Subacute infection with temperature-sensitive vesicular stomatitis virus mutant G41 in the central nervous system of mice. II. Immunofluorescent, morphologic, and immunologic studies.

Inoculation of three- to four-week-old BALB/c mice with temperature-sensitive (ts) vesicular stomatitis virus (VSV) mutant G41 produced a subacute neurological disease mainly localized in the spinal cord. Meningitis and diffuse microglial infiltration of the anterior horns of the spinal cord were seen starting six days after infection when neuronal degenerative changes could be seen. Infection of neurons was demonstrated by immunofluorescence microscopy five days after infection. Two to three weeks after infection, loosening of the neuropil was evident due to neuronal dropout, and the mononuclear infiltration had become perivascularly distributed and had changed in character because of a striking increase in plasma cells. These cells together with Russell bodies became the main inflammatory cellular component about four to five weeks after viral inoculation. Starting eight days after infection, several foci of primary demyelination could be found in the anterior columns of the spinal cord. Immunological responses appeared within four days after infection when both neutralizing antibody and stimulation of specific spleen lymphocytes could be demonstrated. Serum antibody responses peaked at 21-28 days but remained elevated for up to 153 days. Stimulation of spleen lymphocyte cells peaked at 10-21 days and also remained elevated for as long as 116 days. The presence of both inflammatory changes and immunological responses to VSV mutant ts-G41 for prolonged periods is characteristic of persistent viral infections. Infection of BALB/c mice with ts-G41 thus represents the first in vivo example of persistent viral infection utilizing ts mutants of VSV.     

The effect of defective-interfering Semliki Forest virus on the histopathology of infection with virulent Semliki Forest virus in mice

The majority of mice inoculated with a mixture of a lethal dose of virulent Semliki Forest virus (SFV) strain ts+ and defective-interfering (DI) SFV remained completely health, with virus infectivity levels in brain tissue reduced by 99.9%. The results of previous studies had suggested that these effects were primarily the result of the intrinsic interfering capacity of DI virus rather than of host defense responses. Because SFV strain ts+ and an avirulent strain of SFV have clearly distinguishable histopathologic effects in brain tissue, the capability of DI virus to change the virulent into the avirulent form of the disease was examined. Modulation of strain ts+ virus infection by DI virus was accompanied by a complete absence of histopathologic changes despite significant levels of infectious virus and thus differed qualitatively from infection with avirulent SFV. These results provide further evidence that the interference is not mediated through stimulation of an immune cell infiltration.     

The influence of defective-interfering particles of the PR-8 strain of influenza A virus on the pathogenesis of pulmonary infection in mice.

Homologous autointerference mediated by defective-interfering (DI) particles was first described with the PR-8 strain of influenza virus. However, little is actually known about the influence of DI particles of influenza virus on the pathogenesis of pulmonary infection. The present studies were designed to determine (1) the requirements for successful autointerference in vivo with DI particle-enriched PR-8 influenza virus and (2) the effects of DI particle-enriched virus on the development and progression of otherwise lethal pulmonary infection in mice. PR-8 influenza virus passaged in chicken eggs, but not that passaged in Madin-Darby bovine kidney cells, and enriched in DI particles markedly attenuated pulmonary infection in seven-week-old Swiss and four-week-old C57B16/Cr mice but not in three- to four-week-old Swiss mice. In addition, replication of influenza virus, straining of viral antigen, and numbers of infiltrates in lungs of mice infected with DI particle-enriched influenza virus were reduced in comparison with values in mice infected with comparable amounts of wild-type influenza virus. The protection mediated by DI particle-enriched virus appeared to be related to augmented humoral immune responses in infected mice rather than to autointerference with replication of wild-type virus.     

