Differentiation of an adult neuron cell line increases susceptibility to rabies infection

A wide variety of in vitro models have been used for studying rabies infection, however, currently, no central nervous system (CNS) adult neuron cultures are available. The current study determined the susceptibility to rabies infection in an adult CNS neuron cell line (CAD-R1). Cultures of CAD-R1 cells were held for 5 days in medium containing serum (undifferentiated CAD-R1 cells) or in serum-free medium (differentiated CAD-R1 cells). They were then infected with highly neurotropic rabies virus (RV) strain (CVS), obtained from fibroblastic cells (CVS-BHK) or from adult mouse brain (CVS-MB). Undifferentiated and differentiated cells were infected with the two RV strains, but the percentage of infected cells in differentiated cultures was significantly greater (83% and 79%, respectively) than in undifferentiated cells (51% and 60%) (Student's t test<0.05). Susceptibility to infection apparently depended on cellular differentiation state, possibly due to acquisition of additional morphological and biochemical characteristics during the differentiation process that made them more susceptible to RV infection. Therefore, CAD R1 cells may represent a good model for RV infection, making them a useful tool for studying RV neurotropism, infection pathogeny, isolation of street virus or producing safer and most potent vaccines.     

Effects of dexamethasone on rhinovirus infection in cultured human tracheal epithelial cells

To examine the effects of glucocorticoid on rhinovirus (RV) infection, primary cultures of human tracheal epithelial cells were infected with either RV2 or RV14. Viral infection was confirmed by demonstrating that viral RNA in infected cells and viral titers of supernatants and lysates from infected cells increased with time. RV14 infection upregulated the expression of mRNA and protein of intercellular adhesion molecule-1 (ICAM-1), the major RV receptor, on epithelial cells, and it increased the production of interleukin (IL)-1beta, IL-6, IL-8, and tumor necrosis factor-alpha in supernatants. Dexamethasone reduced the viral titers of supernatants and cell lysates, viral RNA of infected cells, and susceptibility of RV14 infection in association with inhibition of cytokine production and ICAM-1 induction. In contrast to RV14 infection, dexamethasone did not alter RV2 infection, a minor group of RVs. These results suggest that dexamethasone may inhibit RV14 infection by reducing the surface expression of ICAM-1 in cultured human tracheal epithelial cells. Glucocorticoid may modulate airway inflammation via reducing the production of proinflammatory cytokines and ICAM-1 induced by rhinovirus infection.     

Bafilomycin A(1) inhibits rhinovirus infection in human airway epithelium: effects on endosome and ICAM-1

To examine the effects of bafilomycin A(1), a blocker of vacuolar H(+)-ATPase, on rhinovirus (RV) infection in the airway epithelium, primary cultures of human tracheal epithelial cells were infected with RV14. Viral infection was confirmed by showing that viral RNA in the infected cells and the viral titers in the supernatants of infected cells increased with time. RV14 infection upregulated the production of cytokines and mRNA of intercellular adhesion molecule (ICAM)-1 in epithelial cells. Bafilomycin A(1) reduced the viral titers of RV14 and inhibited the production of cytokines and ICAM-1 before and after RV14 infection. Bafilomycin A(1) reduced susceptibility of epithelial cells to RV14 infection. RV14 increased activated nuclear factor-kappaB in the cells, and bafilomycin A(1) reduced the activated nuclear factor-kappaB. Bafilomycin A(1) decreased the number of acidic endosomes in the epithelial cells. These results suggest that bafilomycin A(1) may inhibit infection by RV14 by not only blocking RV RNA entry into the endosomes but also reducing ICAM-1 expression in the epithelial cells. Bafilomycin A(1) may therefore modulate airway inflammation after RV infection.     

Overexpression of tumor necrosis factor alpha by a recombinant rabies virus attenuates replication in neurons and prevents lethal infection in mice

The effect of tumor necrosis factor alpha (TNF-alpha) on rabies virus (RV) infection of the mouse central nervous system (CNS) was studied, using recombinant RV engineered to express either soluble TNF-alpha [SPBN-TNF-alpha+] or insoluble membrane-bound TNF-alpha [SPBN-TNF-alpha(MEM)]. Growth curves derived from infections of mouse neuroblastoma NA cells revealed significantly less spread and production of SPBN-TNF-alpha+ than of SPBN-TNF-alpha(MEM) or SPBN-TNF-alpha-, which carries an inactivated TNF-alpha gene. The expression of soluble or membrane-bound TNF-alpha was not associated with increased cell death or induction of alpha/beta interferons. Brains of mice infected intranasally with SPBN-TNF-alpha+ showed significantly less virus spread than did mouse brains after SPBN-TNF-alpha- infection, and none of the SPBN-TNF-alpha+-infected mice succumbed to RV infection, whereas 80% of SPBN-TNF-alpha- -infected mice died. Reduced virus spread in SPBN-TNF-alpha+-infected mouse brains was paralleled by enhanced CNS inflammation, including T-cell infiltration and microglial activation. These data suggest that TNF-alpha exerts its protective activity in the brain directly through an as yet unknown antiviral mechanism and indirectly through the induction of inflammatory processes in the CNS.     

