The uncoupled relationship between the temperature-sensitivity and neurovirulence in mice of mutants of vesicular stomatitis virus.

Inoculation of wild-type (wt) VSV intracerebrally (i.c.) in Swiss weanling mice results in a rapidly fatal illness with death in two to three days. In contrast, i.c. inoculation of temperature-sensitive (ts) VSV mutants G3I and G22, but not ts GII or ts G4I, results in a more slowly progressive central nervous system (CNS) disease with distinct neurological signs. Studies undertaken to evaluate the neurovirulence of ts VSV mutants indicated that the ability of ts mutants to produce pathological changes in the CNS of mice appeared related to their ability to replicate to high titre in brain and spinal cord. However, replication of ts VSV mutants in brain alone was not sufficient to produce clinical illness. More importantly, the ability of ts VSV mutants to replicate at non-permissive temperatures in vitro did not appear to correlate with neurovirulence. VSV harvests from brains and spinal cords of mice infected with each of the ts mutants were temperature-insensitive. In spite of their temperature-insensitivity, the biological behaviour of viruses recovered from CNS tissues was, surprisingly, not that which was characteristic of revertant clones. Virus isolates recovered from infected CNS tissues, despite their temperature-insensitivity, behaved biologically like the orignal stocks of ts mutant virus. These data suggest that temperature-sensitivity is not directly correlated with the unique pathogenesis elicited by infection with ts VSV mutants.     

Murine central nervous system infection by a viral temperature-sensitive mutant: a subacute disease leading to demyelination.

Temperature-sensitive (ts) mutants of viruses may represent an important mechanism for viral persistence. Ts mutants of different complementation groups of vesicular stomatitus virus (VSV) have shown various disease patterns in infected mice which were at variance with the clinical and pathologic features of wild-type virus infection. To investigate whether neurovirulence of different ts mutants was dependent on the individual mutant or on the biochemical defect(s) common to all members of a complementation group, we infected mice with ts G32 VSV, a mutant of the same complementation group III as the previously described ts G31 VSV. Pathologic changes in infected mice were sharply different from those produced by ts G31 VSV and actually similar to those produced by ts G41 VSV, a member of Complementation Group IV, also previously described. These results suggest that the biologic behavior of ts mutants is dependent on the individual characteristics of each mutant. The most important alterations by ts G32 VSV were in the white matter of brain and spinal cord, where extensive inflammatory demyelination was observed. Lack of inflammation and demyelination in similarly infected nude mice would suggest that, in this infection, demyelination is produced by the host immune response rather than by direct viral myelinolytic activity. Such findings are similar to those we described in other viral infections and support the hypothesis of a common host-mediated pathway leading to demyelination in a variety of unrelated viral infections. These conclusions may have relevance to human demyelinating diseases.     

Comparison of central nervous system disease produced by wild-type and temperature-sensitive mutants of vesicular stomatitis virus.

The pathogenicity of infection produced following intracerebral (i.c.) inoculation of wild-type vesicular stomatitis virus (VSV) or temperature-sensitive (ts) mutants of VSV was compared. ts mutants used were ts 31 (VSV complementation group II) and ts 41 (VSV complementation group IV). The i.c. injection of wild-type VSV in weanling Swiss mice produced a rapidly fatal encephalitis with death of mice in 2 to 3 days. Histopathologically, such mice exhibited minimal changes of encephalitis on light microscopy. In contrast to the highly virulent, rapidly fatal central nervous system (CNS) infection seen after i.c. inoculation of wild-type VSV, infection with ts 31 VSV produced a more slowly progressive CNS infection characterized by hind limb paralysis and death 6 to 9 days after infection. Histopathologically, CNS infection with ts 31 is associated with previously unreported extensive spongiform changes in the gray matter of the spinal cord. The inoculation of ts 41 i.c., on the other hand, did not result in either clinical illness or histopathological changes in the spinal cords or brains of infected mice. The absence of clinical and histopathological lesions following i.c. infection of ts 41 VSV suggests that the capacity to alter the pathogenesis of VSV CNS infection may be a function of only certain ts mutants of VSV.     

