CNS expression of B7-H1 regulates pro-inflammatory cytokine production and alters severity of Theiler's virus-induced demyelinating disease

The CNS is a unique organ due to its limited capacity for immune surveillance. As macrophages of the CNS, microglia represent a population originally known for the ability to assist neuronal stability, are now appreciated for their role in initiating and regulating immune responses in the brain. Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease is a mouse model of multiple sclerosis (MS). In response to TMEV infection in vitro, microglia produce high levels of inflammatory cytokines and chemokines, and are efficient antigen-presenting cells (APCs) for activating CD4(+) T cells. However, the regulatory function of microglia and other CNS-infiltrating APCs in response to TMEV in vivo remains unclear. Here we demonstrate that microglia increase expression of proliferating cell nuclear antigen (PCNA), and phenotypically express high levels of major histocompatibility complex (MHC)-Class I and II in response to acute infection with TMEV in SJL/J mice. Microglia increase expression of the inhibitory co-stimulatory molecule, B7-H1 as early as day 5 post-infection, while CNS-infiltrating CD11b(+)CD11c(-)CD45(HIGH) monocytes/macrophages and CD11b(+)CD11c(+)CD45(HIGH) dendritic cells upregulate expression of B7-H1 by day 3 post-infection. Utilizing a neutralizing antibody, we demonstrate that B7-H1 negatively regulates TMEV-specific ex vivo production of interferon (IFN)-γ, interleukin (IL)-17, IL-10, and IL-2 from CD4(+) and CD8(+) T cells. In vivo blockade of B7-H1 in SJL/J mice significantly exacerbates clinical disease symptoms during the chronic autoimmune stage of TMEV-IDD, but only has minimal effects on viral clearance. Collectively, these results suggest that CNS expression of B7-H1 regulates activation of TMEV-specific T cells, which affects protection against TMEV-IDD.     

Differential virus replication, cytokine production, and antigen-presenting function by microglia from susceptible and resistant mice infected with Theiler's virus