Effects of rhinovirus infection on histamine and cytokine production by cell lines from human mast cells and basophils

To understand the biochemical events that occur in the airways after rhinovirus (RV) infection, we developed for the first time a model in which the cell lines from human mast cells (HMC-1) and basophils (KU812) can be infected with RV14, a major group RV. Viral infection was confirmed by demonstrating that viral titers in culture supernatants, and RV RNA increased with time. RV14 infection alone and a combination of PMA plus calcium ionophore A23187, did not increase histamine production by these cells, although IgE plus anti-IgE increased the histamine production. However, histamine content in the supernatants increased in response to PMA plus A23187, or IgE plus anti-IgE after RV14 infection. PMA plus A23187 or IgE plus anti-IgE induced the production of IL-8 and GM-CSF in supernatants of HMC-1 cells and IL-4 and IL-6 in supernatants of KU812 cells. RV14 infection further increased the production of the cytokines, whereas RV14 infection alone did not alter the production of the cytokines by these cells. An Ab to ICAM-1 inhibited RV14 infection of the cells and decreased the production of cytokines and histamine after RV14 infection. RV14 infection enhanced the increases in intracellular calcium concentration and activation of NF-kappaB by PMA plus A23187 in the cells. These findings suggest that RV14 infection may prime the cytokine and histamine production from mast cells and basophils and may cause airway inflammation in asthma.     

Theiler's virus infection induces a specific cytotoxic T lymphocyte response

Theiler's virus, a murine picornavirus, persists in the central nervous system of susceptible mouse strains and causes chronic inflammation and primary demyelination. One of the current hypotheses is that demyelination is, at least in part, mediated by virus-specific cytotoxic T lymphocytes (CTL). However, it is generally assumed that picornaviruses do not induce CTL. In point of fact, their existence has only been demonstrated for Coxsackievirus B-3. To determine whether Theiler's virus induces a CTL response, we generated a murine mastocytoma cell line stably transfected with the coding region of the genome of Theiler's virus strain DA. Using these cells as targets we showed that infected DBA/2 mice, a susceptible strain, produce cytotoxic T lymphocytes. The cytotoxic activity was Theiler's-virus specific. It was for the most part mediated by CD8+ T lymphocytes and H-2 restricted. This is the first demonstration that a specific CTL response is generated during Theiler's virus infection.     

Quantitative comparison of infection of neural cell and fibroblast monolayers by two strains of Toxoplasma gondii

Disease caused by the coccidian Toxoplasma gondii can be confined to the central nervous system, although the parasite is capable of infecting all organ systems. To determine whether neural cells are differentially susceptible to infection and destruction by T. gondii, infection of neonatal mouse brain monolayers was compared to infection of human fibroblast monolayers under the same conditions with equal inocula of two parasite strains. In preliminary experiments there was no difference in total parasite yield or in plaques per monolayer between rodent and human cells. A standardized inoculum of T. gondii RH strain caused 35.6 +/- 6.4 (SD) plaques per well in neural explant monolayers compared to 39.3 +/- 12.5 plaques per well in fibroblasts. T. gondii P strain produced 35.6 +/- 8.9 infected foci per well in neural cells compared to 32.6 +/- 9.3 foci in fibroblasts. Intrinsic properties of neural cells do not appear to cause a higher rate of infection than that in nonneural cells.     

Infections that induce autoimmune diabetes in BBDR rats modulate CD4+CD25+ T cell populations