An ultrastructural study of central nervous system disease produced by wild-type and temperature-sensitive mutants of vesicular stomatitis virus.

Outbred Swiss mice 3 to 4 weeks of age were injected intracerebrally with wild-type vesicular stomatitis virus or its temperature-sensitive (ts) mutants ts 11, ts 22, ts 31, and ts 41. Brain and spinal cord were then studied for pathologic changes by electron microscopy. All mice infected with wild-type vesicular stomatitis virus died within 2 days of inoculation. Diffuse ependymal alterations often culminating in necrosis in brain and especially spinal cord and rare foci of necrosis and mononuclear cell infiltration in the injected hemisphere were the main pathologic changes in these animals. In contrast, mice infected intracerebrally with ts 22 and ts 31 showed their first clinical signs, consisting of hind limb paralysis, on day 4 and did not die until day 7 or 8 after inoculation. Ependymal alterations were of less severe degree in these animals, whereas the most striking changes were those of status spongiosus limited to the gray matter of the spinal cord. Such status spongiosus was mainly due to ballooning of dendrites and astrocytic processes, although myelin and neurons were also occasionally involved. Mice infected with ts 11 and ts 41, on the other hand, remained clinically well and failed to show significant pathologic features at 4 and 8 days after intracerebral inoculation. This study would indicate that some ts mutants of vesicular stomatitis virus are capable of altering the fulminating disease produced by the parent virus and of producing strikingly different pathologic changes.     

Status spongiousus resulting from intracerebral infection of mice with temperature-sensitive mutants of vesicular stomatitis virus.

Mice infected intracerebrally (i.c.) with wild-type (wt) VSV or temperature-sensitive (ts) mutants, ts 11, ts 22, ts 31 and ts 41, were studied for the development of histopathological lesions in the central nervous system (CNS). Mice infected i.c. with wt VSV exhibited histopathological lesions consisting principally of occasional foci of perivascular mononuclear cell infiltration and rare foci of necrosis. All wt VSV infected mice died within 2 days of i.c. inoculation. In contrast, mice infected i.c. with ts 22 and ts 31 developed spongiform lesions limited to the grey matter of the spinal cord beginning 4 days after inoculation. The spongiform lesions rapidly spread to involve the entire grey matter of the spinal cord by 5-7 days after infection. Vacuolar changes were restricted principally to neuronal processes and astrocytes. Ts 22 and ts 31 infected mice developed neurological illness beginning 4 days after infection and the majority of mice died by 7 days after infection. Mice infected with ts 11 and ts 41, on the other hand, remained clinically well and were devoid of neuro-pathological lesions at 4 and 8 days after infection.     

In vivo assembly and maturation of vesicular stomatitis virus.

Previous studies on vesicular stomatitis virus (VSV) maturation in infected cells have utilized in vitro cell cultures. The present study is, to our knowledge, the first in vivo analysis of VSV-cell interaction in the central nervous system of weaning outbred Swiss mice. Intracerebral inoculation of wild-type VSV resulted in rapid viral replication in brain and spinal cord. By immunoflourescence, viral antigens were first seen in ependymal cells of brain and spinal cord and soon thereafter in surrounding neurons. The large anterior horn neurons of spinal cord appeared to be in the most heavily infected. Ultrastructurally, VSV-neuron interaction evolved in three phases. The first phase consisted of viral entry into the cell by fusion and viropexis. The second phase was characterized by nucleocapsid accumulation and resulted in the appearance of large cytoplasmic inclusions. The third phase was maturation and release from the infected cell and was accomplished by viral budding from plasma membranes. Degenerative changes in infected neurons were generally absent. Cells in the area of the central canal seemed to present a different pattern of virus-cell interaction especially at the level of maturation and release. Some of these cells in advanced stages of degeneration showed viral particles free in nucleocapsid material with no virus-membrane association. Viral budding was not observed and, because these cells do eventually die, it is possible that virus was released in the intercellular space at the moment of cellular disruption. These results suggest that VSV-cell interactions may vary depending upon the nature of the infected cells.     

