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.

Coronavirus-induced membrane fusion requires the cysteine-rich domain in the spike protein.

The spike glycoprotein of mouse hepatitis virus strain A59 mediates the early events leading to infection of cells, including fusion of the viral and cellular membranes. The spike is a type I membrane glycoprotein that possesses a conserved transmembrane anchor and an unusual cysteine-rich (cys) domain that bridges the putative junction of the anchor and the cytoplasmic tail. In this study, we examined the role of these carboxyl-terminal domains in spike-mediated membrane fusion. We show that the cytoplasmic tail is not required for fusion but has the capacity to enhance membrane fusion activity. Chimeric spike protein mutants containing substitutions of the entire transmembrane anchor and cys domain with the herpes simplex virus type 1 glycoprotein D (gD-1) anchor demonstrated that fusion activity requires the presence of the A59 membrane-spanning domain and the portion of the cys domain that lies upstream of the cytoplasmic tail. The cys domain is a required element since its deletion from the wild-type spike protein abrogates fusion activity. However, addition of the cys domain to fusion-defective chimeric proteins was unable to restore fusion activity. Thus, the cys domain is necessary but is not sufficient to complement the gD-1 anchor and allow for membrane fusion. Site-specific mutations of conserved cysteine residues in the cys domain markedly reduce membrane fusion, which further supports the conclusion that this region is crucial for spike function. The results indicate that the carboxyl-terminus of the spike transmembrane anchor contains at least two distinct domains, both of which are necessary for full membrane fusion.

Mouse hepatitis virus A59-induced demyelination can occur in the absence of CD8+ T cells.

Mouse hepatitis virus causes a chronic demyelinating disease in C57BL/6 mice. While early studies suggested demyelination is due to direct cytolytic effects of virus on oligodendrocytes, there is increasing evidence for the involvement of the immune system in the mechanism of demyelination. In this study we have asked whether demyelination can occur in the absence of functional MHC class I expression and CD8+ T cells. We infected transgenic mice lacking expression of beta 2 microglobulin (beta 2 M -/- mice) with MHV-A59. In beta 2M-/- mice, virus was much more lethal than in either of the parental strains used to produce the mice; furthermore, while clearance from the CNS did occur in beta 2M-/- mice, it was slower than in C57BL/6 mice. This is consistent with the importance of CD8+ cells in viral clearance. Because of the increased sensitivity of the beta 2M-/- mice to infection, only low levels of virus could be used to evaluate chronic disease. Even at these low levels, demyelination did occur in some animals. To compare infection in beta 2M-/- and C57BL/6 mice we used a higher dose of an attenuated variant of MHV-A59, C12. The attenuated variant induced less demyelination in C57BL/6 mice compared to wild type A59, but the levels observed were not significantly different from those seen in beta 2M-/- mice. Thus, MHV-induced demyelination can occur in some animals in the absence of MHC class I and CD8+ cells.

Hepatitis mutants of mouse hepatitis virus strain A59.

MHV-A59 causes acute meningoencephalitis and hepatitis in susceptible mice, and a persistent productive, but nonlytic, infection of cultured glial cells. We have shown previously that viruses isolated from persistently infected glial cell cultures have a fusion-defective phenotype and were impaired in their abilities to cause hepatitis compared to wild-type MHV-A59. Two mutants chosen for detailed study, B11 and C12, display two distinct hepatitis phenotypes. The ability of B11 to replicate in the liver was dependent on infectious dose and route of inoculation, while C12 consistently displayed decreased liver titers regardless of dose and route of inoculation. Sequence analysis of wild-type, mutant and revertant S proteins indicates that 1) a mutation in the N terminal subunit of S, resulting in a glutamine to leucine amino acid substitution (Q159L), may affect ability to cause hepatitis and 2) a cleavage site mutation (H716D) which determines fusogenicity is not responsible for the altered hepatitis phenotype. Sequence analysis indicated that hepatitis-producing revertants did not revert at mutation Q159L, although it is possible that a mutation in the heptad repeat domain of S2 may compensate for the mutation in S1. Since B11, C12 and a nonattenuated fusion mutant (B12) have identical S protein sequences, there must be additional mutations outside of S which influence both virulence and ability to replicate in the liver.

MHV-A59 fusion mutants are attenuated and display altered hepatotropism.

