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.     

Enterotropic mouse hepatitis virus infection in nude mice

The cause of emaciation and diarrhea in athymic nude mice was found to be hyperplastic typhlocolitis resulting from infection with enterotropic mouse hepatitis virus (MHV). The disease was reproduced in experimentally-inoculated nude mice using intestinal homogenates from affected mice and cell culture-derived virus. Material derived from an experimental mouse was passed into neonatal Swiss mice and caused acute typhlocolitis. Virus failed to grow in NCTC-1469 cells and 17Cl-1 cells, which are normally permissive for MHV, but grew to low titer in a mouse rectal carcinoma cell line, CMT 93. These results show that an enterotropic strain of MHV can cause chronic enteric disease in athymic nude mice. The pattern of infection differs markedly from the more common MHV wasting syndrome in nude mice caused by non-enteric strains of MHV.     

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.     

Characterization of Omental Immune Aggregates during Establishment of a Latent Gammaherpesvirus Infection

Herpesviruses are characterized by their ability to establish lifelong latent infection. The gammaherpesvirus subfamily is distinguished by lymphotropism, establishing and maintaining latent infection predominantly in B lymphocytes. Consequently, gammaherpesvirus pathogenesis is closely linked to normal B cell physiology. Murine gammaherpesvirus 68 (MHV68) pathogenesis in laboratory mice has been extensively studied as a model system to gain insights into the nature of gammaherpesvirus infection in B cells and their associated lymphoid compartments. In addition to B cells, MHV68 infection of macrophages contributes significantly to the frequency of viral genome-positive cells in the peritoneal cavity throughout latency. The omentum, a sheet of richly-vascularized adipose tissue, resides in the peritoneal cavity and contains clusters of immune cell aggregates termed milky spots. Although the value of the omentum in surgical wound-healing has long been appreciated, the unique properties of this tissue and its contribution to both innate and adaptive immunity have only recently been recognized. To determine whether the omentum plays a role in gammaherpesvirus pathogenesis we examined this site during early MHV68 infection and long-term latency. Following intraperitoneal infection, immune aggregates within the omentum expanded in size and number and contained virus-infected cells. Notably, a germinal-center B cell population appeared in the omentum of infected animals with earlier kinetics and greater magnitude than that observed in the spleen. Furthermore, the omentum harbored a stable frequency of viral genome-positive cells through early and into long-term latency, while removal of the omentum prior to infection resulted in a slight decrease in the establishment of splenic latency following intraperitoneal infection. These data provide the first evidence that the omentum is a site of chronic MHV68 infection that may contribute to the maintenance of chronic infection.     

Demyelinating and nondemyelinating strains of mouse hepatitis virus differ in their neural cell tropism

Some strains of mouse hepatitis virus (MHV) can induce chronic inflammatory demyelination in mice that mimics certain pathological features of multiple sclerosis. We have examined neural cell tropism of demyelinating and nondemyelinating strains of MHV in order to determine whether central nervous system (CNS) cell tropism plays a role in demyelination. Previous studies demonstrated that recombinant MHV strains, isogenic other than for the spike gene, differ in the extent of neurovirulence and the ability to induce demyelination. Here we demonstrate that these strains also differ in their abilities to infect a particular cell type(s) in the brain. Furthermore, there is a correlation between the differential localization of viral antigen in spinal cord gray matter and that in white matter during acute infection and the ability to induce demyelination later on. Viral antigen from demyelinating strains is detected initially in both gray and white matter, with subsequent localization to white matter of the spinal cord, whereas viral antigen localization of nondemyelinating strains is restricted mainly to gray matter. This observation suggests that the localization of viral antigen to white matter during the acute stage of infection is essential for the induction of chronic demyelination. Overall, these observations suggest that isogenic demyelinating and nondemyelinating strains of MHV, differing in the spike protein expressed, infect neurons and glial cells in different proportions and that differential tropism to a particular CNS cell type may play a significant role in mediating the onset and mechanisms of demyelination.     

Genetic study of mouse sensitivity to MHV3 infection: influence of the H-2 complex.

