Interaction of mouse hepatitis virus 3 with Kupffer cells explanted from susceptible and resistant mouse strains. Antiviral activity, interleukin-1 synthesis

The genetic sensitivity of mouse strains to mouse hepatitis virus 3 (MHV 3) has been related in vitro to a delay of virus replication in liver sinusoidal cells. In vivo immuno-histochemical studies of the liver from infected mice have demonstrated that mechanisms other than direct viral injury are in operation. To examine potential mechanisms, the interaction of lipopolysaccharide (LPS)-stimulated Kupffer cells with MHV 3 was studied. We first observed a dramatic inhibition in viral replication in LPS-treated Kupffer cells explanted from A/J resistant mice. Second, we demonstrated that MHV 3 induced a dose-dependent interleukin 1 (IL-1) activity in the supernatants of infected Kupffer cells of both strains. These results led us finally to examine the antigen-processing function of the Kupffer cells of both strains of mice. No striking differences were observed in the ability of Kupffer cells from resistant or sensitive mice to collaborate with immunocompetent lymphocytes. Our data suggest that Kupffer cells play a double role which is crucial in the pathogenesis of MHV 3-induced hepatitis. First, they act directly as the genetically determined sensitivity of mice to MHV 3 infection is correlated with the efficiency of the antiviral activity induced in Kupffer cells by LPS. Second, they act indirectly through the synthesis of different amounts of IL-1 induced by MHV 3. This hypothesis is further borne out by the effects of indomethacin treatment on the course of MHV 3 infection in A/J resistant mice in vivo.     

Interaction of mouse hepatitis virus 3 with Kupffer cells explanted from susceptible and resistant mouse strains. Antiviral activity, interleukin-1 synthesis

Abstract The genetic sensitivity of mouse strains to mouse hepatitis virus 3 (MHV 3) has been related in vitro to a delay of virus replication in liver sinusoidal cells. In vivo immuno-histochemical studies of the liver from infected mice have demonstrated that mechanisms other than direct viral injury are in operation. To examine potential mechanisms, the interaction of lipopolysaccharide (LPS)-stimulated Kupffer cells with MHV 3 was studied. We first observed a dramatic inhibition in viral replication in LPS-treated Kupffer cells explanted from A/J resistant mice. Second, we demonstrated that MHV 3 induced a dose-dependent interleukin 1 (IL-1) activity in the supernatants of infected Kupffer cells of both strains. These results led us finally to examine the antigen-proceesing function of the Kupffer cellsof both strains of mice. No striking differences were observed in the ability of Kupffer cells from resistant or sensitive mice to collaborate with immunocompetent lymphocytes. Our data suggest that Kupffer cells play a double role which is crucial in the pathogenesis of MHV 3-induced hepatitis. First, they act directly as the genetically determined sensitivity of mice to MHV 3 infection is correlated with the efficiency of the antiviral activity induced in Kupffer cells by LPS. Second, they act indirectly through the synthesis of different amounts of IL-1 induced by MHV 3. This hypothesis is further borne out by the effects of indomethacin treatment on the course of MHV 3 infection in A/J resistant mice in vivo.     

Selective tropism of a neurotropic coronavirus for ependymal cells, neurons, and meningeal cells.

The ability of a neurotropic virus, mouse hepatitis virus type 3 (MHV3), to invade the central nervous system (CNS) and to recognize cells selectively within the brain was investigated in vivo and in vitro. In vivo, MHV3 induced in C3H mice a genetically controlled infection of meningeal cells, ependymal cells, and neurons. In vitro, purified MHV3 bound to the surface of isolated ependymal cells and cultured cortical neurons but not to oligodendrocytes or cultured astrocytes. MHV3 replicated within cultured cortical neurons and neuroblastoma cells (NIE 115); infected cultured neurons nonetheless survived and matured normally for a 7-day period postinfection. On the other hand, MHV3 had a low affinity for cortical glial cells or glioma cells (C6 line), both of which appear to be morphologically unaltered by viral infection. Finally, MHV3 infected and disrupted cultured meningeal cells. This suggests that differences in the affinity of cells for MHV3 are determinants of the selective vulnerability of cellular subpopulations within the CNS. In vivo, a higher titer of virus was needed for CNS penetration in the genetically resistant (A/Jx) mice than in the susceptible (C57/BL6) mouse strain. However, in spite of viral invasion, no neuropathological lesions developed. In vitro viral binding to adult ependymal cells of susceptible and resistant strains of mice was identical. Genetic resistance to MHV3-CNS infection appeared to be mediated both by a peripheral mechanism limiting viral penetration into the CNS and by intra-CNS mechanisms, presumably at a stage after viral attachment to target cells.     

