Mouse Hepatitis Virus Research Articles

Disrupted CXCR2 Signaling in Oligodendroglia Lineage Cells Enhances Myelin Repair in a Viral Model of Multiple Sclerosis.

CXCR2 is a chemokine receptor expressed on oligodendroglia that has been implicated in the pathogenesis of neuroinflammatory demyelinating diseases as well as enhancement of the migration, proliferation, and myelin production by oligodendroglia. Using an inducible proteolipid protein (Plp) promoter-driven Cre-loxP recombination system, we were able to assess how timed ablation of Cxcr2 in oligodendroglia affected disease following intracranial infection with the neurotropic JHM strain of mouse hepatitis virus (JHMV). Generation of Plp-Cre-ER(T)::Cxcr2flox/flox transgenic mice (termed Cxcr2-CKO mice) allows for Cxcr2 to be silenced in oligodendrocytes in adult mice following treatment with tamoxifen. Ablation of oligodendroglia Cxcr2 did not influence clinical severity in response to intracranial infection with JHMV. Infiltration of activated T cells or myeloid cells into the central nervous system (CNS) was not affected, nor was the ability to control viral infection. In addition, the severity of demyelination was similar between tamoxifen-treated mice and vehicle-treated controls. Notably, deletion of Cxcr2 resulted in increased remyelination, as assessed by g-ratio (the ratio of the inner axonal diameter to the total outer fiber diameter) calculation, compared to that in vehicle-treated control mice. Collectively, our findings argue that CXCR2 signaling in oligodendroglia is dispensable with regard to contributing to neuroinflammation, but its deletion enhances remyelination in a preclinical model of the human demyelinating disease multiple sclerosis (MS).IMPORTANCE Signaling through the chemokine receptor CXCR2 in oligodendroglia is important for developmental myelination in rodents, while chemical inhibition or nonspecific genetic deletion of CXCR2 appears to augment myelin repair in animal models of the human demyelinating disease multiple sclerosis (MS). To better understand the biology of CXCR2 signaling on oligodendroglia, we generated transgenic mice in which Cxcr2 is selectively ablated in oligodendroglia upon treatment with tamoxifen. Using a viral model of neuroinflammation and demyelination, we demonstrate that genetic silencing of CXCR2 on oligodendroglia did not affect clinical disease, neuroinflammation, or demyelination, yet there was increased remyelination. These findings support and extend previous findings suggesting that targeting CXCR2 may offer a therapeutic avenue for enhancing remyelination in patients with demyelinating diseases.

The Curious Case of the Nidovirus Exoribonuclease: Its Role in RNA Synthesis and Replication Fidelity.

Among RNA viruses, the order Nidovirales stands out for including viruses with the largest RNA genomes currently known. Nidoviruses employ a complex RNA-synthesizing machinery comprising a variety of non-structural proteins (nsps). One of the postulated drivers of the expansion of nidovirus genomes is the presence of a proofreading 3′-to-5′ exoribonuclease (ExoN) belonging to the DEDDh family. ExoN may enhance the fidelity of RNA synthesis by correcting nucleotide incorporation errors made by the RNA-dependent RNA polymerase. Here, we review our current understanding of ExoN evolution, structure, and function. Most experimental data are derived from studies of the ExoN domain of coronaviruses (CoVs), which were triggered by the bioinformatics-based identification of ExoN in the genome of severe acute respiratory syndrome coronavirus (SARS-CoV) and its relatives in 2003. Although convincing data supporting the proofreading hypothesis have been obtained, from biochemical assays and studies with CoV mutants lacking ExoN functionality, the features of ExoN from most other nidovirus families remain to be characterized. Remarkably, viable ExoN knockout mutants were obtained only for two CoVs, mouse hepatitis virus (MHV) and SARS-CoV, whose RNA synthesis and replication kinetics were mildly affected by the lack of ExoN function. In several other CoV species, ExoN inactivation was not tolerated, and knockout mutants could not be rescued when launched using a reverse genetics system. This suggests that ExoN is also critical for primary viral RNA synthesis, a property that poorly matches the profile of an enzyme that would merely boost long-term replication fidelity. In CoVs, ExoN resides in a bifunctional replicase subunit (nsp14) whose C-terminal part has (N7-guanine)-methyltransferase activity. The crystal structure of SARS-CoV nsp14 has shed light on the interplay between these two domains, and on nsp14’s interactions with nsp10, a co-factor that strongly enhances ExoN activity in vitro assays. Further elucidation of the structure-function relationships of ExoN and its interactions with other (viral and/or host) members of the CoV replication machinery will be key to understanding the enzyme’s role in viral RNA synthesis and pathogenesis, and may contribute to the design of new approaches to combat emerging nidoviruses.