Mouse hepatitis virus strain A59 causes a persistent productive, but nonlytic, infection of cultured glial cells. We have mutants isolated from persistently infected glial cell cultures which have been shown to be fusion-defective due to a histidine to aspartic acid mutation (H716D) near the cleavage site of the peplomer protein, S. Here, we examine the pathogenicity of these mutants and show differences in hepatotropism and virulence compared to wild-type virus (WT). Two mutants chosen for detailed study, B11 and C12, were impaired in their abilities to cause hepatitis and/or replicate in the liver of susceptible mice. Furthermore, B11 and C12 display two separate hepatotropic phenotypes. The ability of B11 to replicate in the liver was dependent on infectious dose and route of inoculation, while C12 consistently displayed decreased hepatotropism regardless of dose and route of inoculation. However, B11 and C12 were shown to replicate in the CNS of infected animals similarly to WT. Like WT, the mutants produced meningoencephalitis during acute infection, with viral antigen exhibiting a similar distribution in the brain, and demyelination during chronic infection. Sequence analysis of wild-type, mutant, and revertant S proteins indicates that (1) a mutation in the N terminal subunit of S (S1), resulting in a glutamine to leucine amino acid substitution (Q159L), may affect hepatotropism and (2) a cleavage site mutation which determines fusogenicity is not responsible for altered hepatotropism. Furthermore, since B11, C12, and a nonattenuated fusion mutant (B12) have identical S protein sequences, there must be additional mutations outside of S which influence both virulence and hepatotropism.

Fusion-defective mutants of mouse hepatitis virus A59 contain a mutation in the spike protein cleavage signal.

Infection of primary mouse glial cell cultures with mouse hepatitis virus strain A59 results in a productive, persistent infection, but without any obvious cytopathic effect. Mutant viruses isolated from infected glial cultures 16 to 18 weeks postinfection replicate with kinetics similar to those of wild-type virus but produce small plaques on fibroblasts and cause only minimal levels of cell-to-cell fusion under conditions in which wild type causes nearly complete cell fusion. However, since extensive fusion is present in mutant-infected cells at late times postinfection, the defect is actually a delay in kinetics rather than an absolute block in activity. Addition of trypsin to mutant-infected fibroblast cultures enhanced cell fusion a small (two- to fivefold) but significant degree, indicating that the defect could be due to a lack of cleavage of the viral spike (fusion) protein. Sequencing of portions of the spike genes of six fusion-defective mutants revealed that all contained the same single nucleotide mutation resulting in a substitution of aspartic acid for histidine in the spike cleavage signal. Mutant virions contained only the 180-kDa form of spike protein, suggesting that this mutation prevented the normal proteolytic cleavage of the 180-kDa protein into the 90-kDa subunits. Examination of revertants of the mutants supports this hypothesis. Acquisition of fusion competence correlates with the replacement of the negatively charged aspartic acid with either the wild-type histidine or a nonpolar amino acid and the restoration of spike protein cleavage. These data confirm and extend previous reports concluding cleavage of S is required for efficient cell-cell fusion by mouse hepatitis virus but not for virus-cell fusion (infectivity).

Identification of peplomer cleavage site mutations arising during persistence of MHV-A59.

Primary mouse glial cell cultures were infected with mouse hepatitis virus strain A59 (MHV-A59) and maintained over an 18 week period. Viruses isolated from these cultures 16-18 weeks postinfection produce small plaques on fibroblasts and cause only minimal levels of cell-to-cell fusion at times when wild type causes nearly complete cell fusion. However, when mutant-infected cultures were examined 24-36 hours postinfection approximately 90% of the cells were in syncytia showing that the fusion defect is not absolute but rather delayed. Addition of trypsin to mutant-infected cultures enhanced cell fusion a small (2- to 5-fold) but significant degree. Sequencing of portions of the spike genes of six fusion-defective mutants revealed that all contained the same single nucleotide mutation resulting in a substitution of aspartic acid for histidine in the spike cleavage signal. Mutant virions contained only the 180 kDa form of spike protein suggesting that this mutation prevented the normal proteolytic cleavage of the 180 kDa protein into the 90 kDa subunits. Examination of revertants of the mutants supports this hypothesis. Replacement of the negatively-charged aspartic acid with either the wild type histidine or a non-polar amino acid was associated with the restoration of spike protein cleavage and cell fusion.

Mouse hepatitis virus A59 increases steady-state levels of MHC mRNAs in primary glial cell cultures and in the murine central nervous system.