Ultrastructural-immunohistochemical evidence for a maturation defect of temperature-sensitive G31 vesicular stomatitis virus in murine spinal cord neurons.

Ultrastructural immunoperoxidase studies were done in spinal cords of mice infected with wild type vesicular stomatitis virus or its temperature-sensitive (ts) mutant G31. Infected neurons showed subplasmalemmal staining of viral antigen and staining of viral particles budding from the neuronal membrane in wild-type vesicular stomatitis virus infection, whereas diffuse membrane and cytoplasmic staining with no budding virus was observed in ts G31 infection. Such findings suggest rapid viral assembly and release of viral particles from cells infected with wild-type virus. In contrast, maturation of ts G31 appears defective, and this would lead to accumulation of viral antigen in the cytoplasm of infected cells. These results correlate with studies in neuroblastoma cells which investigated the growth cycles of wild type, ts G31, and the spinal cord isolate of ts G31 as well as the viral protein-synthetic capacity of these viruses.     

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.     

Enhancement of Neisseria meningitidis infection in mice by addition of iron bound to transferrin.

Weanling mice were inoculated intracerebrally with selected vesicular stomatitis virus (VSV) complementation group II and III temperature-sensitive (ts) mutants. Of the VSV ts mutants studied, only ts G32, a group III complementation mutant, appeared neurovirulent. Interestingly, neither the capacity to replicate in central nervous system tissue nor the ability to replicate in certain neurally derived continuous cell lines at semipermissive or nonpermissive temperatures appeared different among the VSV ts mutants employed. Finally, the pathological alterations in central nervous system tissue produced by VSV ts G32 were entirely different than those produced by G31 VSV ts in the group III mutant. These studies support the hypothesis that both the virological and neuropathological features produced by different VSV ts mutants are dependent upon the unique characteristics of each mutant, rather than upon a common biochemical defect shared by all members of a complementation group.     

The isolation of interferon-inducing mutants of vesicular stomatitis virus with altered viral P function for the inhibition of total protein synthesis

We have previously reported that T1026, a temperature-sensitive (ts) noncytocidal mutant of VSV, and its ts revertant, T1026-R1, are nonconditional mutants in the VSV function "P" for the inhibition of total protein synthesis (viral plus cellular) in infected cells (C. P. Stanners, A. M. Francoeur, and T. Lam, 1977, Cell 11, 273-281; C. P. Stanners, S. Kennedy, and L. Poliquin, 1987, Virology 160, 255-258). We have also shown that P- mutants such as these are superior interferon inducers relative to their parental P+ wild-type virus, HR, and that P- mutants may be distinguished from P+ virus using the plaque interferon production of PIF assay. (A. M. Francoeur, T. Lam, and C. P. Stanners, 1980, Virology 105, 526-536). In order to carry the analysis of VSV P function further, a number of independent mutants in the VSV P function are required. We show here that the PIF assay may be used to isolate spontaneously occurring interferon-inducing mutants (PIF+ mutants) from wild-type VSV (PIF- virus) populations. About one-half of the PIF+ mutants isolated with the PIF assay were found to have alterations in the VSV P function. As well as mutants that were defective for the inhibition of total protein synthesis, the assay yielded a new class of VSV P function mutants which appear to inhibit protein synthesis more severely than does P+ virus. The majority of newly isolated PIF+ mutants was also found to be temperature sensitive for growth. The ts phenotype, however, could be reverted for most PIF+ mutants with little effect on the PIF or P phenotype. These findings show that interferon induction and P function are related functions of VSV; this fact has allowed the isolation of a repertoire of mutants with widely varying P function.     