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.

A proline insertion-deletion in the spike glycoprotein fusion peptide of mouse hepatitis virus strongly alters neuropathology

Fusion peptides (FPs) in spike proteins are key players mediating early events in cell-to-cell fusion, vital for intercellular viral spread. A proline residue located at the central FP region has often been suggested to have a distinctive role in this fusion event. The spike glycoprotein from strain RSA59 (PP) of mouse hepatitis virus (MHV) contains two central, consecutive prolines in the FP. Here, we report that deletion of one of these proline residues, resulting in RSA59 (P), significantly affected neural cell syncytia formation and viral titers postinfection in vitro Transcranial inoculation of C57Bl/6 mice with RSA59 (PP) or RSA59 (P) yielded similar degrees of necrotizing hepatitis and meningitis, but only RSA59 (PP) produced widespread encephalitis that extended deeply into the brain parenchyma. By day 6 postinfection, both virus variants were mostly cleared from the brain. Interestingly, inoculation with the RSA59 (P)-carrying MHV significantly reduced demyelination at the chronic stage. We also found that the presence of two consecutive prolines in FP promotes a more ordered, compact, and rigid structure in the spike protein. These effects on FP structure were due to proline's unique stereochemical properties intrinsic to its secondary amino acid structure, revealed by molecular dynamics and NMR experiments. We therefore propose that the differences in the severity of encephalitis and demyelination between RSA59 (PP) and RSA59 (P) arise from the presence or absence, respectively, of the two consecutive prolines in FP. Our studies define a structural determinant of MHV entry in the brain parenchyma important for altered neuropathogenesis.

CD4+ T cells regulate Mouse Hepatitis Virus induced activation of microglia

Abstract Multiple Sclerosis (MS) is a human neurological disease characterised by neuroinflammatory demyelination and axonal loss. Studies in MS are focussed to understand the process of demyelination driven by B/T lymphocytes, but we have limited knowledge about direct virus-induced neural cell dystrophy and the role of innate immunity in MS. Our studies employ Mouse Hepatitis Virus (MHV) induced neuroinflammatory model to understand microglia-mediated myelin stripping as a mechanism of demyelination. Microglia are the CNS resident macrophages that lie at the intersection of immune response and neurodegenerative process. They control the initiation, progression and resolution of neuroinflammation in the CNS. One of our studies in MHV model demonstrated that in the absence of CD4+ T cells, mice are more susceptible to acute encephalitis and chronic demyelination. In order to understand the mechanism of anti-viral immune response, we have investigated the role of microglia upon CD4+ T cell modulation. Results from our studies showed that a higher number of Iba1+ microglial cells persist in the brain parenchyma of CD4−/− mice at the chronic infection stage in comparison to the WT mice. Real-time qPCR screening of inflammatory markers like IL6, IL10, IL12, CXCL10, CCL5, CD86 and CD163 presented a consistent proinflammatory microenvironment in the CNS and comparative lower expression of Btk1 and Arg1 in the brains of CD4−/− mice. In conclusion, our findings propose that further in vitro and in vivo studies are necessary to understand the role of CD4+ T cell activation and maturation which might help in favouring a shift towards adaptive immunity, differential microglial activation, viral clearance and maintaining neural homeostasis.