Viruses are believed to contribute to the pathogenesis of autoimmune type 1A diabetes in humans. This pathogenic process can be modeled in the BBDR rat, which develops pancreatic insulitis and type 1A-like diabetes after infection with Kilham's rat virus (RV). The mechanism is unknown, but does not involve infection of the pancreatic islets. We first documented that RV infection of BBDR rats induces diabetes, whereas infection with its close homologue H-1 does not. Both viruses induced similar humoral and cellular immune responses in the host, but only RV also caused a decrease in splenic CD4(+)CD25(+) T cells in both BBDR rats and normal WF rats. Surprisingly, RV infection increased CD4(+)CD25(+) T cells in pancreatic lymph nodes of BBDR but not WF rats. This increase appeared to be due to the accumulation of nonproliferating CD4(+)CD25(+) T cells. The results imply that the reduction in splenic CD4(+)CD25(+) cells observed in RV-infected animals is virus specific, whereas the increase in pancreatic lymph node CD4(+)CD25(+) cells is both virus and rat strain specific. The data suggest that RV but not H-1 infection alters T cell regulation in BBDR rats and permits the expression of autoimmune diabetes. More generally, the results suggest a mechanism that could link an underlying genetic predisposition to environmental perturbation and transform a "regulated predisposition" into autoimmune diabetes, namely, failure to maintain regulatory CD4(+)CD25(+) T cell function.     

CD4+ T cells are required for HSP65 expression in host macrophages and for protection of mice infected with Plasmodium yoelii

We have reported that macrophages expressing heat-shock protein 65 play an essential role in protection of mice infected with Plasmodium yoelii. In this study, we investigated the function and expression mechanism of HSP65 in macrophages of mice infected with P. yoelii. C57BL/6 (B6) mice are susceptible to infection with the lethal (L) strain but resistant to infection with the non-lethal (NL) strain of P. yoelii. The percentage of apoptotic macrophages in mice infected with the L strain was higher than that in mice infected with the NL strain. However, the percentage was low in L strain infected mice if they acquired resistance to the infection by primary infection with the NL strain. That apoptosis was reversely correlated with HSP65 expression in splenic macrophages from mice infected with P. yoelii suggests HSP65 may contribute to protective immunity by preventing apoptosis of macrophages in malarial infection. Cell depletion/transfer experiments showed that CD4+ T cells, but not CD8+ T cells, gammadelta T cells, NK cells or NK T cells, were required for HSP65 expression in macrophages as well as for protection of mice infected with P. yoelii. In conclusion, HSP65 may play a role in preventing apoptosis of macrophages in mice infected with P. yoelii. CD4+ T cells are required for HSP65 expression and for protective immunity against P. yoelii infection.     

Rotavirus stimulates release of serotonin (5-HT) from human enterochromaffin cells and activates brain structures involved in nausea and vomiting

Rotavirus (RV) is the major cause of severe gastroenteritis in young children. A virus-encoded enterotoxin, NSP4 is proposed to play a major role in causing RV diarrhoea but how RV can induce emesis, a hallmark of the illness, remains unresolved. In this study we have addressed the hypothesis that RV-induced secretion of serotonin (5-hydroxytryptamine, 5-HT) by enterochromaffin (EC) cells plays a key role in the emetic reflex during RV infection resulting in activation of vagal afferent nerves connected to nucleus of the solitary tract (NTS) and area postrema in the brain stem, structures associated with nausea and vomiting. Our experiments revealed that RV can infect and replicate in human EC tumor cells ex vivo and in vitro and are localized to both EC cells and infected enterocytes in the close vicinity of EC cells in the jejunum of infected mice. Purified NSP4, but not purified virus particles, evoked release of 5-HT within 60 minutes and increased the intracellular Ca²⁺ concentration in a human midgut carcinoid EC cell line (GOT1) and ex vivo in human primary carcinoid EC cells concomitant with the release of 5-HT. Furthermore, NSP4 stimulated a modest production of inositol 1,4,5-triphosphate (IP₃), but not of cAMP. RV infection in mice induced Fos expression in the NTS, as seen in animals which vomit after administration of chemotherapeutic drugs. The demonstration that RV can stimulate EC cells leads us to propose that RV disease includes participation of 5-HT, EC cells, the enteric nervous system and activation of vagal afferent nerves to brain structures associated with nausea and vomiting. This hypothesis is supported by treating vomiting in children with acute gastroenteritis with 5-HT₃ receptor antagonists.     

High-magnitude, virus-specific CD4 T-cell response in the central nervous system of coronavirus-infected mice

The neurotropic JHM strain of mouse hepatitis virus (MHV) causes acute encephalitis and chronic demyelinating encephalomyelitis in rodents. Previous results indicated that CD8 T cells infiltrating the central nervous system (CNS) were largely antigen specific in both diseases. Herein we show that by 7 days postinoculation, nearly 30% of the CD4 T cells in the acutely infected CNS were MHV specific by using intracellular gamma interferon (IFN-gamma) staining assays. In mice with chronic demyelination, 10 to 15% of the CD4 T cells secreted IFN-gamma in response to MHV-specific peptides. Thus, these results show that infection of the CNS is characterized by a large influx of CD4 T cells specific for MHV and that these cells remain functional, as measured by cytokine secretion, in mice with chronic demyelination.     

