Ceacam1a-/- mice are completely resistant to infection by murine coronavirus mouse hepatitis virus A59

CEACAM1a glycoproteins are members of the immunoglobulin (Ig) superfamily and the carcinoembryonic antigen family. Isoforms expressing either two or four alternatively spliced Ig-like domains in mice have been found in a number of epithelial, endothelial, or hematopoietic tissues. CEACAM1a functions as an intercellular adhesion molecule, an angiogenic factor, and a tumor cell growth inhibitor. Moreover, the mouse and human CEACAM1a proteins are targets of viral or bacterial pathogens, respectively, including the murine coronavirus mouse hepatitis virus (MHV), Haemophilus influenzae, Neisseria gonorrhoeae, and Neisseria meningitidis, as well as Moraxella catarrhalis in humans. We have shown that targeted disruption of the Ceacam1a (MHVR) gene resulting in a partial ablation of the protein in mice (p/p mice) led to reduced susceptibility to MHV-A59 infection of the modified mice in the BALB/c background. We have now engineered and produced a Ceacam1a-/- mouse that exhibits complete ablation of the CEACAM1a protein in every tissue where it is normally expressed. We report that 3-week-old Ceacam1a-/- mice in the C57BL/6 genetic background are fully resistant to MHV-A59 infection by both intranasal and intracerebral routes. Whereas virus-inoculated wild-type +/+ C57BL/6 mice showed profound liver damage and spinal cord demyelination under these conditions, Ceacam1a-/- mice displayed normal livers and spinal cords. Virus was recovered from liver and spinal cord tissues of +/+ mice but not of -/- mice. These results indicate that CEACAM1a is the sole receptor for MHV-A59 in both liver and brain and that its deletion from the mouse renders the mouse completely resistant to infection by this virus.     

Persistence of mouse hepatitis virus A59 RNA in a slow virus demyelinating infection in mice as detected by in situ hybridization.

Mouse hepatitis virus strain A59 produces chronic central nervous system demyelination in rodents. As late as 6 months after intracerebral inoculation of mice 4 to 6 weeks old, when infectious virus cannot be recovered and viral antigens cannot be detected in the central nervous systems and livers of these animals, primary demyelination is still evident. Using cloned virus-specific DNAs and the highly sensitive and specific technique of in situ hybridization, we have detected low levels of mouse hepatitis virus A59 RNA in the central nervous systems and livers of mice 10 months after inoculation. We suggest that viral persistence may play a role in mouse hepatitis virus A59-induced chronic demyelination.     

Targeted Recombination within the Spike Gene of Murine Coronavirus Mouse Hepatitis Virus-A59: Q159 Is a Determinant of Hepatotropism

Previous studies of a group of mutants of the murine coronavirus mouse hepatitis virus (MHV)-A59, isolated from persistently infected glial cells, have shown a strong correlation between a Q159L amino acid substitution in the S1 subunit of the spike gene and a loss in the ability to induce hepatitis and demyelination. To determine if Q159L alone is sufficient to cause these altered pathogenic properties, targeted RNA recombination was used to introduce a Q159L amino acid substitution into the spike gene of MHV-A59. Recombination was carried out between the genome of a temperature-sensitive mutant of MHV-A59 (Alb4) and RNA transcribed from a plasmid (pFV1) containing the spike gene as well as downstream regions, through the 3′ end, of the MHV-A59 genome. We have selected and characterized two recombinant viruses containing Q159L. These recombinant viruses (159R36 and 159R40) replicate in the brains of C57BL/6 mice and induce encephalitis to a similar extent as wild-type MHV-A59. However, they exhibit a markedly reduced ability to replicate in the liver or produce hepatitis compared to wild-type MHV-A59. These viruses also exhibit reduced virulence and reduced demyelination. A recombinant virus containing the wild-type MHV-A59 spike gene, wtR10, behaved essentially like wild-type MHV-A59. This is the first report of the isolation of recombinant viruses containing a site-directed mutation, encoding an amino acid substitution, within the spike gene of any coronavirus. This technology will allow us to begin to map the molecular determinants of pathogenesis within the spike glycoprotein.     