Attenuation of Recombinant Vesicular Stomatitis Viruses Encoding Mutant Glycoproteins Demonstrate a Critical Role for Maintaining a High pH Threshold for Membrane Fusion in Viral Fitness

A plasmid-based recovery system was used to generate four unique vesicular stomatitis virus (VSV) mutants that encode glycoproteins (G proteins) with single or double amino acid substitutions in two conserved acidic residues adjacent to the putative G protein fusion domain. Previously we demonstrated that three of the mutant G proteins (D137-L, E139-L, and DE-SS) have slightly reduced pH thresholds for membrane fusion activity. In this report we show that even though the viruses encoding D137-L, E139-L, and DE-SS were recovered with high efficiency, these mutants were attenuated for growth in cell culture. Plaque formation was significantly delayed with these mutants and the plaques were smaller and more diffuse than those produced by wild-type VSV. In addition, cells infected with these mutants produced approximately 5- to 10-fold less infectious virus than cells infected with a similarly recovered VSV encoding the wild-type G protein. Using R18-labeled virus we found that the mutant G proteins had approximately 50% of the fusion activity of wild-type G at pH 6.3 and only 75% activity at pH 5.8. We also show that the mutant viruses were more sensitive to chloroquine inhibition of infection than either wild-type VSV or the mutant E139-T, which has a fusion phenotype similar to wild-type G protein. Reduced fusion activity and attenuation of infectivity was not due to differences in the amount of G protein incorporated into virions, nor to differences in the amount of virus binding to cells at physiological pH. Although infectivity was assayed at neutral pH, we observed an increase in virus binding with both mutant and wild-type virions as the pH was lowered, and the increase in binding occurred near the pH threshold for membrane fusion activity. From these data we propose a model in which VSV entry involves an increase in virus binding to the inner leaflet of the endosomal membrane during endosome acidification. Concomitant with this higher affinity binding, G protein becomes primed to initiate fusion of the viral envelope with the endosomal membrane. Viruses with mutations that delay the onset of increased binding and fusion lag behind wild-type VSV in their ability to initiate a productive infection, potentially because the location within the cytoplasm where these viruses ultimately fuse is not optimal for either virus uncoating or replication of the viral genome.     

Pathogenicity and immunogenicity for mice of temperature-sensitive mutants of vesicular stomatitis virus

Temperature-sensitive (ts) mutants of vesicular stomatitis (VS) virus were tested for their pathogenicity and immunogenicity in weanling mice. Compared with the wild-type virus (ts(+)), ts mutants representing genetic complementation groups I, II, and IV were considerably less pathogenic for mice infected by the intracerebral route and caused few deaths after intranasal inoculation. Mice were completely resistant to ts(+) and ts mutants by the intraperitoneal route. Resistance to intracerebral challenge with ts(+) VS virus was only minimal in mice vaccinated intraperitoneally with ts(+) or ts mutants and only moderate in mice vaccinated intranasally with three ts mutants. Intranasal vaccination, particularly with group IV mutants, resulted in solid immunity within 3 days to intranasal challenge with ts(+) virus. VS viral neutralizing antibody was present in the bronchial secretions of mice by 12 h after intranasal inoculation of mutant ts IV44; the bronchial antibody titers declined to undetectable levels between 3 and 7 days after vaccination. Neutralizing antibody was detected in the serum of mice by the third day after intranasal vaccination with ts IV44 and persisted at high level for at least 11 days. Certain classes of ts mutants would appear to be promising candidates for use as attenuated, live virus vaccines.     

Sequential disassembly of the cytoskeleton in BHK21 cells infected with vesicular stomatitis virus

