-19 infectious disease, a tensor decomposition (TD)-based unsupervised feature extraction (FE) approach was applied to a gene expression profile dataset of the mouse liver and spleen with experimental infection of mouse hepatitis virus, which is regarded as a suitable model of human coronavirus infection. TD-based unsupervised FE selected 134 altered genes, which were enriched in protein-protein interactions with orf1ab, polyprotein, and 3C-like protease that are well known to play critical roles in coronavirus infection, suggesting that these 134 genes can represent the coronavirus infectious process. We then selected compounds targeting the expression of the 134 selected genes based on a public domain database. The identified drug compounds were mainly related to known antiviral drugs, several of which were also included in those previously screened with an in silico method to identify candidate drugs for treating COVID-19.

RT-PCR Assay for Detection of Enterotropic Strains of Mouse Hepatitis Virus in Faecal Samples

Background: Mouse Hepatitis Virus (MHV) is one of the most important and common viral infections of laboratory mice, due to its highly contagious and subclinical nature, posing threat to the research outcomes. Periodic screening of laboratory mice for MHV is mandatory. Hence, this study was intended to develop sensitive faecal based RT-PCR assays to detect active infection of enterotropic MHV in laboratory mice. Methods: Primers targeting N-gene of MHV were selected and their sensitivity was analysed in tenfold serially diluted gene template in the presence of negative mouse faecal cDNA. Thirty-six weaned mice at the age of 6 to 18 weeks were randomly selected at different periods and the blood and faecal sample were collected for serology and RT-PCR assay respectively. RT-PCR assay in colon samples was carried out for comparison. Result: PCR assay of MHV detected as low as 4 fg of plasmid DNA. Seroprevalence of MHV is very high than the prevalence by RT-PCR assay showing its retrospective nature and also seroprevalence includes both enterotropic and polytropic strain. RT-PCR results in faecal samples are analogous with that of the colon samples, showing the reliability of antemortem testing of mice for enterotropic strain of MHV.

C1qa deficiency in mice increases susceptibility to mouse hepatitis virus A59 infection.

BackgroundMouse hepatitis virus (MHV) A59 is a highly infectious pathogen and starts in the respiratory tract and progresses to systemic infection in laboratory mice. The complement system is an important part of the host immune response to viral infection. It is not clear the role of the classical complement pathway in MHV infection.ObjectivesThe purpose of this study was to determine the importance of the classical pathway in coronavirus pathogenesis by comparing C1qa KO mice and wild-type mice.MethodsWe generated a C1qa KO mouse using CRISPR/Cas9 technology and compared the susceptibility to MHV A59 infection between C1qa KO and wild-type mice. Histopathological and immunohistochemical changes, viral loads, and chemokine expressions in both mice were measured.ResultsMHV A59-infected C1qa KO mice showed severe histopathological changes, such as hepatocellular necrosis and interstitial pneumonia, compared to MHV A59-infected wild-type mice. Virus copy numbers in the olfactory bulb, liver, and lungs of C1qa KO mice were significantly higher than those of wild-type mice. The increase in viral copy numbers in C1qa KO mice was consistent with the histopathologic changes in organs. These results indicate that C1qa deficiency enhances susceptibility to MHV A59 systemic infection in mice. In addition, this enhanced susceptibility effect is associated with dramatic elevations in spleen IFN-γ, MIP-1 α, and MCP-1 in C1qa KO mice.ConclusionsThese data suggest that C1qa deficiency enhances susceptibility to MHV A59 systemic infection, and activation of the classical complement pathway may be important for protecting the host against MHV A59 infection.

Oligodendrocytes that survive acute coronavirus infection induce prolonged inflammatory responses in the CNS

Neurotropic strains of mouse hepatitis virus (MHV), a coronavirus, cause acute and chronic demyelinating encephalomyelitis with similarities to the human disease multiple sclerosis. Here, using a lineage-tracking system, we show that some cells, primarily oligodendrocytes (OLs) and oligodendrocyte precursor cells (OPCs), survive the acute MHV infection, are associated with regions of demyelination, and persist in the central nervous system (CNS) for at least 150 d. These surviving OLs express major histocompatibility complex (MHC) class I and other genes associated with an inflammatory response. Notably, the extent of inflammatory cell infiltration was variable, dependent on anatomic location within the CNS, and without obvious correlation with numbers of surviving cells. We detected more demyelination in regions with larger numbers of T cells and microglia/macrophages compared to those with fewer infiltrating cells. Conversely, in regions with less inflammation, these previously infected OLs more rapidly extended processes, consistent with normal myelinating function. Together, these results show that OLs are inducers as well as targets of the host immune response and demonstrate how a CNS infection, even after resolution, can induce prolonged inflammatory changes with CNS region-dependent impairment in remyelination.