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.     

Demyelination determinants map to the spike glycoprotein gene of coronavirus mouse hepatitis virus

Demyelination is the pathologic hallmark of the human immune-mediated neurologic disease multiple sclerosis, which may be triggered or exacerbated by viral infections. Several experimental animal models have been developed to study the mechanism of virus-induced demyelination, including coronavirus mouse hepatitis virus (MHV) infection in mice. The envelope spike (S) glycoprotein of MHV contains determinants of properties essential for virus-host interactions. However, the molecular determinants of MHV-induced demyelination are still unknown. To investigate the mechanism of MHV-induced demyelination, we examined whether the S gene of MHV contains determinants of demyelination and whether demyelination is linked to viral persistence. Using targeted RNA recombination, we replaced the S gene of a demyelinating virus (MHV-A59) with the S gene of a closely related, nondemyelinating virus (MHV-2). Recombinant viruses containing an S gene derived from MHV-2 in an MHV-A59 background (Penn98-1 and Penn98-2) exhibited a persistence-positive, demyelination-negative phenotype. Thus, determinants of demyelination map to the S gene of MHV. Furthermore, viral persistence is insufficient to induce demyelination, although it may be a prerequisite for the development of demyelination.     

PDGF and FGF2 regulate oligodendrocyte progenitor responses to demyelination

Acute demyelination of adult CNS, resulting from trauma or disease, is initially followed by remyelination. However, chronic lesions with subsequent functional impairment result from eventual failure of the remyelination process, as seen in multiple sclerosis. Studies using animal models of successful remyelination delineate a progression of events facilitating remyelination. A universal feature of this repair process is extensive proliferation of oligodendrocyte progenitor cells (OPs) in response to demyelination. To investigate signals that regulate OP proliferation in response to demyelination we used murine hepatitis virus-A59 (MHV-A59) infection of adult mice to induce focal demyelination throughout the spinal cord followed by spontaneous remyelination. We cultured glial cells directly from demyelinating and remyelinating spinal cords using conditions that maintain the dramatically enhanced OP proliferative response prior to CNS remyelination. We identify PDGF and FGF2 as significant mitogens regulating this proliferative response. Furthermore, we demonstrate endogenous PDGF and FGF2 activity in these glial cultures isolated from demyelinated CNS tissue. These findings correlate well with our previous demonstration of increased in vivo expression of PDGF and FGF2 ligand and corresponding receptors in MHV-A59 lesions. Together these studies support the potential of these pathways to function in vivo as critical factors in regulating remyelination.     

Detection of rabies virus in different tissues of experimentally infected mice at preclinical and postclinical stages of the disease.

Rabies fixed virus (CVS) was passaged 10 times in mice by intramuscular (im) route followed by experimental inoculation of the titrated virus in 4 groups of mice with the dose of 0.1 ml of 1000 mouse (LD50 0.03 ml) using intracerebral (ic), intravenous (iv), intramuscular (im), intraocular (io), and intranasal (in) routes respectively. No marked variation in clinical signs due to variation of routes could be detected. Involvement of brain with io route could be detected even in preclinical stage. Although the virus could be detected in the postclinical stage in all the tissues under study (brain, skin, salivary gland and corneal impression), with io and ic routes spread of the virus was observed in comparatively higher concentrations.     

The Spike Protein of Murine Coronavirus Mouse Hepatitis Virus Strain A59 Is Not Cleaved in Primary Glial Cells and Primary Hepatocytes