Genetic study of acute and chronic mouse hepatitis virus type 3 disease was carried out in segregating generations of a cross involving a susceptible (C57BL/6) and a resistant (A/J) mouse strain. The data obtained indicate that one or two recessive genes may be involved in resistance of acute and chronic diseases but suggest that the genes involved in both diseases are different. In this cross, no correlation was observed between H-2 and acute or chronic disease. In mice of congenic lines, however, A/Sn (H-2a), A.SW (H-2s), A.BY (H-2b), and A.CA (H-2f), it appeared that the presence of the H-2f allele conferred to heterozygote as well as to homozygote animals the capacity to resist the development of chronic disease. It seems, therefore, that MHV3 sensitivity in mice is under the influence of at least two major genes: one for the acute disease and the other, H-2 linked, for the chronic disease.     

Neonatal susceptibility to MHV3 infection in mice. I. Transfer of resistance.

Up to 3 weeks of age, mice of the resistant A/J strain are fully susceptible to mouse hepatitis virus type 3 infection (MHV3). Immune deficiency, however, resulting from neonatal thymectomy or long term ALS administration led A/J animals to remain susceptible when tested at adult age. Whole spleen cells transferred from normal adult A/J donor mice protected suckling syngeneic recipients from i.p. infection with MHV3. Such a protective capacity of spleen cells was abolished after treatment with anti-theta serum and complement. Spleen cell separation by means of adherence to plastic also showed that neither the nonadherent nor the adherent populations injected separately were able to confer resistance to young mice challenged with the virus. Protection was not achieved with peritoneal cells originating from adult syngeneic animals. Transfer of resistance to MHV3 was obtained, however, when peritoneal cells were associated with adherent spleen cells. This study indicated that two types of mature cells, at least, were required for transferring MHV3 resistance into newborn mice of the A/J strain: T lymphocytes and an adherent spleen cell population.     

Lung infection with gamma-herpesvirus induces progressive pulmonary fibrosis in Th2-biased mice

Idiopathic pulmonary fibrosis (IPF) is a progressive, fibrotic lung disease of unknown etiology. A viral pathogenesis in IPF has been suggested since >95% of IPF patients have evidence of chronic pulmonary infection with one or more herpesviruses. To determine whether pulmonary infection with herpesvirus can cause lung fibrosis, we infected mice with the murine gamma-herpesvirus 68 (MHV68). Because IPF patients have a T helper type 2 (Th2) pulmonary phenotype, we used IFN-gammaR-/-, a strain of mice biased to develop Th2 responses. Chronic MHV68 infection of IFN-gammaR-/- mice resulted in progressive deposition of interstitial collagen as shown by light and electron microscopy. A significant decrease in tidal volume paralleled the collagen deposition. Five features typically seen in IPF, increased transforming growth factor-beta expression, myofibroblast transformation, production of Th2 cytokines, hyperplasia of type II cells, and increased expression of matrix metalloproteinase-7, were also present in chronically infected IFN-gammaR-/- mice. There also was altered synthesis of surfactant proteins, which is seen in some patients with familial IPF. MHV68 viral protein was found in type II alveolar epithelial cells, especially in lung areas with extensive alveolar remodeling. In summary, chronic herpesvirus pulmonary infection in IFN-gammaR-/- mice causes progressive pulmonary fibrosis and many of the pathological features seen in IPF.     

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.     

Comparative analysis of RNA genomes of mouse hepatitis viruses.

The RNA genomes of various murine hepatitis virus (MHV) strains were studied by T1-oligonucleotide fingerprinting analysis with regard to their structure and sequence relationship. It was found that the MHV particles contained only positive-stranded 60S RNA which had a "cap" structure at its 5' end. No negative-stranded RNA was found. It was also shown that most of the MHV strains had diverged quite extensively in their genetic sequences. However, MHV-3, a hepatotropic strain, and A59, a nonpathogenic strain, were found to have very similar oligonucleotide fingerprinting patterns. Yet, each of them contained two to four specific oligonucleotides. The MHV-3-specific oligonucleotides were conserved in almost all of the hepatotropic MHV strains studied. In contrast, two of the A59-specific oligonucleotides were absent from the genomes of all hepatotropic strains. These findings suggest that these unique oligonucleotides might be localized at the genetic region(s) associated with viral pathogenicity or other biological properties of the virus. Comparison of viral structural proteins also suggests that MHV-3 and A59 are more closely related than other MHV strains. The significance of these findings is discussed.     