Infection with Theiler''s murine encephalomyelitis virus (TMEV) in the central nervous system (CNS) causes an immune system-mediated demyelinating disease similar to human multiple sclerosis in susceptible but not resistant strains of mice. To understand the underlying mechanisms of differential susceptibility, we analyzed viral replication, cytokine production, and costimulatory molecule expression levels in microglia and macrophages in the CNS of virus-infected resistant C57BL/6 (B6) and susceptible SJL/J (SJL) mice. Our results indicated that message levels of TMEV, tumor necrosis factor alpha, beta interferon, and interleukin-6 were consistently higher in microglia from virus-infected SJL mice than in those from B6 mice. However, the levels of costimulatory molecule expression, as well as the ability to stimulate allogeneic T cells, were significantly lower in TMEV-infected SJL mice than in B6 mice. In addition, microglia from uninfected naïve mice displayed differential viral replication, T-cell stimulation, and cytokine production, similar to those of microglia from infected mice. These results strongly suggest that different levels of intrinsic susceptibility to TMEV infection, cytokine production, and T-cell activation ability by microglia contribute to the levels of viral persistence and antiviral T-cell responses in the CNS, which are critical for the differential susceptibility to TMEV-induced demyelinating disease between SJL and B6 mice.BeAn and DA are members of Theiler''s original subgroup of Theiler''s murine encephalitis virus (TMEV) (52). Intracerebral inoculation of susceptible mice, such as SJL/J (SJL) mice, with either of these viruses results in a biphasic disease characterized by early encephalitis and late chronic demyelination (24). Infection of susceptible mice with these viruses results in a chronic, white matter-demyelinating disease similar to human multiple sclerosis (24). In susceptible strains, infection of the central nervous system (CNS) with TMEV leads to a chronic immune response to viral antigens, which eventually leads to autoimmune responses against myelin autoantigens (29). In contrast, resistant mouse strains, such as C57BL/6 (B6), rapidly clear virus from the CNS and do not develop demyelinating disease, suggesting that viral persistence in these mice corresponds to susceptibility to disease (26, 42, 45). Demyelination in susceptible mice is considered to be immunity mediated, as removal of immune components reduces the clinical onset and severity of demyelinating disease (9, 25, 44, 47).In particular, infiltration of proinflammatory CD4⁺ Th1-type cells has been associated with tissue destruction and demyelination (41, 56). A number of CD4⁺ T cells specific for TMEV during the course of disease in SJL mice recognize four predominant viral capsid epitopes (VP1_233-250, VP2_74-86, VP3_24-37, and VP4_51-70), with one each on the external and internal capsid proteins (10, 19, 55, 56). The external capsid epitopes appear to account for the majority (∼80%) of major histocompatibility complex (MHC) class II-restricted T-cell responses to TMEV capsid proteins (55, 57). Recently, viral capsid epitopes recognized by CNS-infiltrating CD4⁺ T cells from TMEV-infected B6 mice have also been identified (18). When levels of virus capsid-specific CD4⁺ T cells in the CNS are compared between B6 and SJL mice at early stages of viral infection, significantly higher levels are found in the CNS of resistant B6 mice (30), suggesting that virus-specific CD4⁺ T cells are important for protection from demyelinating disease during initial immune responses (2). Similarly, levels of CNS-infiltrating virus-specific CD8⁺ T cells in the CNS are as much as threefold higher in resistant mice at the same time point (28). Therefore, it appears that levels of both initial CD4⁺ and CD8⁺ T-cell responses to the virus are critically important in setting the stage of viral persistence/clearance and consequent susceptibility or resistance to inflammatory demyelinating disease.In order to further understand the potential mechanisms of differences in susceptibility and antiviral immunity levels between SJL and B6 mice, the properties of resident microglial cells and infiltrating macrophages in the CNS during the early stage of viral infection in these mouse strains were investigated. It has previously been established that nonprofessional antigen-presenting cells (APCs; mainly microglial cells and astrocytes) isolated from the CNS of TMEV-infected SJL mice are capable of presenting antigens to both TMEV- and CNS autoantigen-specific T-cell hybridomas and clones (21, 33, 37). Furthermore, microglial cells and/or infiltrating macrophages in the CNS are known to be a major cell population supporting viral persistence during chronic infection (4). Hence, these cells support the replication of TMEV and the activation and/or differentiation of CD4⁺ and CD8⁺ T cells infiltrating the CNS of virus-infected mice. Therefore, CNS APCs involved in triggering T-cell responses and harboring viral persistence may ultimately determine susceptibility/resistance to TMEV-IDD via their effects on virus clearance/persistence as well as on target tissue inflammation.In this study, we compared the potential roles of microglia and macrophages from TMEV-infected susceptible SJL and resistant B6 mice in the innate and adaptive immune responses affecting viral persistence and ultimate disease susceptibility. Our results indicate that viral replication and cytokine production levels are consistently higher in microglia from TMEV-infected SJL mice than in those from B6 mice. In addition, the expression of costimulatory molecules is significantly higher in resistant B6 mice throughout the course of viral infection, suggesting more efficient T-cell activation in resistant B6 mice. On the other hand, both virus replication and type I interferon (IFN) production in microglia from naïve SJL mice are significantly higher than those in such cells from naïve B6 mice. Therefore, differences in the intrinsic properties of microglia in viral replication and virus-induced innate cytokine production are likely to contribute significantly to viral persistence, cellular infiltration to the CNS, and consequent inflammation, leading to the development of demyelinating disease.     

Temporal and anatomic relationship between virus replication and cytokine gene expression after vaginal simian immunodeficiency virus infection

The current knowledge about early innate immune responses at mucosal sites of human immunodeficiency virus (HIV) entry is limited but likely to be important in the design of effective HIV vaccines against heterosexual transmission. This study examined the temporal and anatomic relationship between virus replication, lymphocyte depletion, and cytokine gene expression levels in mucosal and lymphoid tissues in a vaginal-transmission model of HIV in rhesus macaques. The results of the study show that the kinetics of cytokine gene expression levels in the acute phase of infection are positively correlated with virus replication in a tissue. Thus, cytokine responses after vaginal simian immunodeficiency virus (SIV) inoculation are earliest and strongest in mucosal tissues of the genital tract and lowest in systemic lymphoid tissues. Importantly, the early cytokine response was dominated by the induction of proinflammatory cytokines, while the induction of cytokines with antiviral activity, alpha/beta interferon, occurred too late to prevent virus replication and dissemination. Thus, the early cytokine response favors immune activation, resulting in the recruitment of potential target cells for SIV. Further, unique cytokine gene expression patterns were observed in distinct anatomic locations with a rapid and persistent inflammatory response in the gut that is consistent with the gut being the major site of early CD4 T-cell depletion in SIV infection.     