Mouse hepatitis virus strain A59 (MHV-A59) produces meningoencephalitis and severe hepatitis during acute infection. Infection of primary cells derived from the central nervous system (CNS) and liver was examined to analyze the interaction of virus with individual cell types derived from the two principal sites of viral replication in vivo. In glial cell cultures derived from C57BL/6 mice, MHV-A59 produces a productive but nonlytic infection, with no evidence of cell-to-cell fusion. In contrast, in continuously cultured cells, this virus produces a lytic infection with extensive formation of syncytia. The observation of few and delayed syncytia following MHV-A59 infection of hepatocytes more closely resembles infection of glial cells than that of continuously cultured cell lines. For MHV-A59, lack of syncytium formation correlates with lack of cleavage of the fusion glycoprotein, or spike (S) protein. The absence of cell-to-cell fusion following infection of both primary cell types prompted us to examine the cleavage of the spike protein. Cleavage of S protein was below the level of detection by Western blot analysis in MHV-A59-infected hepatocytes and glial cells. Furthermore, no cleavage of this protein was detected in liver homogenates from C57BL/6 mice infected with MHV-A59. Thus, cleavage of the spike protein does not seem to be essential for entry and spread of the virus in vivo, as well as for replication in vitro.     

Hydrocephalus in suckling rats infected intracerebrally with mouse hepatitis virus, MHV-A59

After intracerebral inoculation of mouse hepatitis virus, MHV-A59 strain, into 3- to 5-day-old Wistar rats, some survivors at 14 days postinoculation (p.i.) were found to lack the cerebral cortex and to have an accumulation of considerable amount of cerebrospinal fluid. The virus titer in the brain increased exponentially after inoculation, reaching a maximum 4 to 6 days p.i. when immunofluorescence revealed virus-specific antigen within neurons in the cerebral cortex. A small amount of infectious virus was also detectable 14 days p.i. when the cerebral anomaly was evident. This brain malformation causing hydrocephalus was due to cerebral damage by viral infection.     

Pathogenesis of Neonatal Herpes Simplex 2 Disease in a Mouse Model Is Dependent on Entry Receptor Expression and Route of Inoculation

Herpes simplex virus (HSV) pathogenesis in mice differs based on availability of the principal entry receptors herpesvirus entry mediator (HVEM) and nectin-1 in a manner dependent upon route of inoculation. After intravaginal or intracranial inoculation of adult mice, nectin-1 is a major mediator of neurologic disease, while the absence of either receptor attenuates disease after ocular infection. We tested the importance of receptor availability and route of infection on disease in mouse models of neonatal HSV. We infected 7-day-old mice lacking neither or one principal HSV receptor or both principal HSV receptors with HSV-2 via a peripheral route (intranasal), via a systemic route (intraperitoneal), or by inoculation directly into the central nervous system (intracranial). Mortality, neurologic disease, and visceral dissemination of virus were significantly attenuated in nectin-1 knockout mice compared with HVEM knockout or wild-type mice after intranasal inoculation. Mice lacking both entry receptors (double-knockout mice) showed no evidence of disease after inoculation by any route. Nectin-1 knockout mice had delayed mortality after intraperitoneal inoculation relative to wild-type and HVEM knockout mice, but virus was able to spread to the brain and viscera in all genotypes except double-knockout mice. Unlike in adult mice, HVEM was sufficient to mediate disease in neonatal mice after direct intracranial inoculation, and the absence of HVEM delayed time to mortality relative to that of wild-type mice. Additionally, in wild-type neonatal mice inoculated intracranially, HSV antigen did not primarily colocalize with NeuN-positive neurons. Our results suggest that differences in receptor expression between adults and newborns may partially explain differences in susceptibility to HSV-2.     

Mousepox in inbred mice innately resistant or susceptible to lethal infection with ectromelia virus. I. Clinical responses

Clinical responses to infection with ectromelia virus strain NIH-79 were determined in several strains of inbred mice. All mice were equally susceptible to infection, but mortality was strain dependent. BALB/c AnNCr, A/JNCr, DBA/2NCr and C3H/He/NCr MTV- mice were highly susceptible to lethal infection whereas AKR/NCr and SJL/NCr mice were moderately susceptible and C57BL/6NCr mice were highly resistant. Death rates were influenced strongly by virus dose and by route of inoculation. High doses were associated with early and high mortality. For a given dose, intraperitoneal inoculation resulted in the highest mortality and death rates were progressively reduced in mice inoculated by the footpad, subcutaneous and intranasal routes. Footpad swelling was prominent in resistant mice and in survivors among susceptible strains. Deaths among AKR and SJL mice were sporadic and often occurred late irrespective of virus dose. It is suggested that this pattern could be influenced by secondary contact infections or by immunologic injury associated with host responses to ectromelia virus.     