The cytopathic effects of vesicular stomatitis virus (VSV) that result in the rounding of BHK21 cells have been studied. The results indicate that they are mediated by a sequential alteration in the distribution of the components of the cytoskeleton, an effect that requires the expression of the viral L protein. The constituents of the cytoskeleton of BHK21 cells were analyzed by fluorescence microscopy. Actin filaments were the first component to become disorganized, so that disassembly of stress fibers were detected 1 hr after infection. The distribution of microtubules and intermediate filaments was unchanged at 2 hr after infection; however, both these cytoskeletal elements exhibited an altered distribution at 3-4 hr after infection. Actinomycin D and cycloheximide did not cause the same effects as infection with VSV, suggesting that inhibition of host-cell gene expression was not responsible. However, viral gene expression was required, since cells infected with uv-irradiated VSV showed the same distribution of cytoskeletal constituents as mock-infected controls. Cells infected at 39.5 degrees (the nonpermissive temperature) with mutants of VSV temperature sensitive in the viral NS (ts G22), N(ts G41), M(ts 0 23), and G(ts 0 45) proteins showed the same changes in the cytoskeleton as those detected with wild-type virus. In contrast, cells infected with ts G11 (L-) showed the characteristic effect of VSV on the cytoskeleton when incubated at 34 degrees (the permissive temperature), but not when incubated at 39.5 degrees. The T-1026 R1 mutant of VSV, which has a much less dramatic effect on cell morphology than wild-type virus, also caused a less marked disruption of the cytoskeleton.     

Cellular quiescence modulates and replication of L15 mutants of vesicular stomatitis virus

Infectious particle production by temperature-sensitive (ts) mutants of vesicular stomatitis virus (VSV) was measured in a variety of different host cell types maintained in a state of quiescence or stimulated to proliferate. At permissive temperatures, all ts mutants and the wild-type virus replicated equally well and with the same kinetics in both quiescent and proliferating cells. At semi-permissive temperatures, however, Lts mutants, with temperature-sensitive virion polymerases, showed a delay of about 6 h in infectious particle production relative to wild-type virus in proliferating cells and greater than 16 h in quiescent cells. The effect was specific for the Lts class of mutants and was not seen for representative mutants in any of the four other complementation groups of VSV. Regarding cellular determinants, the effect was correlated only with the growth phase and not with the species of origin, interferon inducibility or with malignant transformation.     

Noninflammatory spongiform polioencephalomyelopathy caused by a neurotropic temperature-sensitive mutant of Moloney murine leukemia virus TB.

Newborn inbred CFW/D mice were inoculated intraperitoneally with ts1, a neurotropic temperature-sensitive mutant of Moloney murine leukemia virus TB (MoMuLV-TB), with the parental wild type (wt) MoMuLV-TB, or with culture medium. A progressive symmetric hindlimb paresis that progressed to paralysis was observed in ts1-infected mice. Wt-infected mice and control mice had no neurologic signs. The severity and progression of neurologic signs correlated with the location, development, and progression of lesions. Lesions consisted of neuronal and glial cell vacuolization in the brain and the anterior horn of the spinal cord, spongiform change in the associated neuropil, spongiform change in lateral and ventral funiculi, and late fibrillary gliosis in the brainstem. There was no inflammation. Lesions were symmetric, increased in severity with time, and consistently arose at specific times in specific nuclei and areas of the brain and spinal cord. Similar, but less severe, histologic lesions were observed in corresponding areas of the central nervous system from wt-infected mice. Ultrastructurally, neuronal and glial cell vacuolization in ts1-infected mice at 31 days after inoculation was caused by dilatation of the endoplasmic reticulum and Golgi complex. Virions were observed in extremely low numbers predominantly in extracellular space and budding from membranes of neurons and glial cells. Virions were not observed in the endoplasmic reticulum or Golgi complex of neurons, nor were there cytoplasmic vacuoles that contained abnormal virions.     

Apoptosis induction after herpes simplex virus infection differs according to cell type in vivo

We compared apoptosis induction in mice following three routes of infection. After intravenous infection, wild-type herpes simplex virus (HSV) types 1 and 2 and US3Δ mutants infected the adrenal gland and caused apoptosis. Corneal infection with wild-type virus resulted in apoptosis in a fraction of infected epithelium cells. Interestingly, many uninfected cells were apoptotic in the retina. Although neurons in the trigeminal ganglion were heavily infected, no apoptotic neurons were observed. Intracranial infection with wild-type virus resulted in HSV-infected cells inside the brain; however, most of the infected neurons escaped apoptosis. In contrast, infection with US3Δ and γ₁34.5Δ mutants caused apoptosis in infected neurons. Cleaved caspase-8 and p53 were detected in apoptotic cells in the adrenal gland and the brain; however, phospho-JNK was detected only in apoptotic cells of the brain. These results suggest that the activation of apoptotic signaling proteins differs depending on the host cell type and modulates the induction of apoptosis in HSV-infected cells.     