PGD2/DP1 signaling deficiency ameliorates experimental autoimmune encephalitis

Abstract D-prostanoid receptor 1 (DP1) is a myeloid cell-expressing receptor of prostaglandin D2 (PGD2), the most abundant prostaglandin in the brain. PGD2/DP1 signaling, which triggers G protein activation and cAMP production, is associated with neuroprotection after ischemia, astrogliosis and demyelination in mice. In our previous study, PGD2/DP1 signaling was found to suppress mouse hepatitis virus (MHV)-induced central nervous system (CNS) inflammasome activation through up-regulating PYDC3 expression in CD11b+ cells. In this study, we now investigated the role of PGD2/DP1 signaling in active experimental autoimmune encephalitis (EAE). In contrast to their enhanced susceptibility to virus-induced CNS inflammation, DP-1−/− mice showed resistance to MOG35–55 peptide-induced EAE, which was characterized by milder demyelination and improved remyelination compared to wild type mice. The amelioration of CNS injury resulted from less infiltration of leukocytes and of activation of microglia and macrophages in the brain and spinal cord. On the other hand, in draining lymph nodes (DLN) of immunized DP-1−/− mice, myeloid cells (macrophages, neutrophils and dendritic cells (DCs)) exhibited highly pro-inflammatory phenotypes, which correlated with activation-induced apoptosis of MOG35–55 peptide-specific T cells. Finally, conditional deletion of DP-1−/− in either microglial (CX3CR1+ cells) or macrophage/monocyte/granulocyte (Lyz2tm1+ cells) failed to ameliorate EAE, suggesting involvement of a different DP1-expressing cell. Ongoing research is directed at determining this critical cell type.

Regulatory role of Ifit2 in mouse hepatitis virus induced neuroinflammation

Abstract Interferons (IFNs) are known to protect from virus dissemination and pathogenesis by means of IFN stimulated genes (ISG) but the mechanisms underlying the antiviral effects are not clearly understood. IFN induced tetratricopeptide repeats (Ifit) genes are a class of ISGs represented in mice by Ifit1, Ifit2 and Ifit3. Intracranial infection of mice with MHV significantly increases mRNA levels of all 3 Ifit genes in the CNS. Moreover, Ifit2 is beneficial in limiting encephalitis caused by neurotropic viruses like VSV, WNV, EMCV, and MHV. To provide better cellular insights into the protective mechanisms of Ifit2, we studied viral spread and inflammation in young Ifit2−/− mice (4–5 weeks) infected with MHV-A59. Compared to wildtype (wt) viral spread was rapid and uncontrolled in Ifit2−/− mice and mortality was reduced to 70% by day 6–8 p.i. at 2000 PFU. Also, the pattern of virus distribution in spinal cords was significantly different. Where virus was restricted prominently to the gray-white matter border in wt mice, viral antigen expanded to both white and gray matter in Ifit2−/− mice, but they are less inflamed and less encephalitic. Surprisingly, despite vastly elevated virus load, microglia/macrophage activation was limited compared to wt mice as examined by anti-Iba-1 staining. Moreover, leukocyte recruitment was reduced despite similar induction of inflammatory cytokines. By contrast, type I IFN induction was significantly reduced. At lower viral doses (500 PFU), Ifit2−/− mice developed severe clinical symptoms and myelin damage at the chronic infection stage. Given the homeostatic interactions between microglia and neurons, we suggest that Ifit2 deficiency may disrupt microglial activation and protective functions to neurons.

Kctd9 Deficiency Impairs Natural Killer Cell Development and Effector Function.