Filovirus infection of STAT-1 knockout mice

We evaluated the susceptibility to Ebola and Marburg virus infection of mice that cannot respond to interferon (IFN)-α/β and IFN-γ because of deletion of the STAT-1 gene. A mouse-adapted Zaire ebolavirus (ZEBOV) caused rapidly lethal disease; wild-type ZEBOV and Sudan Ebolavirus and 4 different Marburg virus strains produced severe, but more slowly progressive illness; and Reston Ebolavirus caused mild disease that was late in onset. The virulence of each agent was mirrored by the pace and severity of pathologic changes in the liver and lymphoid tissues. A virus-like particle vaccine elicited strong antibody responses but did not protect against mouse-adapted ZEBOV challenge.     

Use of temperature-sensitive mutants of mouse cytomegalovirus as vaccines

Three temperature-sensitive (ts) mutants of mouse cytomegalovirus (MCMV) were compared with parent virus for their ability to produce acute infection, to stimulate protection against a lethal challenge with salivary gland MCMV, and to become latent and then be reactivated. During the acute phase of infection, ts mutant virus demonstrated very limited replication. During the later phase of infection (seven to 14 days after challenge), titers of virus in the pancreas and salivary glands in mice infected with parent virus continued to rise, whereas no virus could be detected in mice infected with the ts mutants. Prior infection with parent virus or the ts mutants protected susceptible mice from a lethal dose of salivary gland MCMV. One year after infection, treatment of mice with an immunosuppressive regimen resulted in reactivation of parent and ts mutant virus. Reactivated virus recovered from mice infected with ts mutant virus remained temperature sensitive.     

Modulation of Semliki Forest virus-induced infection of mice by defective-interfering virus

Preparations of defective-interfering (DI) Semliki Forest virus (SFV) differed qualitatively in their ability to protect mice against lethal SFV-induced encephalitis. The preparations fell into three categories: (1) DI virus p13a protected the majority of mice and left them immune to subsequent challenge with 100 50% lethal doses of SFV; (2) DI virus p4 protected mice to a similar extent, but the susceptibility of all surviving mice to challenge suggested that the protection was mediated without the intervention of the adaptive immune response; and (3) DI virus p5 did not protect mice even though its interference titer was similar to that of the protective preparations. How DI viruses p4 and p13a modulate this lethal infection is not clear; the failure of p4 to stimulate protective immunity suggests that nonadaptive host responses are important, but neither p4 nor p13a altered the course of infection with a heterologous neurotropic virus. Compared with avirulent SFV infection, both DI virus-modulated infections were poorly immunogenic with regard to the humoral immune response, although a minority of mice did have high levels of neutralizing antibody. Other, unknown factors are evidently at work and remain to be elucidated.     

Vesicular stomatitis virus glycoprotein is necessary for H-2-restricted lysis of infected cells by cytotoxic T lymphocytes.

Vesicular stomatitis virus (VSV) elicited cytotoxic thymus-derived lymphocytes (CTLs) in mice of the BALB/c and three congenic strains (BALB.b, BALB.k, BALB.HTG). CTL lysis of VSV-infected fibroblasts from the four strains was restricted by the target cells' major histocompatibility complex (H-2). Target cells were also infected with two temperature-sensitive mutants of VSV, tsM and tsG in which, respectively, the viral matrix protein and glycoprotein are not expressed at 39 degrees (restrictive temperature) on the infected cell's surface membrane. At the restrictive temperature, cells infected with wild-type VSV or tsM were lysed by CTLs, but cells infected with tsG were not. The requirement for the glycoprotein on the target cell was also evident from the ability of antisera to the glycoprotein to block completely CTL lysis of VSV-infected cells.     

Role of early genes in pathogenesis of adenovirus pneumonia.