Replicon vectors derived from Sindbis virus and Semliki forest virus that establish persistent replication in host cells

Alphavirus replicon vectors are well suited for applications where transient, high-level expression of a heterologous gene is required. Replicon vector expression in cells leads to inhibition of host macromolecular synthesis, culminating in eventual cell death by an apoptotic mechanism. For many applications, including gene expression studies in cultured cells, a longer duration of transgene expression without resulting cytopathic effects is useful. Recently, noncytopathic Sindbis virus (SIN) variants were isolated in BHK cells, and the mutations responsible were mapped to the protease domain of nonstructural protein 2 (nsP2). We report here the isolation of additional variants of both SIN and Semliki Forest virus (SFV) replicons encoding the neomycin resistance gene that can establish persistent replication in BHK cells. The SIN and SFV variant replicons resulted from previously undescribed mutations within one of three discrete regions of the nsP2 gene. Differences among the panel of variants were observed in processing of the nonstructural polyprotein and in the ratios of subgenomic to genomic RNAs. Importantly, high-level expression of a heterologous gene was retained with most replicons. Finally, in contrast to previous studies, efficient packaging was obtained with several of the variant replicons. This work expands the utility of noncytopathic replicons and the understanding of how alphavirus replicons establish persistent replication in cultured cells.     

Evidence that low-level viremias during effective highly active antiretroviral therapy result from two processes: expression of archival virus and replication of virus

Episodes of low-level viremia (LLV), with plasma human immunodeficiency virus type 1 (HIV-1) RNA levels ranging from 50 to 400 copies (c)/ml, occur commonly during highly active antiretroviral therapy (HAART). LLV has been associated with virologic failure of HAART in some studies, while in others LLV did not appear to affect the clinical outcome. To understand the processes leading to LLV, genetic analyses were used to determine whether plasma virions emanated from archived or from newly evolved viral genomes. Episodes of LLV (plasma HIV-1 RNA, 50 to 379 [median, 77] c/ml) were detected in 21/37 (57%) HIV-1-infected children with median plasma HIV-1 RNA levels of <50 c/ml during 79 patient years of HAART. Viral sequences were derived by direct sequencing of PCR products from 21 plasma specimens diluted to end point. In phylogenetic analysis, LLV viral sequences grouped with virus from early in the course of infection in 8/11 subjects. Six specimens had multiple identical viral sequences, suggesting origin from clonally expanded infected cells. LLV plasma virus evolved over time, indicating viral replication, in 3/11 subjects. Two of these had frequent LLV, including the selection of drug-resistant mutants. In summary, plasma virus from episodes of LLV during effective HAART appeared to originate from two distinct processes, (i) clonal outgrowth from long-lived HIV-1-infected cells, presumably following activation and proliferation of these cells, and (ii) ongoing viral replication that included the selection of new drug-resistant mutants. These observations provide a plausible explanation for the divergent clinical outcomes previously associated with LLV.     

Innate resistance to herpes simplex virus infection. Human lymphocyte and monocyte inhibition of viral replication

Peripheral blood monocytes and lymphocytes isolated from most humans are resistant to HSV infection in vitro. Viral replication is inhibited very early in the cycle, prior to the onset of alpha-protein synthesis; no viral protein or DNA synthesis is detectable even up to 1 week later. The enhanced expression of two 62-kDa and 57-kDa cellular proteins, however, is induced in the lymphocyte population within 3 to 5 h after infection. A 30-kDa protein is induced in the monocyte population immediately after infection. The induced expression of 62-kDa and 57-kDa lymphocyte proteins appears to be virus-mediated because: a) HSV and pseudorabies virus (although not vaccinia virus) induce the expression of 62-kDa and 57-kDa proteins, b) heat shock or exposure of lymphocytes to uninfected cell extracts does not induce expression of either protein, c) 62-kDa protein is not induced in lymphocytes stimulated with a mitogenic concentration of PHA. UV-inactivated HSV induces expression of 62-kDa and 57-kDa proteins in a manner similar to that observed with untreated virus. In contrast, expression of 30-kDa monocyte protein is induced nonspecifically by either uninfected cell extracts or cell extracts containing virus. Sixty-two-kilodalton and 57-kDa protein induction appears to be a marker for human lymphocytes that express profound intracellular resistance to infection with HSV. Induced expression of these proteins occurs only in lymphocytes that inhibit viral replication very early in the growth cycle, prior to the onset of alpha-protein synthesis. Expression of 62-kDa and 57-kDa proteins is not induced in lymphocytes that are permissive or partially permissive to infection with HSV.     