We previously showed that potassium channel tetramerization domain containing 9 (KCTD9) is aberrantly expressed in natural killer (NK) cells in patients with hepatitis B virus-associated acute-on-chronic liver failure and mice with experimental fulminant hepatitis. However, the mechanism underlying the regulation of NK cell function and fulminant hepatitis progression by KCTD9 is unknown. Here, we investigated the role of Kctd9 in regulation of early development, maturation, and function of NK cells using Kctd9-knockout mice. Compared to wild-type mice, Kctd9-deficient mice exhibited impaired NK cell lineage commitment, as evidenced by selective reduction in the refined NK progenitors, and incomplete NK cell maturation, as manifested by a higher proportion of CD11b− NK cells and a lower percentage of CD11b+ NK cells with high proliferative potential. Moreover, Kctd9-depleted NK cells displayed insufficient IFN-γ production, degranulation, and granzyme B production in response to cytokine stimulation, and attenuated cytotoxicity to tumor cells in vitro. The defect in NK cells was further supported by ameliorated liver damage and improved survival in Kctd9-deficient mice following murine hepatitis virus strain-3 (MHV-3) infection, which otherwise leads to immune-mediated fulminant hepatitis, a phenotype homologous to that caused by NK cell depletion in wild-type mice. Further investigation to identify the underlying mechanism revealed that Kctd9 deficiency hindered the expression of transcription factors, including Ets1, Nfil3, Eomes, and Id2 in NK cells. Collectively, our data reveal that Kctd9 acts as a novel regulator for NK cell commitment, maturation, and effector function.

Antagonism of dsRNA-Induced Innate Immune Pathways by NS4a and NS4b Accessory Proteins during MERS Coronavirus Infection.

Middle East respiratory syndrome coronavirus (MERS-CoV) was first identified in 2012 as a novel etiological agent of severe respiratory disease in humans. As during infection by other viruses, host sensing of viral double-stranded RNA (dsRNA) induces several antiviral pathways. These include interferon (IFN), oligoadenylate synthetase (OAS)-RNase L, and protein kinase R (PKR). Coronaviruses, including MERS-CoV, potently suppress the activation of these pathways, inducing only modest host responses. Our study describes the functions of two accessory proteins unique to MERS-CoV and related viruses, NS4a and NS4b, during infection in human airway epithelium-derived A549 cells. NS4a has been previously characterized as a dsRNA binding protein, while NS4b is a 2',5'-phosphodiesterase with structural and enzymatic similarity to NS2 encoded by mouse hepatitis virus (MHV). We found that deletion of NS4a results in increased interferon lambda (IFNL1) expression, as does mutation of either the catalytic site or nuclear localization sequence of NS4b. All of the mutant viruses we tested exhibited slight decreases in replication. We previously reported that, like MHV NS2, NS4b antagonizes OAS-RNase L, but suppression of IFN is a previously unidentified function for viral phosphodiesterases. Unexpectedly, deletion of NS4a does not result in robust activation of the PKR or OAS-RNase L pathways. Therefore, MERS-CoV likely encodes other proteins that contribute to suppression or evasion of these antiviral innate immune pathways that should be an important focus of future work. This study provides additional insight into the complex interactions between MERS-CoV and the host immune response.IMPORTANCE Middle East respiratory syndrome coronavirus (MERS-CoV) is the second novel zoonotic coronavirus to emerge in the 21st century and cause outbreaks of severe respiratory disease. More than 2,200 cases and 800 deaths have been reported to date, yet there are no licensed vaccines or treatments. Coronaviruses encode unique accessory proteins that are not required for replication but most likely play roles in immune antagonism and/or pathogenesis. Our study describes the functions of MERS-CoV accessory proteins NS4a and NS4b during infection of a human airway-derived cell line. Loss of these accessory proteins during MERS-CoV infection leads to host antiviral activation and modestly attenuates replication. In the case of both NS4a and NS4b, we have identified roles during infection not previously described, yet the lack of robust activation suggests much remains to be learned about the interactions between MERS-CoV and the infected host.