Intranasal inoculation of type 5 adenovirus into the cotton rat Sigmodon hispidus produces a pneumonia pathologically similar to that in humans, and it, therefore, provides an excellent animal model to investigate the pathogenesis of this disease. The goal of this study was to test the hypothesis that accumulation of viral structural proteins is responsible for a major portion of the cell-damage-producing disease. Since viral DNA replication is essential for synthesis of the viral structural proteins, which are products of late genes, the hypothesis was tested using mutants defective in genes required for DNA synthesis. Most experiments were done with the conditionally lethal temperature-sensitive (ts) mutant H5ts125, which contains a mutation in the early region 2A (E2A) gene encoding the DNA-binding protein. The data show that infection with 1 x 10(9.0) plaque-forming units of H5ts125 induced a pneumonia that was as extensive and qualitatively the same as that after wild-type adenovirus type 5 infection, although H5ts125 did not replicate to produce infectious virus. When cotton rats were infected with 1 x 10(8.0) plaque-forming units of wild-type adenovirus type 5 or H5ts125, the pneumonias that followed were pathologically similar; in the latter phases, however, wild-type virus produced slightly more extensive pneumonia than did H5ts125, probably because its replication permitted infection of more susceptible cells.     

Subacute infection with temperature-sensitive vesicular stomatitis virus mutant G41 in the central nervous system of mice. I. Clinical and virologic studies.

Inoculation of three- to four-week-old BALB/c mice with temperature-sensitive (ts) vesicular stomatitis virus mutant G41 produced a subacute neurological disease, initially characterized by development of lethargy, hunched posture, and ruffled fur within five to seven days after infection. More than 90% of infected mice developed these clinical signs. In approximately 60% of infected mice, the initial neurological signs proceeded to striking hind-limb paralysis and weight loss. These signs usually appeared by seven to nine days after infection and lasted for 21-28 days. Only 16% of the mice died as a result of infection; death usually occurred eight to 12 days after infection. Most of the infected mice recovered from the acute phase of disease and appeared normal by four weeks after infection. However, hind-limb paralysis persisted in 4% of the mice for as long as the mice were observed, i.e., 42 days. The mutant ts-G41 was recovered from the brains and spinal cords of infected mice for the first seven days after infection. Peak titers of virus were modest, 10(4)-10(5) pfu/ml in brain tissue and 10(3)-10(4) pfu/ml in spinal cord tissue. Virus isolated after in vivo infection was temperature-sensitive and thus not revertant wild-type virus. Although virus was recoverable by homogenization for only the first seven days of infection, use of cocultivation techniques permitted the detection of ts-G41 in brains and spinal cords of infected animals for as long as 21 days after infection. Virus recovered by cocultivation was also temperature-sensitive.     

The locus coeruleus neurotoxin,DSP4, and/or a high sugar diet induce behavioral and biochemical alterations in wild-type mice consistent with Alzheimers related pathology

Alzheimer’s disease (AD) is the sixth leading cause of death in the United States where it is estimated that one in three seniors dies with AD or another dementia. Are modern lifestyle habits a contributing factor? Increased carbohydrate (sugar) consumption, stress and disruption of sleep patterns are quickly becoming the norm rather than the exception. Interestingly, seven months on a non-invasive high sucrose diet (20% sucrose in drinking water) has been shown to induce behavioral, metabolic and pathological changes consistent with AD in wild-type mice. As chronic stress and depression are associated with loss of locus coeruleus (LC) noradrenergic neurons and projections (source of anti-inflammatory and trophic factor control), we assessed the ability for a selective LC neurotoxin (DSP4) to accelerate and aggravate a high-sucrose mediated AD-related phenotype in wild-type mice. Male C57/Bl6 mice were divided into four groups: 1) saline injected, 2) DSP4 injected, 3) high sucrose drinking water (20%) or 4) DSP4 injected and high sucrose drinking water. We demonstrate that high sucrose consumption and DSP4 treatment promote an early-stage AD-related phenotype after only 3–4 months, as evidenced by elevated fecal corticosterone, increased despair, spatial memory deficits, increased AChE activity, elevated NO production, decreased pGSK3β and increased pTau. Combined treatment appears to accelerate and aggravate pathological processes consistent with Alzheimer disease and dementia. Developing a simple model in wild-type mice will highlight environmental and lifestyle factors that need to be addressed to slow, prevent or even reverse the rising trend in dementia patient numbers and cost.     