Differential human immunodeficiency virus expression in CD4+ cloned lymphocytes: from viral latency to replication.

By using cloning methodology, 13 CD4+, CD8-, CD45RO+, and CD29+ clones, isolated from human immunodeficiency virus (HIV)-negative donors, have been characterized and tested regarding their susceptibility to two strains of HIV type 1 (HIV-1). Infected clones possess integrated provirus. Only six are able to replicate HIV-1, while seven may normally grow without cytopathic effect and without viral replication. These results argue that all CD4+ lymphocyte clones may be infectable but that a heterogeneity exists regarding their abilities to replicate HIV-1.     

Human serum facilitates hepatitis C virus infection, and neutralizing responses inversely correlate with viral replication kinetics at the acute phase of hepatitis C virus infection

The factors leading to spontaneous clearance of hepatitis C virus (HCV) or to viral persistence are elusive. Understanding virus-host interactions that enable acute HCV clearance is key to the development of more effective therapeutic and prophylactic strategies. Here, using a sensitive neutralization assay based on infectious HCV pseudoparticles (HCVpp), we have studied the kinetics of humoral responses in a cohort of acute-phase patients infected during a single nosocomial outbreak in a hemodialysis center. The 17 patients were monitored for the spontaneous outcome of HCV infection for 6 months before a treatment decision was made. Blood samples were taken frequently (15 +/- 4 per patient). Phylogenetic analysis of the predominant virus(es) revealed infection by only one of two genotype 1b strains. While all patients seroconverted, their sera induced two opposing effects in HCVpp infection assays: inhibition and facilitation. Furthermore, the ability of sera to facilitate or inhibit infection correlated with the presence of either infecting HCV strain and divided the patients into two groups. In group 1, the progressive emergence of a relatively strong neutralizing response correlated with a fluctuating decrease in high initial viremia, leading to control of viral replication. Patients in group 2 failed to reduce viremia within the acute phase, and no neutralizing responses were detected despite seroconversion. Strikingly, sera of group 2, as well as naive sera, facilitated infection by HCVpp displaying HCV glycoproteins from different genotypes and strains, including those retrieved from patients. These results provide new insights into the mechanisms of viral persistence and immune control of viremia.     

Monoclonal antibodies against Aleutian disease virus distinguish virus strains and differentiate sites of virus replication from sites of viral antigen sequestration.

Monoclonal antibodies (mAbs) were used to study antigenic differences among strains of Aleutian disease virus (ADV) and to characterize viral proteins in vitro and in vivo. A number of ADV field strains could be discriminated, and highly virulent Utah I ADV was clearly delineated from the tissue culture-adapted avirulent ADV-G strain. This specificity could be demonstrated by indirect immunofluorescence against infected cultures of Crandell feline kidney cells or against tissues of Utah I ADV-infected mink. Viral antigens were demonstrated in both the nuclei and the cytoplasm of infected tissue culture cells. However, in mink mesenteric lymph node, spleen, and liver, viral antigen was observed only in the cytoplasm. Absence of nuclear fluorescence suggested that the detected antigen represented phagocytized viral antigens rather than replicating virus. This conclusion was supported by the finding that mAbs reactive only against low-molecular-weight polypeptides derived from intact viral proteins gave the same pattern of in vivo fluorescence as mAbs with broad reactivity for large or small (or both) viral polypeptides. The distribution of infected cells was the same as that described for macrophages in these tissues and suggested that cells of the reticuloendothelial system had sequestered viral antigens.     