The crystal structure of main protease from mouse hepatitis virus A59 in complex with an inhibitor

Mouse hepatitis virus A59 (MHV-A59) is a representative member of the genus betacoronavirus within the subfamily Coronavirinae, which infects the liver, brain and respiratory tract. Through different inoculation routes, MHV-A59 can provide animal models for encephalitis, hepatitis and pneumonia to explore viral life machinery and virus-host interactions. In viral replication, non-structural protein 5 (Nsp5), also termed main protease (Mpro), plays a dominant role in processing coronavirus-encoded polyproteins and is thus recognized as an ideal target of anti-coronavirus agents. However, no structure of the MHV-A59 Mpro has been reported, and molecular exploration of the catalysis mechanism remains hindered. Here, we solved the crystal structure of the MHV-A59 Mpro complexed with a Michael acceptor-based inhibitor, N3. Structural analysis revealed that the Cβ of the vinyl group of N3 covalently bound to C145 of the catalytic dyad of Mpro, which irreversibly inactivated cysteine protease activity. The lactam ring of the P1 side chain and the isobutyl group of the P2 side chain, which mimic the conserved residues at the same positions of the substrate, fit well into the S1 and S2 pockets. Through a comparative study with Mpro of other coronaviruses, we observed that the substrate-recognition pocket and enzyme inhibitory mechanism is highly conservative. Altogether, our study provided structural features of MHV-A59 Mpro and indicated that a Michael acceptor inhibitor is an ideal scaffold for antiviral drugs.

Molecular detection of mouse hepatitis virus in laboratory mouse colonies.

The animal health monitoring is required to issue health certificates. The viral hepatitis virus is one of the most important infectious agents in mice breeding colonies. This research used RT-PCR to identify contaminations to mouse hepatitis virus. 18 out of 29 specimens were found to be infected, a prevalence of 62%. PCR product was purified and sequenced. Phylogenetic analysis revealed that the identified strain in this study was closely related to a strain reported from France. In the conventional system, contamination with different infectious agents is inevitable, thus it is better to replace the contaminated colonies with clean animals.

Simultaneous rapid detection of Hantaan virus and Seoul virus using RT-LAMP in rats.

BackgroundHemorrhagic fever with renal syndrome is in most cases caused by the Hantaan virus (HTNV) and Seoul virus (SEOV). To develop and apply reverse transcription loop-mediated isothermal amplification (RT-LAMP) to detect HTNV and SEOV simultaneously, which was faster, more cost effective, and easier to perform as the target gene amplified rapidly. In this article an assay based on LAMP is demonstrated, which only employs such apparatus as a water bath or a heat block.MethodsA chromogenic method using the calcein/Mn2+ complex and real-time turbidity monitoring method were used to assess reaction progress of the reaction, and the specificity of the RT-LAMP-based assay was assessed by detecting cDNAs/cRNAs generated from Coxsackievirus A16, Influenza virus, lymphocytic choriomeningitis virus, mouse poxvirus, rotavirus, mouse hepatitis virus. In addition, 23 clinical specimens were used to determine the agreement between the RT-LAMP assay with reverse transcriptase polymerase chain reaction (RT-PCR) and immunofluorescence (IFT) method.ResultsThe detection limit of RT-LAMP to HNTV and SEOV was as low as 10 copies/μL with optimized reaction conditions, which was much more sensitive than the RT-PCR method (100–1,000 copies/μL). At the same time, the detection results of 23 clinical specimens have also illustrated the agreement between this the RT-LAMP assay with RT-PCR and IFT.DiscussionThis RT-LAMP assay could be used to perform simultaneous and rapid detection of HTNV and SEOV to the clinical specimens.