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.     

Mouse susceptibility to mouse hepatitis virus infection is linked to viral receptor genotype.

We have reported that the receptor for mouse hepatitis virus (MHV) expressed in MHV-susceptible BALB/c mice (MHVR1) has 10 to 30 times the virus-binding activity of the MHV receptor expressed in MHV-resistant SJL mice (MHVR2) (N. Ohtsuka, Y. K. Yamada, and F. Taguchi, J. Gen. Virol. 77:1683-1992, 1996). This fact indicates the possibility that the difference in MHV susceptibility between BALB/c and SJL mice is determined by the virus-binding activity of the receptor. To test this possibility, we have examined MHV susceptibility in mice with the homozygous MHVR1 gene (R1/R1 genotype), mice with the MHVR1 and MHVR2 genes (R1/R2 genotype), and mice with the homozygous MHVR2 gene (R2/R2 genotype) produced by cross and backcross mating between BALB/c and SJL mice. All 63 F2 and backcrossed mice with the MHVR1 gene (R1/R1 and R1/R2) were susceptible to MHV infection, and all 57 with the homozygous MHVR2 gene (R2/R2) were resistant. We have also examined the MHV receptor genotypes of several mouse strains that were reported to be susceptible to MHV infection. All of those mice had the MHVR1 gene. These results suggest the possibility that the viral receptor determines the susceptibility of the whole animal to MHV infection.     

Sequential bottlenecks drive viral evolution in early acute hepatitis C virus infection

Hepatitis C is a pandemic human RNA virus, which commonly causes chronic infection and liver disease. The characterization of viral populations that successfully initiate infection, and also those that drive progression to chronicity is instrumental for understanding pathogenesis and vaccine design. A comprehensive and longitudinal analysis of the viral population was conducted in four subjects followed from very early acute infection to resolution of disease outcome. By means of next generation sequencing (NGS) and standard cloning/Sanger sequencing, genetic diversity and viral variants were quantified over the course of the infection at frequencies as low as 0.1%. Phylogenetic analysis of reassembled viral variants revealed acute infection was dominated by two sequential bottleneck events, irrespective of subsequent chronicity or clearance. The first bottleneck was associated with transmission, with one to two viral variants successfully establishing infection. The second occurred approximately 100 days post-infection, and was characterized by a decline in viral diversity. In the two subjects who developed chronic infection, this second bottleneck was followed by the emergence of a new viral population, which evolved from the founder variants via a selective sweep with fixation in a small number of mutated sites. The diversity at sites with non-synonymous mutation was higher in predicted cytotoxic T cell epitopes, suggesting immune-driven evolution. These results provide the first detailed analysis of early within-host evolution of HCV, indicating strong selective forces limit viral evolution in the acute phase of infection.     

Expression of cellular oncogene Bcl-xL prevents coronavirus-induced cell death and converts acute infection to persistent infection in progenitor rat oligodendrocytes

Murine coronavirus mouse hepatitis virus (MHV) causes persistent infection of the central nervous system (CNS) in rodents, which has been associated with demyelination. However, the precise mechanism of MHV persistence in the CNS remains elusive. Here we show that the progenitor oligodendrocytes (central glial 4 [CG-4] cells) derived from newborn rat brain were permissive to MHV infection, which resulted in cell death, although viral replication was restricted. Interestingly, treatment with fetal bovine serum or exogenous expression of cellular oncogene Bcl-xL prevented CG-4 cells from MHV-induced cell death. Significantly, overexpression of Bcl-xL alone was sufficient to convert acute to persistent, nonproductive infection in CG-4 cells. This finding indicates that intracellular factors rather than viral components play a critical role in establishing viral persistence in CNS cells. Although viral genomic RNAs continuously persisted in Bcl-xL-expressing CG-4 cells over 10 passages, infectious virus could no longer be isolated beyond 2 passages of the cell. Such a phenomenon resembles the persistent MHV infection in animal CNS. Thus, the establishment of a persistent, nonproductive infection in CG-4 cells may provide a useful in vitro model for studying viral persistence in animal CNS. The data also suggest that direct virus-host cell interaction is one of the underlying mechanisms that regulate viral persistence in CNS cells.     