Crucial role for prion protein membrane anchoring in the neuroinvasion and neural spread of prion infection

In nature prion diseases are usually transmitted by extracerebral prion infection, but clinical disease results only after invasion of the central nervous system (CNS). Prion protein (PrP), a host-encoded glycosylphosphatidylinositol (GPI)-anchored membrane glycoprotein, is necessary for prion infection and disease. Here, we investigated the role of the anchoring of PrP on prion neuroinvasion by studying various inoculation routes in mice expressing either anchored or anchorless PrP. In control mice with anchored PrP, intracerebral or sciatic nerve inoculation resulted in rapid CNS neuroinvasion and clinical disease (154 to 156 days), and after tongue, ocular, intravenous, or intraperitoneal inoculation, CNS neuroinvasion was only slightly slower (193 to 231 days). In contrast, in anchorless PrP mice, these routes resulted in slow and infrequent CNS neuroinvasion. Only intracerebral inoculation caused brain PrPres, a protease-resistant isoform of PrP, and disease in both types of mice. Thus, anchored PrP was an essential component for the rapid neural spread and CNS neuroinvasion of prion infection.     

Single-amino-acid substitutions in open reading frame (ORF) 1b-nsp14 and ORF 2a proteins of the coronavirus mouse hepatitis virus are attenuating in mice

A reverse genetic system was recently established for the coronavirus mouse hepatitis virus strain A59 (MHV-A59), in which cDNA fragments of the RNA genome are assembled in vitro into a full-length genome cDNA, followed by electroporation of in vitro-transcribed genome RNA into cells with recovery of viable virus. The "in vitro-assembled" wild-type MHV-A59 virus (icMHV-A59) demonstrated replication identical to laboratory strains of MHV-A59 in tissue culture; however, icMHV-A59 was avirulent following intracranial inoculation of C57BL/6 mice. Sequencing of the cloned genome cDNA fragments identified two single-nucleotide mutations in cloned genome fragment F, encoding a Tyr6398His substitution in open reading frame (ORF) 1b p59-nsp14 and a Leu94Pro substitution in the ORF 2a 30-kDa protein. The mutations were repaired individually and together in recombinant viruses, all of which demonstrated wild-type replication in tissue culture. Following intracranial inoculation of mice, the viruses encoding Tyr6398His/Leu94Pro substitutions and the Tyr6398His substitution alone demonstrated log10 50% lethal dose (LD50) values too great to be measured. The Leu94Pro mutant virus had reduced but measurable log10 LD5), and the "corrected" Tyr6398/Leu94 virus had a log10 LD50 identical to wild-type MHV-A59. The experiments have defined residues in ORF 1b and ORF 2a that attenuate virus replication and virulence in mice but do not affect in vitro replication. The results suggest that these proteins serve roles in pathogenesis or virus survival in vivo distinct from functions in virus replication. The study also demonstrates the usefulness of the reverse genetic system to confirm the role of residues or proteins in coronavirus replication and pathogenesis.     

Clinical complications of Mycoplasma pneumoniae disease--central nervous system

The mechanism of the neurologic complications associated with primary atypical pneumonia is unknown. To examine the ability of Mycoplasma pneumoniae to enter the brain of experimental animals, the organism was inoculated into adult and suckling mice by various routes. After intranasal infection, M. pneumoniae was isolated from brains and lungs of both groups of mice. After intracerebral inoculation, the high levels of the mycoplasma persisted for two months or more in the brains of suckling mice. In addition, after intravenous infection, the systemic spread of infection occurred in the mice treated with high doses of cyclophosphamide. Our results suggest that M. pneumoniae may be able to reach the brain via blood and it may occur with relative ease in compromised hosts.     