Depletion of RIPK1 in hepatocytes exacerbates liver damage in fulminant viral hepatitis

The protein kinase RIPK1 plays a crucial role at the crossroad of stress-induced signaling pathways that affects cell’s decision to live or die. The present study aimed to define the role of RIPK1 in hepatocytes during fulminant viral hepatitis, a worldwide syndrome mainly observed in hepatitis B virus (HBV) infected patients. Mice deficient for RIPK1, specifically in liver parenchymal cells (Ripk1LPC-KO) and their wild-type littermates (Ripk1fl/fl), were challenged by either the murine hepatitis virus type 3 (MHV3) or poly I:C, a synthetic analog of double-stranded RNA mimicking viral pathogen-associated molecular pattern. Ripk1LPC-KO mice developed more severe symptoms at early stage of the MHV3-induced fulminant hepatitis. Similarly, administration of poly I:C only triggered increase of systemic transaminases in Ripk1LPC-KO mice, reflecting liver damage through induced apoptosis as illustrated by cleaved-caspase 3 labeling of liver tissue sections. Neutralization of TNF-α or prior depletion of macrophages were able to prevent the appearance of apoptosis of hepatocytes in poly I:C-challenged Ripk1LPC-KO mice. Moreover, poly I:C never induced direct hepatocyte death in primary culture whatever the murine genotype, while it always stimulated an anti-viral response. Our investigations demonstrated that RIPK1 protects hepatocytes from TNF-α secreted from macrophages during viral induced fulminant hepatitis. These data emphasize the potential worsening risks of an HBV infection in people with polymorphism or homozygous amorphic mutations already described for the RIPK1 gene.

Fine Tuning the Cytokine Storm by IFN and IL-10 Following Neurotropic Coronavirus Encephalomyelitis.

The central nervous system (CNS) is vulnerable to several viral infections including herpes viruses, arboviruses and HIV to name a few. While a rapid and effective immune response is essential to limit viral spread and mortality, this anti-viral response needs to be tightly regulated in order to limit immune mediated tissue damage. This balance between effective virus control with limited pathology is especially important due to the highly specialized functions and limited regenerative capacity of neurons, which can be targets of direct virus cytolysis or bystander damage. CNS infection with the neurotropic strain of mouse hepatitis virus (MHV) induces an acute encephalomyelitis associated with focal areas of demyelination, which is sustained during viral persistence. Both innate and adaptive immune cells work in coordination to control virus replication. While type I interferons are essential to limit virus spread associated with early mortality, perforin, and interferon-γ promote further virus clearance in astrocytes/microglia and oligodendrocytes, respectively. Effective control of virus replication is nonetheless associated with tissue damage, characterized by demyelinating lesions. Interestingly, the anti-inflammatory cytokine IL-10 limits expansion of tissue lesions during chronic infection without affecting viral persistence. Thus, effective coordination of pro- and anti-inflammatory cytokines is essential during MHV induced encephalomyelitis in order to protect the host against viral infection at a limited cost.

Clara Cell 10 kDa Protein Alleviates Murine Hepatitis Virus Strain 3-Induced Fulminant Hepatitis by Inhibiting Fibrinogen-Like Protein 2 Expression.

Background: Fulminant hepatitis (FH) is a serious threat to human life, accompanied by massive and rapid necroinflammation. Kupffer cells, the major immune cell population involved in innate immune responses, are considered to be central for FH. Fibrinogen-like protein 2 (Fgl2) is a pro-coagulant protein that is substantially induced in macrophages upon viral infection, and Fgl2 depletion represses murine hepatitis virus strain 3 (MHV-3) infection. Clara cell 10 kDa (CC10) protein is a secretory protein with anti-inflammatory properties in allergic rhinitis and asthma. However, its mechanisms of action and pathogenic roles in other disease are still unclear. In this study, we aimed to determine the role of CC10 in FH and the regulation of Fgl2 by CC10.Methods: A mouse FH model was established by peritoneal injection of MHV-3. The mice received CC10 protein through tail vein injection before viral infection. Survival rate, liver function, liver histology, fibrin deposition, and necrosis were examined. The regulatory effect of CC10 on Fgl2 expression was investigated using THP-1 cells and mouse peritoneal macrophages in vitro.Results: In the mouse FH model induced by MHV-3, the survival rate increased from 0 to 12.5% in the CC10 group compared to that in the saline-only control group. Meanwhile, the levels of ALT and AST in serum were significantly decreased and liver damage was reduced. Furthermore, hepatic Fgl2, TNF-α, and IL-1β expression was obviously downregulated together with fibrin deposition, and hepatocyte apoptosis was reduced after administration of CC10 protein. In vitro, CC10 was found to significantly inhibit the expression of Fgl2 in IFN-γ-treated THP-1 cells and MHV-3-infected mouse peritoneal macrophages by western blot and real-time PCR. However, there was no direct interaction between CC10 and Fgl2 as shown by co-immunoprecipitation. Microarray investigations suggested that HMG-box transcription factor 1 (HBP1) was significantly low in CC10-treated and IFN-γ-primed THP-1 cells. HBP1-siRNA treatment abrogated the inhibitory effect of CC10 on Fgl2 expression in Human Umbilical Vein Endothelial cells (HUVECs).Conclusion:CC10 protects against MHV-3-induced FH via suppression of Fgl2 expression in macrophages. Such effects may be mediated by the transcription factor HBP1.