A thiopurine drug inhibits West Nile virus production in cell culture, but not in mice

Many viruses within the Flavivirus genus cause significant disease in humans; however, effective antivirals against these viruses are not currently available. We have previously shown that a thiopurine drug, 6-methylmercaptopurine riboside (6MMPr), inhibits replication of distantly related viruses within the Flaviviridae family in cell culture, including bovine viral diarrhea virus and hepatitis C virus replicon. Here we further examined the potential antiviral effect of 6MMPr on several diverse flaviviruses. In cell culture, 6MMPr inhibited virus production of yellow fever virus, dengue virus-2 (DENV-2) and West Nile virus (WNV) in a dose-dependent manner, and DENV-2 was significantly more sensitive to 6MMPr treatment than WNV. We then explored the use of 6MMPr as an antiviral against WNV in an immunocompetent mouse model. Once a day treatment of mice with 0.5 mg 6MMPr was just below the toxic dose in our mouse model, and this dose was used in subsequent studies. Mice were treated with 6MMPr immediately after subcutaneous inoculation with WNV for eight consecutive days. Treatment with 6MMPr exacerbated weight loss in WNV-inoculated mice and did not significantly affect mortality. We hypothesized that 6MMPr has low bioavailability in the central nervous system (CNS) and examined the effect of pre-treatment with 6MMPr on viral loads in the periphery and CNS. Pre-treatment with 6MMPr had no significant effect on viremia or viral titers in the periphery, but resulted in significantly higher viral loads in the brain, suggesting that the effect of 6MMPr is tissue-dependent. In conclusion, despite being a potent inhibitor of flaviviruses in cell culture, 6MMPr was not effective against West Nile disease in mice; however, further studies are warranted to reduce the toxicity and/or improve the bioavailability of this potential antiviral drug.     

Signaling through the prostaglandin I2 receptor IP protects against respiratory syncytial virus-induced illness

The role of prostanoids in modulating respiratory syncytial virus (RSV) infection is unknown. We found that RSV infection in mice increases production of prostaglandin I(2) (PGI(2)). Mice that overexpress PGI(2) synthase selectively in bronchial epithelium are protected against RSV-induced weight loss and have decreased peak viral replication and gamma interferon levels in the lung compared to nontransgenic littermates. In contrast, mice deficient in the PGI(2) receptor IP have exacerbated RSV-induced weight loss with delayed viral clearance and increased levels of gamma interferon in the lung compared to wild-type mice. These results suggest that signaling through IP has antiviral effects while protecting against RSV-induced illness and that PGI(2) is a potential therapeutic target in the treatment of RSV.     

Natural cytotoxicity against mouse hepatitis virus-infected target cells. I. Correlation of cytotoxicity with virus binding to leukocytes

Spleen cells from uninfected control mice selectively lysed BALB/c 3T3 fibroblasts infected with mouse hepatitis virus (MHV), a murine coronavirus. Lysis of infected cells occurred within 3 hr, and histocompatibility between effector and target cells was not required. This natural, cell-mediated, virus-associated cytotoxicity differed from NK cell- and T cell-mediated lysis. Spleen cells from animals infected with MHV were enriched in NK activity and were more cytotoxic to YAC-1 target cells, but did not show enhanced cytotoxicity for MHV-infected target cells. Spleen cells from beige mice, which are deficient in NK cell activity, were able to lyse MHV-infected target cells, as were spleen cells from nude mice, which are deficient in T cell activity. Lysis of MHV-infected target cells could be mediated by cells from the spleen and, to a lesser extent, by cells from the bone marrow, but not by resident peritoneal cells or thymocytes. We suggest the term "virus killer (VK) activity" for this phenomenon. VK activity of splenocytes from different mouse strains correlated with the ability of the splenocytes to bind purified radiolabeled MHV virions. MHV virions caused agglutination of spleen leukocytes from susceptible mouse strains, indicating that leukocyte agglutination or adsorption may provide a useful assay for coronaviruses such as MHV which lack hemagglutinating activity. SJL mouse splenocytes did not bind MHV and did not lyse infected targets. MHV bound relatively well to splenocytes of other mouse strains, but poorly to thymocytes and erythrocytes. Binding of MHV to leukocytes was not influenced by 6 mM EDTA or EGTA, indicating a lack of requirement for Mg++ or Ca++. VK activity was also resistant to EDTA and EGTA, in contrast to NK activity, which was sensitive to those chelating agents. VK activity was also unaffected by actinomycin D, cycloheximide, or puromycin, indicating that new protein synthesis was not required for lysis. Antibody to interferon-alpha/beta did not block lysis, nor was there substantially enhanced lysis mediated by leukocytes from mice infected with virus and thus exposed to high levels of interferon. VK activity was blocked by antibody directed against the peplomeric glycoprotein E2 of MHV. VK activity required infected target cells, because cells with adsorbed MHV virions were not lysed by splenocytes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Efficacy of combined therapy with amantadine, oseltamivir, and ribavirin in vivo against susceptible and amantadine-resistant influenza A viruses