Infection of mixed glial cell cultures with mouse hepatitis virus (MHV)-A59 results in an approximately six-fold increase in the level of major histocompatibility complex (MHC) class I mRNA. In situ hybridization of glial cell cultures infected with MHV-A59 again showed enhanced MHC mRNA expression, both in infected and uninfected cells. These results extend our earlier finding that MHC surface antigens are enhanced on astrocytes and oligodendrocytes after MHV-A59 infection and suggest that this enhancement is a result of an increase in the steady-state level of MHC mRNA. We further demonstrate that increases in MHC mRNA occur in the murine central nervous system (CNS) following infection in vivo. Northern blot analysis of RNA from the brains of infected animals showed transient expression of both MHC class I and class II mRNA over the first 14 days of infection. Expression coincided with viral replication and clearance. In situ hybridization of brain sections from infected animals showed that class I and class II expression was widespread throughout all portions of the brain and in uninfected as well as infected cells. Viral RNA, in contrast, was observed in small foci of cells and mostly within the limbic system. Thus enhancement of MHC mRNA was not restricted either to areas of infection or inflammation. The spatial relationship between viral and MHC expression supports our hypothesis that a soluble mediator is involved in the mechanism of the increase in MHC levels. The fact that MHC induction occurs in vivo as well as in vitro suggests MHC may be important in the mechanism of MHV-induced disease.

Rotavirus temperature-sensitive mutants are genetically stable and participate in reassortment during mixed infection of mice.

Mixed and single infections of 7-day-old suckling mice with SA11 temperature-sensitive (ts) mutants and RRV wild-type were examined to determine if selection against ts mutations occurred in the suckling mouse model. Single infections with ts mutants indicated that mutant replication was restricted relative to wild-type and that disease was similarly reduced. Revertant (ts+) progeny did not appear to be selected during infection. Mixed infection with ts mutant and RRV wild-type revealed a reduction in the replication of RRV suggesting that the ts mutants displayed an interference phenotype in vivo similar to that observed in vitro. However, reduced replication of the RRV parent in mixed infection did not result in a significant reduction in disease relative to RRV infection. When progeny from the mixed infections were isolated at the permissive temperature both ts and ts+ progeny were observed, and the genome segments of these progeny segregated in a manner consistent with the temperature phenotype of each progeny clone and the location of the ts mutation determined in vitro. Selection of ts+ progeny from mixed infected mice at nonpermissive temperature yielded either the RRV parent or ts+ reassortants. The segregation of genome segments in these ts+ reassortant progeny was consistent with the location of the ts lesion determined previously in vitro. These results indicate the following with respect to infection of suckling mice with ts mutants: (1) ts lesions are genetically stable and are not selected against during in vivo infection, (2) ts mutants cause disease with reduced severity, (3) ts mutants interfere with the replication of wild-type virus in vivo but not with the severity of disease, and (4) mixed infection of suckling mice may be useful in genetic studies with rotaviruses not adapted to growth in cultured cells.

Intracellular RNA synthesis directed by temperature-sensitive mutants of simian rotavirus SA11.

The kinetics of intracellular synthesis of single-stranded (ss) RNA and double-stranded (ds) RNA directed by prototype temperature-sensitive (ts) mutants representing the 10 mutant groups of rotavirus SA11 were examined. Cells were infected with individual mutants or wild type under one-step growth conditions and maintained at permissive temperature (31 degrees) or nonpermissive temperature (39 degrees). At various times postinfection, infected cells were pulse-labeled, ssRNA and dsRNA were purified, RNA species were resolved by electrophoresis and autoradiography, and RNA synthesis was quantitated by computer-assisted densitometry. The mutants representing all groups synthesized significantly less ssRNA and dsRNA at both 31 degrees and 39 degrees, when compared to wild type. When the ratio of synthesis at 39 degrees/31 degrees was determined for ssRNA and dsRNA of each mutant, three RNA synthesis phenotypes were evident. The tsB(339), tsC(606), and tsE(1400) mutants synthesized both ssRNA and dsRNA in a temperature-dependent manner. The group G mutant, tsG(2130), synthesized ssRNA in temperature-independent fashion but was temperature-dependent for the synthesis of dsRNA. The remaining mutants, tsA(778), tsD(975), tsF(2124), tsH(2384), tsI(2403), and tsJ(2131), synthesized both ssRNA and dsRNA in a temperature-independent fashion. The RNA synthesis phenotypes of the ts mutants are discussed in terms of what is known of the function(s) of the protein species to which ts lesions have been assigned.

Passive immunity modulates genetic reassortment between rotaviruses in mixedly infected mice.