Murine Coronavirus Infection Activates the Aryl Hydrocarbon Receptor in an Indoleamine 2,3-Dioxygenase-Independent Manner, Contributing to Cytokine Modulation and Proviral TCDD-Inducible-PARP Expression.

The aryl hydrocarbon receptor (AhR) is a cytoplasmic receptor/transcription factor that modulates several cellular and immunological processes following activation by pathogen-associated stimuli, though its role during virus infection is largely unknown. Here, we show that AhR is activated in cells infected with mouse hepatitis virus (MHV), a coronavirus (CoV), and contributes to the upregulation of downstream effector TCDD-inducible poly(ADP-ribose) polymerase (TiPARP) during infection. Knockdown of TiPARP reduced viral replication and increased interferon expression, suggesting that TiPARP functions in a proviral manner during MHV infection. We also show that MHV replication induced the expression of other genes known to be downstream of AhR in macrophages and dendritic cells and in livers of infected mice. Further, we found that chemically inhibiting or activating AhR reciprocally modulated the expression levels of cytokines induced by infection, specifically, interleukin 1β (IL-1β), IL-10, and tumor necrosis factor alpha (TNF-α), consistent with a role for AhR activation in the host response to MHV infection. Furthermore, while indoleamine 2,3-dioxygenase (IDO1) drives AhR activation in other settings, MHV infection induced equal expression of downstream genes in wild-type (WT) and IDO1-/- macrophages, suggesting an alternative pathway of AhR activation. In summary, we show that coronaviruses elicit AhR activation by an IDO1-independent pathway, contributing to upregulation of downstream effectors, including the proviral factor TiPARP, and to modulation of cytokine gene expression, and we identify a previously unappreciated role for AhR signaling in CoV pathogenesis.IMPORTANCE Coronaviruses are a family of positive-sense RNA viruses with human and agricultural significance. Characterizing the mechanisms by which coronavirus infection dictates pathogenesis or counters the host immune response would provide targets for the development of therapeutics. Here, we show that the aryl hydrocarbon receptor (AhR) is activated in cells infected with a prototypic coronavirus, mouse hepatitis virus (MHV), resulting in the expression of several effector genes. AhR is important for modulation of the host immune response to MHV and plays a role in the expression of TiPARP, which we show is required for maximal viral replication. Taken together, our findings highlight a previously unidentified role for AhR in regulating coronavirus replication and the immune response to the virus.

The autoimmune response elicited by mouse hepatitis virus (MHV-A59) infection is modulated by liver tryptophan-2,3-dioxygenase (TDO)

In a previous work we demonstrated that inhibition of mouse indoleamine 2,3-dioxygenase (IDO) by methyltryptophan (MT) exacerbated the pathological actions of mouse hepatitis virus (MHV-A59) infection, suggesting that tryptophan (TRP) catabolism was involved in viral effects. Since there is a second enzyme that dioxygenates TRP, tryptophan-2, 3-dioxygenase (TDO), which is mainly located in liver, we decided to study its role in our model of MHV-infection.Results showed that in vivo TDO inhibition by LM10, a derivative of 3-(2-(pyridyl) ethenyl) indole, resulted in a decrease of anti- MHV Ab titers induced by the virus infection. Besides, a reduction of some alarmin release, i.e, uric acid and high-mobility group box1 protein (HMGB1), was observed. Accordingly, since alarmin liberation was related to the expression of autoantibodies (autoAb) to fumarylacetoacetate hydrolase (FAH), these autoAb also diminished. Moreover, PCR results indicated that TDO inhibition did not abolish viral replication. Furthermore, histological liver examination did not reveal strong pathologies, whereas mouse survival was hundred percent in control as well as in MHV-infected mice treated with LM10.Data presented in this work indicate that in spite of the various TDO actions already described, specific TDO blockage could also restrain some MHV actions, mainly suppressing autoimmune reactions. Such results should prompt further experiments with various viruses to confirm the possible use of a TDO inhibitor such as LM-10 to treat either viral infections or even autoimmune diseases triggered by a viral infection.