Cells that express all five proteins of vesicular stomatitis virus from cloned cDNAs support replication, assembly, and budding of defective interfering particles.

An alternative approach to structure-function analysis of vesicular stomatitis virus (VSV) gene products and their interactions with one another during each phase of the viral life cycle is described. We showed previously by using the vaccinia virus-T7 RNA polymerase expression system that when cells expressing the nucleocapsid protein (N), the phosphoprotein (NS), and the large polymerase protein (L) of VSV were superinfected with defective interfering (DI) particles, rapid and efficient replication and amplification of (DI) particle RNA occurred. Here, we demonstrate that all five VSV proteins can be expressed simultaneously when cells are contransfected with plasmids containing the matrix protein (M) gene and the glycoprotein (G) gene of VSV in addition to plasmids containing the genes for the N, NS, and L proteins. When cells coexpressing all five VSV proteins were superinfected with DI particles, which because of their defectiveness are unable to express any viral proteins or to replicate, DI particle replication, assembly, and budding were observed and infectious DI particles were released into the culture fluids. Omission of either the M or G protein expression resulted in no DI particle budding. The vector-supported DI particles were similar in size and morphology to the authentic DI particles generated from cells coinfected with DI particles and helper VSV and their infectivity could be blocked by anti-VSV or anti-G antiserum. The successful replication, assembly, and budding of DI particles from cells expressing all five VSV proteins from cloned cDNAs provide a powerful approach for detailed structure-function analysis of the VSV gene products in each step of the replicative cycle of the virus.     

Interruption of ganglioside synthesis produces central nervous system degeneration and altered axon-glial interactions

Gangliosides, which are sialylated glycosphingolipids, are the major class of glycoconjugates on neurons and carry the majority of the sialic acid within the central nervous system (CNS). To determine the role of ganglioside synthesis within the CNS, mice carrying null mutations in two critical ganglioside-specific glycosyltransferase genes, Siat9 (encoding GM3 synthase) and Galgt1 (encoding GM2 synthase), were generated. These double-null mice were unable to synthesize gangliosides of the ganglio-series of glycosphingolipids, which are the major ganglioside class in the CNS. Soon after weaning, viable mice developed a severe neurodegenerative disease that resulted in death. Histopathological examination revealed striking vacuolar pathology in the white matter regions of the CNS with axonal degeneration and perturbed axon-glia interactions. These results indicate that ganglioside synthesis is essential for the development of a stable CNS, possibly by means of the promotion of interactions between axon and glia.     

Loss of leukemogenicity caused by mutations in the membrane glycoprotein structural gene of Friend spleen focus-forming virus.

Friend virus infection of mice causes progressive leukemogenesis--a rapid splenic erythroblastosis that develops weeks later into a disseminating erythroleukemia. Furthermore, the replication-defective Friend spleen focus-forming virus (F-SFFV) encodes a membrane glycoprotein with an apparent Mr of 55,000 (designated gp55), which is structurally and immunologically related to the membrane envelope glycoproteins of dual tropic murine leukemia viruses. We now have isolated three spontaneous F-SFFV mutants that encode abnormally sized gp55-related glycoproteins with apparent Mrs of 40,000, 54,000, and 58,000, respectively. RNA blot and Southern blot analyses indicate that the mutant nucleic acids do not have substantial deletions or insertions in their glycoprotein gene regions. Protein fragmentation patterns indicate that the mutations affect nonoverlapping domains of the glycoprotein. Furthermore, these mutant glycoproteins seem to be defective in their processing to the plasma membranes. Although transmitted efficiently between cultured cells, the mutants have dramatically reduced leukemogenicities compared with the same titers of wild-type F-SFFV. We conclude that the gp55 structural gene is necessary for initiating the erythroblast proliferative phase of Friend disease and that changes in membranes can be primary causes rather than only secondary consequences of tumor progression.     