The limited efficacy of existing antiviral therapies for influenza--coupled with widespread baseline antiviral resistance--highlights the urgent need for more effective therapy. We describe a triple combination antiviral drug (TCAD) regimen composed of amantadine, oseltamivir, and ribavirin that is highly efficacious at reducing mortality and weight loss in mouse models of influenza infection. TCAD therapy was superior to dual and single drug regimens in mice infected with drug-susceptible, low pathogenic A/H5N1 (A/Duck/MN/1525/81) and amantadine-resistant 2009 A/H1N1 influenza (A/California/04/09). Treatment with TCAD afforded >90% survival in mice infected with both viruses, whereas treatment with dual and single drug regimens resulted in 0% to 60% survival. Importantly, amantadine had no activity as monotherapy against the amantadine-resistant virus, but demonstrated dose-dependent protection in combination with oseltamivir and ribavirin, indicative that amantadine's activity had been restored in the context of TCAD therapy. Furthermore, TCAD therapy provided survival benefit when treatment was delayed until 72 hours post-infection, whereas oseltamivir monotherapy was not protective after 24 hours post-infection. These findings demonstrate in vivo efficacy of TCAD therapy and confirm previous reports of the synergy and broad spectrum activity of TCAD therapy against susceptible and resistant influenza strains in vitro.     

Primary HIV-1 infection of human CD4+ T cells passaged into SCID mice leads to selection of chronically infected cells through a massive fas-mediated autocrine suicide of uninfected cells

We have recently shown that a human CD4+ T cell line (CEM-SS) acquires the permissiveness to M-tropic strains and primary isolates of HIV-1 after transplantation into SCID mice. This permissiveness was associated with the acquisition of a memory (CD45RO+) phenotype as well as of a functional CCR5 coreceptor. In this study, we have used this model for invest-igating in vivo the relationships between HIV-1 infection, apoptosis and T cell differentiation. When an in vivo HIV-1 infection was performed, the CEM cell tumors grew to a lower extent than the uninfected controls. CEM cells explanted from uninfected SCID mice (ex vivo CEM) underwent a significant level of spontaneous apoptosis and proved to be CD45RO+, Fas+ and Fas-L+, while Bcl-2 expression was significantly reduced as compared to the parental cells. Acute HIV-1 infection markedly increased apoptosis of uninfected ex vivo CEM cells, through a Fas/Fas-L-mediated autocrine suicide/fratricide, while parental cells did not undergo apoptosis following viral infection. The susceptibility to apoptosis of ex vivo CEM cells infected with the NSI strain of HIV-1, was progressively lost during culture, in parallel with the loss of Fas-L and marked changes in the Bcl-2 cellular distribution. On the whole, these results are strongly reminiscent of a series of events possibly occurring during HIV-1 infection. After an initial depletion of bystander CD4+ memory T cells during acute infection, latently or chronically infected CD4+ T lymphocytes are progressively selected and are protected against spontaneous apoptosis through the development of an efficient survival program. Studies with human cells passaged into SCID mice may offer new opportunities for an in vivo investigation of the mechanisms involved in HIV-1 infection and CD4+ T cell depletion.     

Antisense morpholino-oligomers directed against the 5' end of the genome inhibit coronavirus proliferation and growth

Conjugation of a peptide related to the human immunodeficiency virus type 1 Tat represents a novel method for delivery of antisense morpholino-oligomers. Conjugated and unconjugated oligomers were tested to determine sequence-specific antiviral efficacy against a member of the Coronaviridae, Mouse hepatitis virus (MHV). Specific antisense activity designed to block translation of the viral replicase polyprotein was first confirmed by reduction of luciferase expression from a target sequence-containing reporter construct in both cell-free and transfected cell culture assays. Peptide-conjugated morpholino-oligomers exhibited low toxicity in DBT astrocytoma cells used for culturing MHV. Oligomer administered at micromolar concentrations was delivered to >80% of cells and inhibited virus titers 10- to 100-fold in a sequence-specific and dose-responsive manner. In addition, targeted viral protein synthesis, plaque diameter, and cytopathic effect were significantly reduced. Inhibition of virus infectivity by peptide-conjugated morpholino was comparable to the antiviral activity of the aminoglycoside hygromycin B used at a concentration fivefold higher than the oligomer. These results suggest that this composition of antisense compound has therapeutic potential for control of coronavirus infection.     