Coronavirus neurovirulence correlates with the ability of the virus to induce proinflammatory cytokine signals from astrocytes and microglia

The molecular and cellular basis of coronavirus neurovirulence is poorly understood. Since neurovirulence may be determined at the early stages of infection of the central nervous system (CNS), we hypothesize that it may depend on the ability of the virus to induce proinflammatory signals from brain cells for the recruitment of blood-derived inflammatory cells. To test this hypothesis, we studied the interaction between coronaviruses (mouse hepatitis virus) of different neurovirulences with primary cell cultures of brain immune cells (astrocytes and microglia) and mouse tissues. We found that the level of neurovirulence of the virus correlates with its differential ability to induce proinflammatory cytokines (interleukin 12 [IL-12] p40, tumor necrosis factor alpha, IL-6, IL-15, and IL-1beta) in astrocytes and microglia and in mouse brains and spinal cords. These findings suggest that coronavirus neurovirulence may depend on a novel discriminatory ability of astrocytes and microglia to induce a proinflammatory response in the CNS.     

CD8(+) T cells mediate viral clearance and disease pathogenesis during acute hepatitis B virus infection

Although the CD4(+)- and CD8(+)-T-cell responses to the hepatitis B virus (HBV) are thought to be crucial for the control of HBV infection, the relative contribution of each T-cell subset as an effector of viral clearance is not known. To examine this question, we monitored the course of HBV infection in control, CD4-depleted, and CD8-depleted chimpanzees. Our results demonstrate that CD8(+) cells are the main effector cells responsible for viral clearance and disease pathogenesis during acute HBV infection, and they suggest that viral clearance is mediated by both noncytolytic and cytolytic effector functions of the CD8(+)-T-cell response.     

Antiretroviral therapy prior to acute viral replication preserves CD4 T cells in the periphery but not in rectal mucosa during acute simian immunodeficiency virus infection

The rectal mucosa is a major site for human immunodeficiency virus entry and CD4 T-cell depletion. The early and near-total loss of these cells from the rectal mucosa severely compromises the ability of the mucosal immune system to control various opportunistic infections. Protecting these cells from infection and destruction can delay disease progression, leading to a better long-term outcome. Here we show that effective suppression of viral infection in memory CD4 T cells from the rectal mucosa and peripheral blood to a very low level with antiretroviral therapy (ART) initiated prior to the peak of infection is associated with opposite outcomes in these tissues. A near-total loss of CD4 T cells in the rectal mucosa contrasted with preservation of most memory CD4 T cells in peripheral blood during the course of treatment. Interestingly, ART significantly reduced viral infection in memory CD4 T cells from both rectal mucosa and peripheral blood. Although early ART was of limited value in protecting the CD4 T cells in the rectal mucosa, the significant preservation of peripheral CD4 T cells could contribute to maintaining immune competence, leading to a better long-term outcome.     

IgM anti-hepatitis C virus in patients with chronic non-A, non-B hepatitis and their relationship to viral replication

Patients with hepatitis C virus (HCV) infection may have different patterns of antibody response to various structural and non-structural viral antigens. We have correlated the serological patterns to the clinical features of chronic infection and to viral replication in 68 HCV-Ab-positive patients with chronic liver disease at different stages (19 with cirrhosis-hepatocellular carcinoma, 38 with chronic active hepatitis and 11 with chronic persistent hepatitis). Serum samples from each patient were assayed for HCV-IgM by enzyme immunoassay and for HCV-RNA by the polymerase chain reaction using primer sets derived from the 5'-non-coding region. The prevalence of HCV-IgM was high (54 patients (79.4%)) and the study showed a good correlation between high values of anti-HCV-IgM and the presence of HCV-RNA in serum, since HCV-RNA was detected in 35 of the 54 IgM-positive patients (64.8%) and notably in 19 of the 20 subjects with high levels of specific IgM. Conversely, all the 35 sera containing HCV-RNA were also reactive for HCV-IgM, while none of the HCV-IgM-negative sera was HCV-RNA reactive. Positivity rates for both HCV-RNA and IgM anti-HCV were higher in the more advanced stages of disease; thus, the clinical pattern of HCV chronic hepatitis seems to be strictly related to the serological pattern and the presence of HCV-RNA.     