Global mRNA degradation during lytic gammaherpesvirus infection contributes to establishment of viral latency

During a lytic gammaherpesvirus infection, host gene expression is severely restricted by the global degradation and altered 3' end processing of mRNA. This host shutoff phenotype is orchestrated by the viral SOX protein, yet its functional significance to the viral lifecycle has not been elucidated, in part due to the multifunctional nature of SOX. Using an unbiased mutagenesis screen of the murine gammaherpesvirus 68 (MHV68) SOX homolog, we isolated a single amino acid point mutant that is selectively defective in host shutoff activity. Incorporation of this mutation into MHV68 yielded a virus with significantly reduced capacity for mRNA turnover. Unexpectedly, the MHV68 mutant showed little defect during the acute replication phase in the mouse lung. Instead, the virus exhibited attenuation at later stages of in vivo infections suggestive of defects in both trafficking and latency establishment. Specifically, mice intranasally infected with the host shutoff mutant accumulated to lower levels at 10 days post infection in the lymph nodes, failed to develop splenomegaly, and exhibited reduced viral DNA levels and a lower frequency of latently infected splenocytes. Decreased latency establishment was also observed upon infection via the intraperitoneal route. These results highlight for the first time the importance of global mRNA degradation during a gammaherpesvirus infection and link an exclusively lytic phenomenon with downstream latency establishment.     

Identification of a novel neuropathogenic Theiler's murine encephalomyelitis virus

Theiler's murine encephalitis viruses (TMEV) are divided into two subgroups based on their neurovirulence. Persistent strains resemble Theiler's original viruses (referred to as the TO subgroup), which largely induce a subclinical polioencephalomyelitis during the acute phase of the disease and can persist in the spinal cord of susceptible animals, inducing a chronic demyelinating disease. In contrast, members of the neurovirulent subgroup cause an acute encephalitis characterized by the rapid onset of paralysis and death within days following intracranial inoculation. We report herein the characterization of a novel neurovirulent strain of TMEV, identified using pyrosequencing technology and referred to as NIHE. Complete coverage of the NIHE viral genome was obtained, and it shares <90% nucleotide sequence identity to known TMEV strains irrespective of subgroup, with the greatest sequence variability being observed in genes encoding the leader and capsid proteins. The histopathological analysis of infected brain and spinal cord demonstrate inflammatory lesions and neuronal necrosis during acute infection with no evidence of viral persistence or chronic disease. Intriguingly, genetic analysis indicates the putative expression of the L protein, considered a hallmark of strains within the persistent subgroup. Thus, the identification and characterization of a novel neurovirulent TMEV strain sharing features previously associated with both subgroups will lead to a deeper understanding of the evolution of TMEV strains and new insights into the determinants of neurovirulence.     

Duration and strain-specificity of immunity to enterotropic mouse hepatitis virus.

We examined the duration and strain-specificity of immunity to enterotropic mouse hepatitis virus (MHV). Two strains of enterotropic MHV (MHV-Y and MHV-RI) were determined to be distinct virus strains by serum neutralization and by enzyme immunoassay. BALB/cByJ mice immunized by oral infection with either MHV-Y or MHV-RI developed serum MHV IgG titers that remained stable for more than 6 months. The animals were protected from reinfection with the homologous virus strain at 1 and 6 months after an initial immunizing infection, based on intestinal histology and polymerase chain reaction for a 375-base-pair segment of the membrane glycoprotein gene. Immunity was also fully protective against challenge with the heterologous strain 1 month after initial immunization and partly protective after 6 months. Maternally-derived passive immunity prevented MHV infection in 1-week-old pups challenged with the homologous strain of MHV, and pups challenged with the heterologous virus strain were partially protected.