Attenuation of Influenza A Virus Disease Severity by Viral Coinfection in a Mouse Model

Influenza viruses and rhinoviruses are responsible for a large number of acute respiratory viral infections in human populations and are detected as copathogens within hosts. Clinical and epidemiological studies suggest that coinfection by rhinovirus and influenza virus may reduce disease severity and that they may also interfere with each other's spread within a host population. To determine how coinfection by these two unrelated respiratory viruses affects pathogenesis, we established a mouse model using a minor serogroup rhinovirus (rhinovirus strain 1B [RV1B]) and mouse-adapted influenza A virus (A/Puerto Rico/8/1934 [PR8]). Infection of mice with RV1B 2 days before PR8 reduced the severity of infection by a low or medium, but not high, dose of PR8. Disease attenuation was associated with an early inflammatory response in the lungs and enhanced clearance of PR8. However, coinfection by RV1B did not reduce PR8 viral loads early in infection or inhibit replication of PR8 within respiratory epithelia or in vitro Inflammation in coinfected mice remained focal compared to diffuse inflammation and damage in the lungs of mice infected by PR8. The timing of RV1B coinfection was a critical determinant of protection, suggesting that sufficient time is needed to induce this response. Finally, disease attenuation was not unique to RV1B: dose-dependent coinfection by a murine coronavirus (mouse hepatitis virus strain 1 [MHV-1]) also reduced the severity of PR8 infection. Unlike RV1B, coinfection with MHV-1 reduced early PR8 replication, which was associated with upregulation of beta interferon (IFN-β) expression. This model is critical for understanding the mechanisms responsible for influenza disease attenuation during coinfection by unrelated respiratory viruses.IMPORTANCE Viral infections in the respiratory tract can cause severe disease and are responsible for a majority of pediatric hospitalizations. Molecular diagnostics have revealed that approximately 20% of these patients are infected by more than one unrelated viral pathogen. To understand how viral coinfection affects disease severity, we inoculated mice with a mild viral pathogen (rhinovirus or murine coronavirus), followed 2 days later by a virulent viral pathogen (influenza A virus). This model demonstrated that rhinovirus can reduce the severity of influenza A virus, which corresponded with an early but controlled inflammatory response in the lungs and early clearance of influenza A virus. We further determined the dose and timing parameters that were important for effective disease attenuation and showed that influenza disease is also reduced by coinfection with a murine coronavirus. These findings demonstrate that coinfecting viruses can alter immune responses and pathogenesis in the respiratory tract.