The central nervous system-associated immune response to parainfluenza type I virus in mice.

The immune response in the cerebrospinal fluid (CSF) and serum of BALB/c mice was compared after intracerebral (i.c.) inoculation with inactivated parainfluenza type 1 virus. The antiviral antibody response in CSF peaked approximately 11 days after primary i.c. inoculation coinciding with or even slightly preceding the response in the serum. Prior extracerebral priming of the mice by the intranasal or i.v. route did not alter the kinetics of the response in CSF. However, the antibody response in CSF after i.c. inoculation was accelerated if the mice were primed previously by the i.c. route. In all instances, CSF and serum differed markedly with regard to the isotype composition, which was characterized by a 20- to 80-fold increase in IgA over IgG1 and IgG2 in CSF. Taken together, the results prove that part of the antiviral antibodies in CSF are locally produced. In addition, the results indicate that after primary i.c. inoculation with virus, the CNS acquired immunocompetence with regard to the secondary anti-parainfluenza response.     

Pathogenesis of street rabies virus infections in resistant and susceptible strains of mice.

Seven strains of mice were examined to determine why susceptibility differences and variations in clinical central nervous system (CNS) disease occurred among these animals after intraperitoneal inoculation of street rabies virus (SRV). Trace experiments for infectious virus indicated that these differences were associated with restriction of virus replication within the CNS. Limitation of viral replication appeared to correlate with the antibody response in that prominent serum anti-SRV neutralizing antibody titers were detected in resistant strains, whereas susceptible strains produced minimal amounts of antibody until their death. The importance of the immune response was reaffirmed with cyclophosphamide studies in that all resistant SJL/J mice died after immunosuppressive treatment. In contrast, cyclophosphamide-treated SJL/J mice whose immune systems were reconstituted with either unfractionated immune spleen cells or with sera 24 h after SRV inoculation survived a lethal dose of SRV. More importantly, immunosuppressed SJL/J and immunodeficient athymic mice were protected when reconstituted with immune serum 72 h after SRV inoculation, a time in which infectious virus was detected in the spinal cords of some mice but was not present in the peritoneal cavity. Additional studies showed that antibody in the cerebrospinal fluid was unimportant in the resistance of mouse strains which remained clinically asymptomatic, but it appeared to be associated with the survival of mice which developed clinical CNS disease. Furthermore, CNS resistance to intranasal or intracerebral inoculation with challenge virus standard rabies virus developed as early as 5 days post-intraperitoneal inoculation of SRV.     

Influence of Statolon on Resistance of Mice to Influenza

Various interferon inducers are known to elicit protection against lethal or infecting doses of certain viral agents. Because of the relatively high morbidity rate of influenza and its seasonal occurrence, we wished to determine whether statolon-induced interferon might be effective in controlling this disease. Mice were treated intraperitoneally with statolon and challenged with influenza A(2) virus by the intranasal route. Although interferon was present in the serum at the time of virus administration, no change in mortality rate was observed. There was, however, a significant increase in the mean survival time of treated animals. Similar results were obtained when Newcastle disease virus was used as the interferon inducer. To determine the effect of the route of challenge, other mice were treated with statolon or Newcastle disease virus and inoculated with mengovirus by the intranasal or intraperitoneal route. The results demonstrated that the treated mice were protected to similar degree against challenge by either route. It is suggested that the relative ineffectiveness of interferon in protecting mice against influenza is due to an intrinsic characteristic of the virus itself rather than the type of interferon induced or the route of virus challenge.     