Epitope-specific antibody response to murine hepatitis virus-4 (strain JHM)

Monoclonal hybridoma antibodies to the structural proteins of murine hepatitis virus-4, strain JHM (MHV-4) were used in a competition binding enzyme immunoassay to analyze at the epitope level the antibody response of mice after infection with MHV-4. Colonized mice often had pre-existing MHV antibodies directed against epitopes on the E2 glycoprotein, the E1 glycoprotein, and the nucleocapsid protein. These mice generated a secondary antibody response after virus inoculation, reaching peak levels 7 days after infection. In contrast, Nude/+ mice raised in a pathogen-free colony had no detectable circulating MHV antibodies and generated a primary antibody response which gradually increased to peak levels 14 to 28 days after infection. Kinetics of antibody responses against specific epitopes usually correlated well with measured total virus-specific antibody responses, but variation was observed. Mice injected with three antigenically distinct strains of MHV made antibody responses to conserved epitopes but not to an antigenic determinant absent in these strains. Measurement of epitope-specific responses in a polyclonal population of viral specific antibodies is feasible and a valuable adjunct in understanding viral immunity.     

Coronavirus non-structural protein 1 is a major pathogenicity factor: implications for the rational design of coronavirus vaccines

Attenuated viral vaccines can be generated by targeting essential pathogenicity factors. We report here the rational design of an attenuated recombinant coronavirus vaccine based on a deletion in the coding sequence of the non-structural protein 1 (nsp1). In cell culture, nsp1 of mouse hepatitis virus (MHV), like its SARS-coronavirus homolog, strongly reduced cellular gene expression. The effect of nsp1 on MHV replication in vitro and in vivo was analyzed using a recombinant MHV encoding a deletion in the nsp1-coding sequence. The recombinant MHV nsp1 mutant grew normally in tissue culture, but was severely attenuated in vivo. Replication and spread of the nsp1 mutant virus was restored almost to wild-type levels in type I interferon (IFN) receptor-deficient mice, indicating that nsp1 interferes efficiently with the type I IFN system. Importantly, replication of nsp1 mutant virus in professional antigen-presenting cells such as conventional dendritic cells and macrophages, and induction of type I IFN in plasmacytoid dendritic cells, was not impaired. Furthermore, even low doses of nsp1 mutant MHV elicited potent cytotoxic T cell responses and protected mice against homologous and heterologous virus challenge. Taken together, the presented attenuation strategy provides a paradigm for the development of highly efficient coronavirus vaccines.     

Murine Gammaherpesvirus 68 Encodes a Second PML-Modifying Protein

The ORF75c tegument protein of murine gammaherpesvirus 68 (MHV68) promotes the degradation of the antiviral promyelocytic leukemia (PML) protein. Surprisingly, MHV68 expressing a degradation-deficient ORF75c replicated in cell culture and in mice similar to the wild-type virus. However, in cells infected with this mutant virus, PML formed novel track-like structures that are induced by ORF61, the viral ribonucleotide reductase large subunit. These findings may explain why ORF75c mutant viruses unable to degrade PML had no demonstrable phenotype after infection.     

A hypervariable region within the 3' cis-acting element of the murine coronavirus genome is nonessential for RNA synthesis but affects pathogenesis

The 3' cis-acting element for mouse hepatitis virus (MHV) RNA synthesis resides entirely within the 301-nucleotide 3' untranslated region (3' UTR) of the viral genome and consists of three regions. Encompassing the upstream end of the 3' UTR are a bulged stem-loop and an overlapping RNA pseudoknot, both of which are essential to MHV and common to all group 2 coronaviruses. At the downstream end of the genome is the minimal signal for initiation of negative-strand RNA synthesis. Between these two ends is a hypervariable region (HVR) that is only poorly conserved between MHV and other group 2 coronaviruses. Paradoxically, buried within the HVR is an octanucleotide motif (oct), 5'-GGAAGAGC-3', which is almost universally conserved in coronaviruses and is therefore assumed to have a critical biological function. We conducted an extensive mutational analysis of the HVR. Surprisingly, this region tolerated numerous deletions, rearrangements, and point mutations. Most striking, a mutant deleted of the entire HVR was only minimally impaired in tissue culture relative to the wild type. By contrast, the HVR deletion mutant was highly attenuated in mice, causing no signs of clinical disease and minimal weight loss compared to wild-type virus. Correspondingly, replication of the HVR deletion mutant in the brains of mice was greatly reduced compared to that of the wild type. Our results show that neither the HVR nor oct is essential for the basic mechanism of MHV RNA synthesis in tissue culture. However, the HVR appears to play a significant role in viral pathogenesis.     