Measles virus nucleocapsid transport to the plasma membrane requires stable expression and surface accumulation of the viral matrix protein

In measles virus (MV)-infected cells the matrix (M) protein plays a key role in virus assembly and budding processes at the plasma membrane because it mediates the contact between the viral surface glycoproteins and the nucleocapsids. By exchanging valine 101, a highly conserved residue among all paramyxoviral M proteins, we generated a recombinant MV (rMV) from cloned cDNA encoding for a M protein with an increased intracellular turnover. The mutant rMV was barely released from the infected cells. This assembly defect was not due to a defective M binding to other matrix- or nucleoproteins, but could rather be assigned to a reduced ability to associate with cellular membranes, and more importantly, to a defective accumulation at the plasma membrane which was accompanied by the deficient transport of nucleocapsids to the cell surface. Thus, we show for the first time that M stability and accumulation at intracellular membranes is a prerequisite for M and nucleocapsid co-transport to the plasma membrane and for subsequent virus assembly and budding processes.     

Hepatitis B virus X protein stimulates viral genome replication via a DDB1-dependent pathway distinct from that leading to cell death

The hepatitis B virus (HBV) X protein (HBx) is essential for virus infection and has been implicated in the development of liver cancer associated with chronic infection. HBx can interact with a number of cellular proteins, and in cell culture, it exhibits pleiotropic activities, among which is its ability to interfere with cell viability and stimulate HBV replication. Previous work has demonstrated that HBx affects cell viability by a mechanism that requires its binding to DDB1, a highly conserved protein implicated in DNA repair and cell cycle regulation. We now show that an interaction with DDB1 is also needed for HBx to stimulate HBV genome replication. Thus, HBx point mutants defective for DDB1 binding fail to complement the low level of replication of an HBx-deficient HBV genome when provided in trans, and one such mutant regains activity when directly fused to DDB1. Furthermore, DDB1 depletion by RNA interference specifically compromises replication of wild-type HBV, indicating that HBx produced from the viral genome also functions in a DDB1-dependent fashion. We also show that HBx in association with DDB1 acts in the nucleus and stimulates HBV replication mainly by enhancing viral mRNA levels, regardless of whether the protein is expressed from the HBV genome itself or supplied in trans. Interestingly, whereas HBx induces cell death in both HepG2 and Huh-7 hepatoma cell lines, it enhances HBV replication only in HepG2 cells, suggesting that the two activities involve distinct DDB1-dependent pathways.     

Role of haemodialysis and hepatitis c virus infection in spontaneous and induced cytokine production of patients with chronic renal disease

Cytokines modulate general and virus infection-related host immune responses. We have investigated cytokine responses in chronic renal disease patients with regard to haemodialysis and hepatitis C virus (HCV) infection. Compared with healthy subjects with normal renal function (n=15), non-dialyzed/renal disease individuals without HCV infection (n=11) showed increased production of tumour necrosis factor (TNF)-alpha, interleukin (IL-)6, IL-10, interferon (IFN-)gamma and IL-12 by blood mononuclear cells (P<0.05). These inflammatory cytokine responses were abolished in haemodialysis patients (n=37;P<0.05), except for IL-12. This hyporesponsiveness in haemodialysis patients was more evident in stimulatory conditions, as shown by the consistent inhibition of IFN-gamma production, and the failure of exogenous IFN-gamma to prime for IL-12 inducibility (P<0.01). The disturbed cytokine response appeared to focus in the T-helper lymphocyte phenotype 1 (Th(1)) because the stimulation of IL-6 and IL-10 (Th(2)phenotype cytokines) was not impaired. The pattern of response was similar among haemodialysis patients with (n=24) or without (n=13) HCV infection. However, HCV-positive haemodialysis patients had a blunted TNF-alpha response (P<0.05) and failed to increase the stimulated IFN-gamma and IL-12 production (P<0.01) compared with chronic hepatitis C patients without renal disease (n=25). On the contrary, IL-10 stimulation was higher in HCV-positive haemodialysis patients (P<0.01). These results disclose the presence in haemodialysis patients of markedly abnormal general and HCV infection-related cytokine responses; the inhibitory alterations appear to affect predominantly the stimulated responses via the Th(1)subset and its relationship with monocyte response with possible pathogenic and therapeutic implications.