Pathogenic role of CD4+ T cells in the progression of neurotropic mouse hepatitis virus induced neuroinflammation

Abstract Neurotropic Mouse hepatitis virus (MHV) induced demyelination serves as a model to understand the mechanisms of demyelination and concomitant axonal loss in CNS, which mimics the pathological symptoms of the human neurological disease Multiple Sclerosis (MS). Inflammation triggered by MHV consists of mixed population of inflammatory cells, predominantly microglia/macrophages, which differs from autoimmune model of MS, where infiltrating T cells are the major contributors to the pathology. Though the acute phase of MHV inflammation is controlled by microglia/microglia, the role of T and B cells in viral induced axonal degeneration and demyelination is controversial. In order to explore the role of CD4+ T cells during transition from an early protective innate response to a highly regulated adaptive response following MHV infection, MHV-infected CD4−/− mice were compared with MHV-infected CD4+/+ (wildtype) mice at days 5/6, 10, 15 and 30 post infection. Viral titre estimation and nucleocapsid gene amplification confirm enhanced replication of the virions in the absence of functional CD4 + T cells in brain tissue. Viral anti-nucleocapsid antigen staining further shows the persistence of virus at day 30 p.i. in CD4−/− mice unlike CD4+/+ mice. Moreover, histopathological analysis of liver, brain and spinal cord showed an elevated susceptibility of CD4−/− mice to encephalitis and axonal degeneration with augmented progression of acute poliomyelitis, bulbar vacuolation, and dorsal root ganglionic inflammation rarely observed in CD4+/+ infected mice. In summary, current study suggests that CD4 T cell is critical for viral clearance, resolving microglial activation, and controlling chronic progressive axonal degeneration.

Proliferative Typhlocolitis With Multinucleated Giant Cells: A Nonspecific Enteropathy in Immunodeficient Sentinel Mice.

Beginning in 2015, athymic nude sentinel mice from conventional, medium-, and high-security facilities presented to the Comparative Pathology Laboratory (CPL) with weight loss, diarrhea, and/or rectal prolapse. Regardless of whether clinical signs were present or absent, the gross observation of ceco-colonic thickening corresponded histologically to pleocellular typhlocolitis with mucosal hyperplasia and lamina proprial multinucleated cells. A subset of affected sentinels exhibited granulomatous serositis and hepatosplenic necrosis with multinucleated cells. Initial suspicion of mouse hepatitis virus infection was excluded by polymerase chain reaction, electron microscopy, and serology. Multinucleated giant cells were confirmed as macrophages by positive immunoreactivity to Mac-3 and Iba-1 and negative immunoreactivity to pancytokeratin. From conventional and medium-security facilities, Helicobacter species were identified in 40 of 143 (27.9%) mice, with H. hepaticus accounting for 72.5% of identified Helicobacter species. Other agents included opportunistic bacterial infection (41/145, 28.3%), murine norovirus (16/106, 15.1%), and pinworms (2/146, 1.4%). From high-security facilities, only Enterobacter cloacae was identified (2/13, 15.4%), and no evidence of Helicobacter sp., murine norovirus, or pinworms was present. No potentially infectious disease agent(s) was identified in 71 of 146 (48.6%) affected nude sentinels from conventional and medium-security facilities and 11 of 13 (84.6%) affected nude sentinels from high-security facilities. No statistically significant differences in histologic lesion scores were identified between Helicobacter-positive and Helicobacter-negative mice. Thus, proliferative typhlocolitis with multinucleated giant cells was considered a nonspecific histologic pattern associated with a variety of primary and opportunistic pathogens in athymic nude mice.

Loss of Cx43-Mediated Functional Gap Junction Communication in Meningeal Fibroblasts Following Mouse Hepatitis Virus Infection.

Mouse hepatitis virus (MHV) infection causes meningoencephalitis by disrupting the neuro-glial and glial-pial homeostasis. Recent studies suggest that MHV infection alters gap junction protein connexin 43 (Cx43)-mediated intercellular communication in brain and primary cultured astrocytes. In addition to astrocytes, meningeal fibroblasts also express high levels of Cx43. Fibroblasts in the meninges together with the basal lamina and the astrocyte endfeet forms the glial limitans superficialis as part of the blood–brain barrier (BBB). Alteration of glial-pial gap junction intercellular communication (GJIC) in MHV infection has the potential to affect the integrity of BBB. Till date, it is not known if viral infection can modulate Cx43 expression and function in cells of the brain meninges and thus affect BBB permeability. In the present study, we have investigated the effect of MHV infection on Cx43 localization and function in mouse brain meningeal cells and primary meningeal fibroblasts. Our results show that MHV infection reduces total Cx43 levels and causes its intracellular retention in the perinuclear compartments reducing its surface expression. Reduced trafficking of Cx43 to the cell surface in MHV-infected cells is associated with loss functional GJIC. Together, these data suggest that MHV infection can directly affect expression and cellular distribution of Cx43 resulting in loss of Cx43-mediated GJIC in meningeal fibroblasts, which may be associated with altered BBB function observed in acute infection.