Invariant NKT Cells Regulate the CD8 T Cell Response during Theiler's Virus Infection

Invariant NKT cells are innate lymphocytes with a broad tissue distribution. Here we demonstrate that iNKT cells reside in the central nervous system (CNS) in the absence of inflammation. Their presence in the CNS dramatically augments following inoculation of C57Bl/6 mice with the neurotropic Theiler''s murine encephalomyelitis virus (TMEV). At the peak of inflammation the cellular infiltrate comprises 45 000 iNKT cells for 1 250 CD8 T cells specific for the immunodominant TMEV epitope. To study the interaction between these two T cell subsets, we infected both iNKT cell deficient Jα18^-/- mice and iNKT cell enriched Vα14 transgenic mice with TMEV. The CD8 T cell response readily cleared TMEV infection in the iNKT cell deficient mice. However, in the iNKT cell enriched mice TMEV infection persisted and was associated with significant mortality. This was caused by the inhibition of the CD8 T cell response in the cervical lymph nodes and spleen after T cell priming. Taken together we demonstrate that iNKT cells reside in the CNS in the absence of inflammation and that their enrichment is associated with the inhibition of the anti-viral CD8 T cell response and an augmented mortality during acute encephalomyelitis.     

Antiviral CD8 T cells recognize borna disease virus antigen transgenically expressed in either neurons or astrocytes

Borna disease virus (BDV) can persistently infect the central nervous system (CNS) of mice. The infection remains nonsymptomatic as long as antiviral CD8 T cells do not infiltrate the infected brain. BDV mainly infects neurons which reportedly carry few, if any, major histocompatibility complex class I molecules on the surface. Therefore, it remains unclear whether T cells can recognize replicating virus in these cells or whether cross-presentation of viral antigen by other cell types is important for immune recognition of BDV. To distinguish between these possibilities, we used two lines of transgenic mice that strongly express the N protein of BDV in either neurons (Neuro-N) or astrocytes (Astro-N). Since these animals are tolerant to the neo-self-antigen, we adoptively transferred T cells with specificity for BDV N. In nontransgenic mice persistently infected with BDV, the transferred cells accumulated in the brain parenchyma along with immune cells of host origin and efficiently induced neurological disease. Neurological disease was also observed if antiviral T cells were injected into the brains of Astro-N or Neuro-N but not nontransgenic control mice. Our results demonstrate that CD8 T cells can recognize foreign antigen on neurons and astrocytes even in the absence of infection or inflammation, indicating that these CNS cell types are playing an active role in immune recognition of viruses.     

轮状病毒感染成年小鼠的研究

目的研究成年昆明种小鼠对实验感染人轮状病毒(rotavirus,RV)的敏感性。方法在实验条件下,用A组人Wa和恒河猴SA11株RV感染成年昆明种小鼠,观察小鼠的临床反应和排毒情况。结果成年昆明种小鼠感染Wa和SA11RV第二天后出现明显的临床腹泻症状,第四天达到高峰;至少在感染后连续6天的动物大便中可检测到较高滴度的RV抗原。结论成年昆明种小鼠对RV感染有很高的敏感性,可做为动物模型,在RV感染的药物治疗效果评价和疫苗保护性效果评价中具有重要价值。     

Interaction of rotavirus with human myeloid dendritic cells

We have previously shown that very few rotavirus (RV)-specific T cells that secrete gamma interferon circulate in recently infected and seropositive adults and children. Here, we have studied the interaction of RV with myeloid immature (IDC) and mature dendritic cells (MDC) in vitro. RV did not induce cell death of IDC or MDC and induced maturation of between 12 and 48% of IDC. Nonetheless, RV did not inhibit the maturation of IDC or change the expression of maturation markers on MDC. After treatment with RV, few IDC expressed the nonstructural viral protein NSP4. In contrast, a discrete productive viral infection was shown in MDC of a subset of volunteers, and between 3 and 46% of these cells expressed NSP4. RV-treated IDC secreted interleukin 6 (IL-6) (but not IL-1beta, IL-8, IL-10, IL-12, tumor necrosis factor alpha, or transforming growth factor beta), and MDC released IL-6 and small amounts of IL-10 and IL-12p70. The patterns of cytokines secreted by T cells stimulated by staphylococcal enterotoxin B presented by MDC infected with RV or uninfected were comparable. The frequencies and patterns of cytokines secreted by memory RV-specific T cells evidenced after stimulation of peripheral blood mononuclear cells (PBMC) with RV were similar to those evidenced after stimulation of PBMC with RV-infected MDC. Finally, IDC treated with RV strongly stimulated naive allogeneic CD4+ T cells to secrete Th1 cytokines. Thus, although RV does not seem to be a strong maturing stimulus for DC, it promotes their capacity to prime Th1 cells.     