Critical role for protein tyrosine phosphatase SHP-1 in controlling infection of central nervous system glia and demyelination by Theiler's murine encephalomyelitis virus

We previously characterized the expression and function of the protein tyrosine phosphatase SHP-1 in the glia of the central nervous system (CNS). In the present study, we describe the role of SHP-1 in virus infection of glia and virus-induced demyelination in the CNS. For in vivo studies, SHP-1-deficient mice and their normal littermates received an intracerebral inoculation of an attenuated strain of Theiler's murine encephalomyelitis virus (TMEV). At various times after infection, virus replication, TMEV antigen expression, and demyelination were monitored. It was found that the CNS of SHP-1-deficient mice uniquely displayed demyelination and contained substantially higher levels of virus than did that of normal littermate mice. Many infected astrocytes and oligodendrocytes were detected in both brains and spinal cords of SHP-1-deficient but not normal littermate mice, showing that the virus replicated and spread at a much higher rate in the glia of SHP-1-deficient animals. To ascertain whether the lack of SHP-1 in the glia was primarily responsible for these differences, glial samples from these mice were cultured in vitro and infected with TMEV. As in vivo, infected astrocytes and oligodendrocytes of SHP-1-deficient mice were much more numerous and produced more virus than did those of normal littermate mice. These findings indicate that SHP-1 is a critical factor in controlling virus replication in the CNS glia and virus-induced demyelination.     

Antibody is required for clearance of infectious murine hepatitis virus A59 from the central nervous system, but not the liver.

Intracerebral inoculation with mouse hepatitis virus strain A59 results in viral replication in the CNS and liver. To investigate whether B cells are important for controlling mouse hepatitis virus strain A59 infection, we infected muMT mice who lack membrane-bound IgM and therefore mature B lymphocytes. Infectious virus peaked and was cleared from the livers of muMT and wild-type mice. However, while virus was cleared from the CNS of wild-type mice, virus persisted in the CNS of muMT mice. To determine how B cells mediate viral clearance, we first assessed CD4(+) T cell activation in the absence of B cells as APC. CD4(+) T cells express wild-type levels of CD69 after infection in muMT mice. IFN-gamma production in response to viral Ag in muMT mice was also normal during acute infection, but was decreased 31 days postinfection compared with that in wild-type mice. The role of Ab in viral clearance was also assessed. In wild-type mice plasma cells appeared in the CNS around the time that virus is cleared. The muMT mice that received A59-specific Ab had decreased virus, while mice with B cells deficient in Ab secretion did not clear virus from the CNS. Viral persistence was not detected in FcR or complement knockout mice. These data suggest that clearance of infectious mouse hepatitis virus strain A59 from the CNS requires Ab production and perhaps B cell support of T cells; however, virus is cleared from the liver without the involvement of Abs or B cells.     

Passive immunization against transmissible gastroenteritis virus in piglets by ingestion of milk of sows inoculated with attenuated virus.

Pregnant sows were inoculated with the attenuated strain, TO--163, of swine transmissible gastroenteritis virus. Suckling piglets born from them received challenge inoculation with the virulent virus at 3 days after birth, and examined for ability to prevent infection and the immunoglobulin (Ig) classes of antibody in milk. A pregnant sow was inoculated intramuscularly with a dose of 10(8.0) TCID50 and intranasally with a dose of 10(9.3) TCID50 of attenuated virus. Piglets born from it suffered from diarrhea after challenge inoculation, but none of them died eventually. Their dam was also affected with diarrhea for 4 to 7 days after challenge inoculation of them. Another pregnant sow was inoculated twice with 10(9.3) TCID50 of attenuated virus, first by the intramuscular and secondly by the intranasal route. Of nine piglets born from it, one excreted soft feces after challenge inoculation, but all survived to grow normally. Their dam manifested no clinical symptoms at all after challenge inoculation of them. The higher the titer of virus inoculated into pregnant sows, the higher the neutralizing antibody titer in serum and milk of the sows after farrowing. The puerperal sow which had received two doses of 10(9.3) TCID50 each of attenuated virus by the intramuscular and intranasal route, respectively, presented the highest neutralizing antibody titer of all the inoculated sows. This titer was 2,048 in serum and 14,183 in colostrum immediately after farrowing. In that sow IgG was the main class of immunoglobulins in neutralizing antibody in milk. Even the IgA antibody titer of that sow was higher than that of any other sow which had been administered with virus of low titer. It was 392 and 19 3 and 9 days, respectively, after farrowing.