Evaluation of five temperature-sensitive mutants of respiratory syncytial virus in primates: II. Genetic analysis of virus recovered during infection.

Five temperature-sensitive (ts) mutants of respiratory syncytial (RS) virus (ts-1, ts-1 NG-1, ts-1 NG-16, ts-2, and ts-7), previously evaluated forinfectivity and virulence in chimpanzees and owl monkeys, were also assayed for in vivo genetic stability. None of the five mutants tested was completely stable genetically. Thus, virus which had lost some or all of the ts property was recovered from each infected chimpanzee. Significantly, each ts-1 NG-1 isolate retained some degree of temperature sensitivity and hence was not true wild-type virus. Clonal analysis of viruses shed by ts-1, ts-1 NG-1, ts-1 NG-16, or ts-7 infected chimpanzees indicated that in most instances only a minority of the virus shed was altered genetically. Of five chimpanzees infected with the ts-2 mutant, three shed only ts virus, and the remaining two chimpanzees shed only ts+ virus. Such ts+ virus proved to be avirulent when evaluated in chimpanzees or owl monkeys, indicating that loss of the ts property did not restore virulence. Based upon these findings, the ts-2 mutant appears to be a suitable candidate for clinical trials in man.     

Temperature-sensitive mutants of mouse hepatitis virus strain A59: isolation, characterization and neuropathogenic properties

Twenty 5-fluorouracil-induced temperature-sensitive (ts) mutants of mouse hepatitis virus strain A59 were isolated from 1284 virus clones. Mutants were preselected on the basis of their inability to induce syncytia in infected cells at the restrictive temperature (40 degrees) vs the permissive temperature (31 degrees). Of these mutants, only those with a relative plating efficiency 40 degrees/31 degrees of 3 x 10(-3) or smaller were kept. Virus yields at 40 degrees compared to 37 degrees and 31 degrees (leakiness) were determined. Most mutants (16) were RNA-, i.e., unable to synthesize virus-specific RNA at the restrictive temperature. The other four were RNA+. No qualitative differences were detected in the virus-specific RNAs in cells infected with RNA+ ts-mutants, both at 31 degrees and 40 degrees. Virus-specific proteins present in cells infected with ts-171 (RNA-) and the RNA+-mutants (ts-43, ts-201, ts-209, and ts-279) were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitates. No qualitative differences in the pattern of virus-specific cellular proteins were detected among the mutants except for an additional polypeptide of about 46,000 daltons in ts-209-infected cells. Finally, the neuropathogenic properties of eight of the mutants were investigated. Whereas 10(2) PFU of wild-type virus injected intracerebrally killed 50 to 100% of 4-week-old Balc/c mice within 1 week, the mutants were highly attenuated. A dose of 10(5) PFU lead to no or transient disease. However, 4 weeks after infection with ts-342, ts-43, or ts-201 obvious histological changes were observed in brain and spinal cord of clinically healthy mice.     

Neural pathway of Powassan virus spread in the central nervous system of white mice]

Electron microscopic investigation of the brains and lumbar spinal cords of adult albino mice infected with Powassan virus was carried out. Virus particles were found within all parts of neurons (perikarya, dendrites, axon), as well as within synaptic apparatus and intercellular gaps of the central nervous tissue. The possibility of the virus spread both throughout the cytoplasm of nerve cells and their processes and the extracellular spaces of the brain was confirmed. Localization of virions within neurons, synapses and myelinated fibers of the spinal cord after intracerebral inoculation suggests that virus spread in the CNS can occur through the CNS parenchyma and also through the nervous conduction pathways. The possible mechanisms of virus dissemination in the CNS of albino mice with experimental Powassan virus encephalomyelitis are discussed.