IL-18 and IL-12 induce intestinal inflammation and fatty liver in mice in an IFN-gamma dependent manner

BACKGROUND: In murine models of inflammatory bowel disease, colonic inflammation is considered to be caused by an aberrant Th1-type immune response. AIM: To investigate if systemic administration of interleukin (IL)-12 and IL-18 to wild-type BALB/c mice induces liver injury and intestinal inflammation, and if pathological changes are observed, what cytokines are involved. METHODS: Mice (BALB/c-wild-type (wt), MRL-lpr/lpr, BALB/c-interferon gamma knock out (IFN-gamma KO), C57BL/6-inducible nitric oxide synthase (iNOS) KO, and BALB/c tumour necrosis factor alpha (TNF-alpha) KO) were injected intraperitoneally each day with IL-12 (20 ng/g/mouse) and/or IL-18 (200 ng/g/mouse). RESULTS: Administration of IL-12 and IL-18 to BALB/c-wt mice induced prominent intestinal mucosal inflammation and fatty liver, leading to piloerection, bloody diarrhoea, and weight loss. IL-12 and IL-18 induced striking elevations in serum levels of IFN-gamma that caused NO production, although increased NO had no exacerbating effect on mice. Moreover, iNOS KO mice, or MRL lpr/lpr mice lacking functional Fas were equally susceptible to IL-12 and IL-18. Administration of IL-12 and IL-18 did not induce TNF-alpha production in wild-type mice, and the same treatment to TNF-alpha KO mice induced intestinal mucosal inflammation. Furthermore, they had diffuse and dense infiltration of small fat droplets in their hepatocytes associated with an increase in serum levels of liver enzymes. In contrast, the same treatment in IFN-gamma KO BALB/c mice and iNOS KO mice did not induce these changes. CONCLUSIONS: Our study strongly indicates that IL-18 together with IL-12 induces intestinal mucosal inflammation in an IFN-gamma dependent but TNF-alpha, NO, and Fas ligand independent manner, and fatty liver is dependent on IFN-gamma and NO.     

Pertussis toxin and extracytoplasmic adenylate cyclase as virulence factors of Bordetella pertussis

A wild-type strain of Bordetella pertussis and a series of transposon Tn5-induced mutants deficient in the production of various factors believed to play a role in pertussis (whooping cough) were tested for virulence in infant mice. The 50% lethal dose of the wild-type strain in these animals was 2 X 10(3) bacteria. A mutant deficient in the production of the filamentous hemagglutinin was almost as virulent as the wild type. Avirulent phase mutants (i.e., deficient in the production of all toxins), a pertussis-toxin mutant, and a double mutant deficient in both hemolysin and adenylate cyclase were severely impaired in the ability to cause pertussis. These data underscore the role of pertussis toxin in the disease and provide the first direct evidence that adenylate cyclase and hemolysin are important in the pathogenesis of the infection.     

Identification of Mycobacterial genes that alter growth and pathology in macrophages and in mice

Mycobacterium tuberculosis lives intracellularly, and many facets of its interactions with host cells are not well understood. We screened an M. tuberculosis transposon library for mutants exhibiting reduced ability to kill eukaryotic cells. Four of the mutants identified had insertions in 3 adjacent genes: single insertions in each of 2 acyl-coenzyme A dehydrogenases (fadE) plus 2 unique insertions in a cytochrome P450 homolog. A mutant in which these genes were replaced by allelic exchange was powerfully attenuated in our macrophage viability assay, and there was a striking defect in its ability to grow intracellularly. Interestingly, the difference between wild-type and mutant growth was minimized in activated macrophages. Aerosol infection of mice revealed a lag in growth, delayed dissemination to the spleen, and reduced lung pathology but no difference in persistence. Thus, these genes may be particularly important for events early during infection.     