A peroxynitrite-dependent pathway is responsible for blood-brain barrier permeability changes during a central nervous system inflammatory response: TNF-alpha is neither necessary nor sufficient

Elevated blood-brain barrier (BBB) permeability is associated with both the protective and pathological invasion of immune and inflammatory cells into CNS tissues. Although a variety of processes have been implicated in the changes at the BBB that result in the loss of integrity, there has been no consensus as to their induction. TNF-alpha has often been proposed to be responsible for increased BBB permeability but there is accumulating evidence that peroxynitrite (ONOO(-))-dependent radicals may be the direct trigger. We demonstrate here that enhanced BBB permeability in mice, whether associated with rabies virus (RV) clearance or CNS autoimmunity, is unaltered in the absence of TNF-alpha. Moreover, the induction of TNF-alpha expression in CNS tissues by RV infection has no impact on BBB integrity in the absence of T cells. CD4 T cells are required to enhance BBB permeability in response to the CNS infection whereas CD8 T cells and B cells are not. Like CNS autoimmunity, elevated BBB permeability in response to RV infection is evidently mediated by ONOO(-). However, as opposed to the invading cells producing ONOO(-) that have been implicated in the pathogenesis of CNS inflammation, during virus clearance ONOO(-) is produced without pathological sequelae by IFN-gamma-stimulated neurovascular endothelial cells.     

Biological basis of rabies virus neurovirulence in mice: comparative pathogenesis study using the immunoperoxidase technique.

The CVS strain of fixed rabies virus causes acute, fatal encephalomyelitis in young adult ICR mice. Variant RV194-2, which was selected from CVS virus in cell culture with a neutralizing antiglycoprotein monoclonal antibody, has a single amino acid change in the glycoprotein. The infections caused by CVS virus and RV194-2 virus were compared in mice for 14 days postinoculation of 5 x 10(7) PFU into the right masseter muscle. All CVS virus-infected mice died (mean time to death, 7.9 days), compared with a mortality rate of 8.5% for RV194-2 virus-infected mice. RV194-2 virus spread to the ipsilateral trigeminal ganglion during the first 2 days postinoculation, and both viruses spread to the ipsilateral motor nucleus of the trigeminal nerve in the pons. Both viruses spread centrifugally and caused infection of bilateral trigeminal ganglia on day 3. The viruses spread throughout the central nervous system (CNS) at similar rates, but CVS virus infected many more neurons than did RV194-2 virus. Rabies virus antigen was observed in only occasional CNS neurons after day 6 of RV194-2 virus infection. By this time, CVS virus had caused severe widespread infection. In this model, virulence depends on improved efficiency of viral spread between CNS neurons rather than the rate of spread or topographical distribution of the infection.     

Protective intestinal anti-rotavirus B cell immunity is dependent on alpha 4 beta 7 integrin expression but does not require IgA antibody production

Rotavirus (RV) is the main cause of severe gastroenteritis in young children; protection has been correlated with intestinal Ab responses. Using a mouse model of RV infection and beta(7)-deficient (beta(7)(-/-)) mice, which do not express alpha(4)beta(7) integrin, we demonstrated the importance of alpha(4)beta(7) integrin in B cell-mediated anti-RV immunity. beta(7)(-/-) mice acutely infected with murine RV resolved infection and developed normal serum IgG Abs but had diminished intestinal IgA responses. alpha(4)beta(7)(-/-) immune B cells did not resolve RV infection when adoptively transferred into RV-infected Rag-2-deficient mice. Fewer RV-specific B cells were found in the intestine of Rag-2-deficient mice transferred with beta(7)(-/-) B cells compared with wild type. The absence of alpha(4)beta(7) expression and/or a lower frequency of IgA-producing cells among transferred beta(7)(-/-) B cells could have accounted for the inability of these cells to resolve RV infection following passive transfer. To distinguish between these possibilities, we studied the importance of IgA production in RV infection using IgA-deficient (IgA(-/-)) mice. IgA(-/-) mice depleted of CD8(+) T cells were able to clear primary RV infection. Similarly, adoptive transfer of immune IgA(-/-) B cells into chronically infected Rag-2-deficient mice resolved RV infection. We further demonstrated in both wild-type and IgA(-/-) mice that, following oral RV infection, protective B cells reside in the alpha(4)beta(7)(high) population. Our findings suggest that alpha(4)beta(7) integrin expression is necessary for B cell-mediated immunity to RV independent of the presence of IgA.