Genetic reassortment between simian rotavirus SA11 and rhesus rotavirus (RRV) occurs with high frequency following mixed infection of nonimmune suckling mice (J. L. Gombold and R. F. Ramig, J. Virol. 57:110-116, 1986). We examined the effects of passively acquired homotypic or heterotypic immunity on reassortment in vivo. Passively immune suckling mice obtained from dams immune to either serotype 3 simian rotavirus (SA11) or serotype 6 bovine rotavirus (NCDV) were infected orally with either SA11 or RRV or a mixture of SA11 and RRV (both serotype 3 viruses). At various times postinfection, signs of disease were noted and the intestines of individual mice were removed and homogenized for titration of infectious virus and isolation of progeny plaques. Electrophoresis of genomic RNA was used to identify reassortants among the viral progeny isolated from infected animals. No reassortants (less than 0.45%) were detected among 224 clones examined from mixedly infected, homotypically immune mice. Twenty-nine reassortants (10.66%) were identified among 272 progeny clones from mixedly infected, heterotypically immune mice. Thus, reassortment was reduced more than 50-fold by homotypic immunity and approximately threefold by heterotypic immunity compared with prior data obtained from mixed infections of nonimmune mice. In addition, reassortment between SA11 and RRV in nonimmune mice was shown to be dependent on the virus dose. Taken together, these results suggest that immune responses may modulate the frequency of reassortment by reducing the effective multiplicity of infection (by neutralization or other immune mechanisms), thereby preventing efficient mixed infection of enterocytes.

Assignment of simian rotavirus SA11 temperature-sensitive mutant groups A, C, F, and G to genome segments.

Crosses were performed between prototype temperature-sensitive (ts) mutants of simian rotavirus SA11 representing reassortment groups A, C, F, and G and ts mutants of rhesus rotavirus RRV that belonged to different reassortment groups. Wild-type (ts+) reassortant progeny were identified by plaque formation at nonpermissive temperature (39 degrees), picked, and grown to high titer. The ts+ phenotype of the resulting progeny clones was verified by titration at 39 degrees and 31 degrees. The electropherotypes of the ts+ clones were determined by electrophoresis, and parental origin of each genome segment was assigned by comparison of segment mobility to parental markers. Analysis of the parental origin of genome segments in the ts+ reassortants derived from SA11 ts X RRV ts crosses revealed the following map locations of the SA11 prototype ts mutants: tsA(778), segment 4; tsC(606), segment 1; tsF(2124), segment 2; and tsG(2130), segment 6. The assignment of tsA was made on the basis of genome segment segregation in two independent crosses with each of two independent RRV ts mutants. The assignment of tsC was made on the basis of segregation in only a single cross with an RRV ts mutant; however, a larger number of progeny clones were examined from this cross. The lesion of tsF was mapped with data from three independent crosses using two different RRV ts mutants. The assignment of tsG was made on the basis of segregation in three independent crosses, two with RRV ts mutants and one with Wa. The assignments of tsA, tsC, and tsF were confirmed in crosses between RRV ts mutants representing those reassortment groups, and SA11 ts mutants in other reassortment groups.

Analysis of reassortment of genome segments in mice mixedly infected with rotaviruses SA11 and RRV.

Seven-day-old CD-1 mice born to seronegative dams were orally inoculated with a mixture of wild-type simian rotavirus SA11 and wild-type rhesus rotavirus RRV. At various times postinfection, progeny clones were randomly isolated from intestinal homogenates by limiting dilution. Analysis of genome RNAs by polyacrylamide gel electrophoresis was used to identify and genotype reassortant progeny. Reassortment of genome segments was observed in 252 of 662 (38%) clones analyzed from in vivo mixed infections. Kinetic studies indicated that reassortment was an early event in the in vivo infectious cycle; more than 25% of the progeny clones were reassortant by 12 h postinfection. The frequency of reassortant progeny increased to 80 to 100% by 72 to 96 h postinfection. A few reassortants with specific constellations of SA11 and RRV genome segments were repeatedly isolated from different litters or different animals within single litters, suggesting that these genotypes were independently and specifically selected in vivo. Analysis of segregation of individual genome segments among the 252 reassortant progeny revealed that, although most segments segregated randomly, segments 3 and 5 nonrandomly segregated from the SA11 parent. The possible selective pressures active during in vivo reassortment of rotavirus genome segments are discussed.

Assignment of simian rotavirus SA11 temperature-sensitive mutant groups B and E to genome segments.

Recombinant (reassortant) viruses were selected from crosses between temperature-sensitive (ts) mutants of simian rotavirus SA11 and wild-type human rotavirus Wa. The double-stranded genome RNAs of the reassortants were examined by electrophoresis in Tris-glycine-buffered polyacrylamide gels and by dot hybridization with a cloned DNA probe for genome segment 2. Analysis of replacements of genome segments in the reassortants allowed construction of a map correlating genome segments providing functions interchangeable between SA11 and Wa. The reassortants revealed a functional correspondence in order of increasing electrophoretic mobility of genome segments. Analysis of the parental origin of genome segments in ts+ SA11/Wa reassortants derived from the crosses SA11 tsB(339) X Wa and SA11 tsE(1400) X Wa revealed that the group B lesion of tsB(339) was located on genome segment 3 and the group E lesion of tsE(1400) was on segment 8.