Expression of CXC chemokine ligand 10 from the mouse hepatitis virus genome results in protection from viral-induced neurological and liver disease

Using the recombinant murine coronavirus mouse hepatitis virus (MHV) expressing the T cell-chemoattractant CXCL10 (MHV-CXCL10), we demonstrate a potent antiviral role for CXCL10 in host defense. Instillation of MHV-CXCL10 into the CNS of CXCL10-deficient (CXCL10(-/-)) mice resulted in viral infection and replication in both brain and liver. Expression of virally encoded CXCL10 within the brain protected mice from death and correlated with increased infiltration of T lymphocytes, enhanced IFN-gamma secretion, and accelerated viral clearance when compared with mice infected with an isogenic control virus, MHV. Similarly, viral clearance from the livers of MHV-CXCL10-infected mice was accelerated in comparison to MHV-infected mice, yet was independent of enhanced infiltration of T lymphocytes and NK cells. Moreover, CXCL10(-/-) mice infected with MHV-CXCL10 were protected from severe hepatitis as evidenced by reduced pathology and serum alanine aminotransferase levels compared with MHV-infected mice. CXCL10-mediated protection within the liver was not dependent on CXC-chemokine receptor 2 (CXCR2) signaling as anti-CXCR2 treatment of MHV-CXCL10-infected mice did not modulate viral clearance or liver pathology. In contrast, treatment of MHV-CXCL10-infected CXCL10(-/-) mice with anti-CXCL10 Ab resulted in increased clinical disease correlating with enhanced viral recovery from the brain and liver as well as increased serum alanine aminotransferase levels. These studies highlight that CXCL10 expression promotes protection from coronavirus-induced neurological and liver disease.     

Expression in a recombinant murid herpesvirus 4 reveals the in vivo transforming potential of the K1 open reading frame of Kaposi's sarcoma-associated herpesvirus

Murid herpesvirus 4 (commonly called MHV-68) is closely related to Kaposi's sarcoma-associated herpesvirus (KSHV) and provides an excellent model system for investigating gammaherpesvirus-associated pathogenesis. MHV-76 is a naturally occurring deletion mutant of MHV-68 that lacks 9,538 bp of the left end of the unique portion of the genome encoding nonessential pathogenesis-related genes. The KSHV K1 protein has been shown to transform rodent fibroblasts in vitro and common marmoset T lymphocytes in vivo. Using homologous recombination techniques, we successfully generated recombinants of MHV-76 that encode green fluorescent protein (MHV76-GFP) and KSHV K1 (MHV76-K1). The replication of MHV76-GFP and MHV76-K1 in cell culture was identical to that of MHV-76. However, infection of BALB/c mice via the intranasal route revealed that MHV76-K1 replicated to a 10-fold higher titer than MHV76-GFP in the lungs at day 5 postinfection (p.i.). We observed type 2 pneumocyte proliferation in areas of consolidation and interstitial inflammation of mice infected with MHV76-K1 at day 10 p.i. MHV76-K1 established a 2- to 3-fold higher latent viral load than MHV76-GFP in the spleens of infected mice on days 10 and 14 p.i., although this was 10-fold lower than that established by wild-type MHV-76. A salivary gland tumor was present in one of four mice infected with MHV76-K1, as well as an increased inflammatory response in the lungs at day 120 p.i. compared with that of mice infected with MHV-76 and MHV76-GFP.     

In vivo RNA-RNA recombination of coronavirus in mouse brain.

RNA-RNA recombination between different strains of the murine coronavirus mouse hepatitis virus (MHV) occurs at a very high frequency in tissue culture. To demonstrate that RNA recombination may play a role in the evolution and pathogenesis of coronaviruses, we sought to determine whether MHV recombination could occur during replication in the animal host of the virus. By using two selectable markers, i.e., temperature sensitivity and monoclonal antibody neutralization, we isolated several recombinant viruses from the brains of mice infected with two different strains of MHV. The recombination frequency was very high, and recombination occurred at multiple sites on the viral RNA genome. This finding suggests that RNA-RNA recombination may play a significant role in natural evolution and neuropathogenesis of coronaviruses.