Herpes simplex virus 1 interaction with Toll-like receptor 2 contributes to lethal encephalitis

Human neonates infected with herpes simplex virus 1 (HSV-1) develop one of three distinct patterns of infection: (i) infection limited to the skin, eye or mouth; (ii) infection of the CNS; or (iii) disseminated infection. The disseminated form usually involves the liver, adrenal gland, and lung, and resembles the clinical picture of bacterial sepsis. This spectrum of symptoms in HSV-1-infected neonates suggests that inflammatory cytokines play a significant role in the pathogenesis of the disease. Recent studies suggest that the Toll-like receptors (TLRs) may play an important role in the induction of inflammatory cytokines in response to viruses. TLRs are mammalian homologues of Toll, a Drosophila protein that is essential for host defense against infection. Engagement of TLRs by bacterial, viral, or fungal components leads to the production and release of cytokines and other antimicrobial products. Here, we demonstrate that TLR2 mediates the inflammatory cytokine response to HSV-1 by using both transfected cell lines and knockout mice. Studies of infected mice revealed that HSV-1 induced a blunted cytokine response in TLR2(-/-) mice. Brain levels of monocyte chemoattractant protein 1 chemokine were significantly lower in TLR2(-/-) mice than in either wild-type or TLR4(-/-) mice. TLR2(-/-) mice had reduced mortality compared with wild-type mice. The differences between TLR2(-/-) mice and both wild-type and TLR4(-/-) mice in the induction of monocyte chemoattractant protein 1, brain inflammation, or mortality could not be accounted for on the basis of virus levels. Thus, these studies suggest the TLR2-mediated cytokine response to HSV-1 is detrimental to the host.     

Oncogenicity in newborn and adult Syrian hamsters of SV40 temperature-sensitive (ts) mutants.

Newborn and adult Syrian hamsters were injected with wild-type SV40 and its temperature-sensitive (ts) mutants A30, A209, A239, B201 and BC210. In contrast to wild-type SV40, ts A30, ts A 239 and ts BC210 were oncogenic in adult hamsters inducing tumors after almost the same latent period as wild type SV40 in newborns but in lower number of animals. Study of TSTA in some tumors (1 generation) induced in newborn and adult hamsters by wild type SV40 and its ts mutants (with the use of immunogenicity and immunosensitivity tests) revealed no significant difference among compared tumors: most of them were immunogenic and immunosensitive. In contrast hamster embryo cells in vitro transformed by SV40 ts A30, ts A239 and ts A209 mutants, studied at different passage levels were all immunoresistant during about 30 in vitro passages and in most cases 10--100 times less immunogenic than hamster embryo cells in vitro transformed by wild type SV40. At higher passage level in some of these lines expression of TSTA improved. The data obtained are discussed in connection with the recently demonstrated [5] significant quantitative difference in tumor specific transplantation antigen activity in hamster cells in vivo and vitro infected, or transformed by wild type SV40 and its ts A mutants.     

Temperature-sensitive cell mutations that inhibit adenovirus 2 replication.

Five temperature-sensitive growth mutants of the hamster cell line BHK-21 were tested for the ability to support adenovirus 2 multiplication at 39 degrees and 33 degrees. Wild-type BHK-21 and mutants ts 422E and ts BCH yielded comparable amounts of virus at 33 degrees and 39 degrees, whereas in three other mutants, ts T22, ts T23, and ts AF8, virus production at 39 degrees was reduced to about 1% of that at 33 degrees. Virus yield in the three mutants was not reduced because of a delay in virus production; for all cells tested maximal virus yield at 39 degrees was obtained by 40-50 hr after infection. Normal yields of infectious virus were not obtained from ts AF8 even with a very high multiplicity of infection. In contrast, the virus yield from ts T22 and ts T23 was multiplicity-dependent. Shiftup experiments demonstrated that in ts AF8, a cell cycle mutant which at 39 degrees becomes arrested in G1, virus multiplication was thermosensitive for the first 40 hr of infection. In ts T22 and ts T23, the thermosensitivity was only for the first 3-4 hr of the infection. In all three mutants viral DNA synthesis was reduced by at least 95% at the higher temperature. The cell function specified by the ts AF8 mutation seems to be required for the early period of adenovirus 2 replication, after virus entry into the cell but before the onset of viral DNA replication.