Interplay between coronavirus, a cytoplasmic RNA virus, and nonsense-mediated mRNA decay pathway

Coronaviruses (CoVs), including severe acute respiratory syndrome CoV and Middle East respiratory syndrome CoV, are enveloped RNA viruses that carry a large positive-sense single-stranded RNA genome and cause a variety of diseases in humans and domestic animals. Very little is known about the host pathways that regulate the stability of CoV mRNAs, which carry some unusual features. Nonsense-mediated decay (NMD) is a eukaryotic RNA surveillance pathway that detects mRNAs harboring aberrant features and targets them for degradation. Although CoV mRNAs are of cytoplasmic origin, the presence of several NMD-inducing features (including multiple ORFs with internal termination codons that create a long 3' untranslated region) in CoV mRNAs led us to explore the interplay between the NMD pathway and CoVs. Our study using murine hepatitis virus as a model CoV showed that CoV mRNAs are recognized by the NMD pathway as a substrate, resulting in their degradation. Furthermore, CoV replication induced the inhibition of the NMD pathway, and N protein (a viral structural protein) had an NMD inhibitory function that protected viral mRNAs from rapid decay. Our data further suggest that the NMD pathway interferes with optimal viral replication by degrading viral mRNAs early in infection, before sufficient accumulation of N protein. Our study presents clear evidence for the biological importance of the NMD pathway in controlling the stability of mRNAs and the efficiency of replication of a cytoplasmic RNA virus.

Chlordecone potentiates auto-immune hepatitis and promotes brain entry of MHV3 during viral hepatitis in mouse models

Chlordecone is an organochlorine used in the 1970’s as a pesticide in banana plantations. It has a long half-life in the soil and can potentially contaminate humans and animals through food. Chlordecone targets, and mainly accumulates in, the liver, leading to hepatomegaly and neurological signs in mammals. Chlordecone does not cause liver injuries or any inflammation by itself at low doses, but it can potentiate the hepatotoxic effects of other chemicals and drugs. We studied the impact of chlordecone on the progression of acute hepatitis in mouse models of co-exposure to chlordecone with Concanavalin A or murine hepatitis virus type 3. We examined the progression of these two types of hepatitis by measuring hepatic transaminase levels in the serum and inflammatory cells in the liver, liver histological studies. Amplified tremors presented in the MHV3- chlordecone mouse model had led us to study the expression of specific genes in the brain. We show that chlordecone amplifies the auto-immune hepatitis induced by Concanavalin A by increasing the number of liver NKT cells, which are involved in liver damage. Chlordecone also accelerated the death of mice infected by murine hepatitis virus and enhanced the entry of the virus into the cervical spinal cord in infected mice, leading to considerable neurological damage. In conclusion, chlordecone potentiates both the Concanavalin A-induced hepatitis and brain damage caused by an hepatotropic/neurotropic virus.

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.

Promoting remyelination through cell transplantation therapies in a model of viral-induced neurodegenerative disease.

Multiple sclerosis (MS) is a central nervous system (CNS) disease characterized by chronic neuroinflammation, demyelination, and axonal damage. Infiltration of activated lymphocytes and myeloid cells are thought to be primarily responsible for white matter damage and axonopathy. Several United States Food and Drug Administration-approved therapies exist that impede activated lymphocytes from entering the CNS thereby limiting new lesion formation in patients with relapse-remitting forms of MS. However, a significant challenge within the field of MS research is to develop effective and sustained therapies that allow for axonal protection and remyelination. In recent years, there has been increasing evidence that some kinds of stem cells and their derivatives seem to be able to mute neuroinflammation as well as promote remyelination and axonal integrity. Intracranial infection of mice with the neurotropic JHM strain of mouse hepatitis virus (JHMV) results in immune-mediated demyelination and axonopathy, making this an excellent model to interrogate the therapeutic potential of stem cell derivatives in evoking remyelination. This review provides a succinct overview of our recent findings using intraspinal injection of mouse CNS neural progenitor cells and human neural precursors into JHMV-infected mice. JHMV-infected mice receiving these cells display extensive remyelination associated with axonal sparing. In addition, we discuss possible mechanisms associated with sustained clinical recovery. Developmental Dynamics 248:43-52, 2019. © 2018 Wiley Periodicals, Inc.