The viral polymerase mediates adaptation of an avian influenza virus to a mammalian host.

Mammalian influenza viruses are descendants of avian strains that crossed the species barrier and underwent further adaptation. Since 1997 in southeast Asia, H5N1 highly pathogenic avian influenza viruses have been causing severe, even fatal disease in humans. Although no lineages of this subtype have been established until now, such repeated events may initiate a new pandemic. As a model of species transmission, we used the highly pathogenic avian influenza virus SC35 (H7N7), which is low-pathogenic for mice, and its lethal mouse-adapted descendant SC35M. Specific mutations in SC35M polymerase considerably increase its activity in mammalian cells, correlating with high virulence in mice. Some of these mutations are prevalent in chicken and mammalian isolates, especially in the highly pathogenic H5N1 viruses from southeast Asia. These activity-enhancing mutations of the viral polymerase complex demonstrate convergent evolution in nature and, therefore, may be a prerequisite for adaptation to a new host paving the way for new pandemic viruses.

Cortical cholinergic decline parallels the progression of Borna virus encephalitis.

Borna disease virus (BDV)-induced meningoencephalitis is associated with the dysfunction of the cholinergic system. Temporal development of this cholinergic decline during pre-encephalitic and encephalitic stages of BDV infection remains however elusive. Changes in choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activities were therefore determined in the cerebral cortex, hippocampus, striatum, amygdala and cholinergic basal forebrain nuclei (ChBFN) of rats infected with BDV. Immunocytochemistry for ChAT and vesicular acetylcholine transporter (VAChT) was employed to identify morphological consequences of BDV infection on cholinergic neurons. Whereas both ChAT and AChE activities changed only slightly under pre-encephalitic conditions, the encephalitic stage was characterized by a significant decrease of ChAT activity in the cerebral cortex, horizontal diagonal band of Broca (hDBB), hippocampus and amygdala concomitant with a marked reduction of AChE activity in the cerebral cortex, hDBB and hippocampus. The striatum and medial septum remained unaffected. ChAT and VAChT immunocytochemistry revealed prominent axonal degeneration in affected cortical and limbic projection areas of ChBFN. In summary, our data indicate progressive deterioration of forebrain cholinergic systems that parallels the progression of BDV encephalitis.

High-dose Borna disease virus infection induces a nucleoprotein-specific cytotoxic T-lymphocyte response and prevention of immunopathology.

Experimental Borna disease virus (BDV) infection of rats and natural infection of horses and sheep leads to severe central nervous system disease based on immunopathological pathways. The virus replicates slowly, and the cellular immune response results in immunopathology. CD8(+) T cells exert effector cell functions, and their activity results in the destruction of virus-infected cells. Previously, Oldach and colleagues (D. Oldach, M. C. Zink, J. M. Pyper, S. Herzog, R. Rott, O. Narayan, and J. E. Clements, Virology 206:426-434, 1995) have reported protection against Borna disease after inoculation of high-dose cell-adapted BDV. Here we show that the outcome of the infection, i.e., immunopathology versus protection, is simply dependent on the amount of virus used for infection. High-dose BDV (10(6) FFU) triggers an early virus-specific reaction of the immune system, as demonstrated by strong cellular and humoral responses. In particular, the early presence and function of nucleoprotein-specific CD8(+) T cells could be demonstrated in the brain. We present evidence that in a noncytolytic and usually persistent virus infection, high-dose input virus mediates early control of the pathogen due to an efficient induction of an antiviral immune mechanism. From these data, we conclude that immune reactivity, in particular the cytotoxic T-cell response, determines whether the virus is controlled with prevention of the ensuing immunopathological disease or whether a persistent infection is established.

MEK-specific inhibitor U0126 blocks spread of Borna disease virus in cultured cells.

Borna disease virus (BDV) is a highly neurotropic virus that causes Borna disease, a virus-induced immune-mediated encephalomyelitis, in a variety of warm-blooded animals. Recent studies reported that BDV can be detected in patients with psychiatric disorders. BDV is noncytopathic, replicates in the nucleus of infected cells, and spreads intraaxonally in vivo. Upon infection of susceptible cultured cells, virus can be detected in foci. Little is known about the cellular components required for BDV replication. Here, we show that the cellular Raf/MEK/ERK signaling cascade is activated upon infection with BDV. In the presence of the MEK-specific inhibitor U0126, cells get infected with BDV; however, there is a block in virus spread to neighboring cells. The effect of the inhibitor on virus spread was still observed when the compound was added 2 h postinfection but not if treatment was initiated as late as 4 h after infection. Our results provide new insights into the BDV-host cell interaction and show that virus infection can be controlled with drugs interfering with a cellular signaling pathway. Since concentrations of the MEK inhibitor required to block BDV focus formation are not toxic for the host cells, our finding may be important with respect to antiviral drug development.

A naturally processed rat major histocompatibility complex class I-associated viral peptide as target structure of borna disease virus-specific CD8+ T cells.

The first naturally processed peptide synthesized by a virus and recognized by classical CD8(+) T cells in association with the RT1.A(l) major histocompatibility complex class I molecule of the Lewis rat is reported. Borna disease virus-specific CD8(+) T cells recognize syngeneic target cells pulsed with peptides extracted from Borna disease virus-infected cells. The predicted peptide sequence ASYAQMTTY from the viral p40 protein coeluted with the cytotoxic T-lymphocyte-reactive fraction was identified among natural ligands by tandem mass spectrometry. Numerous naturally processed peptides derived from intracellular bacteria, viruses, or tumors and recognized by CD8(+) T cells of man and mice are known, leading to a better understanding of cellular immune mechanisms against pathogens in these two species. In contrast, for the rat little information exists with regard to the function and role of CD8(+) T cells as part of their cellular immune defense system. This first naturally processed viral epitope in the rat contributes to the understanding of the rat cellular immune response and might trigger the identification of more cytotoxic T-lymphocyte epitopes in this animal.

Influenza virus propagation is impaired by inhibition of the Raf/MEK/ERK signalling cascade.

Influenza A viruses are important worldwide pathogens in humans and different animal species. The functions of most of the ten different viral proteins of this negative-strand RNA virus have been well elucidated. However, little is known about the virus-induced intracellular signalling events that support viral replication. The Raf/MEK/ERK cascade is the prototype of mitogen-activated protein (MAP) kinase cascades and has an important role in cell growth, differentiation and survival. Investigation of the function of this pathway has been facilitated by the identification of specific inhibitors such as U0126, which blocks the cascade at the level of MAPK/ERK kinase (MEK). Here we show that infection of cells with influenza A virus leads to biphasic activation of the Raf/MEK/ERK cascade. Inhibition of Raf signalling results in nuclear retention of viral ribonucleoprotein complexes (RNPs), impaired function of the nuclear-export protein (NEP/NS2) and concomitant inhibition of virus production. Thus, signalling through the mitogenic cascade seems to be essential for virus production and RNP export from the nucleus during the viral life cycle.

Neutralizing antibodies in persistent borna disease virus infection: prophylactic effect of gp94-specific monoclonal antibodies in preventing encephalitis.

Borna disease virus (BDV) infection triggers an immune-mediated encephalomyelitis and results in a persistent infection. The immune response in the acute phase of the disease is characterized by a cellular response in which CD8(+) T cells are responsible for the destruction of virus-infected brain cells. CD4(+) T cells function as helper cells and support the production of antiviral antibodies. Antibodies generated in the acute phase of the disease against the nucleoprotein and the phosphoprotein are nonneutralizing. In the chronic phase of the disease, neutralizing antibodies directed against the matrix protein and glycoprotein are synthesized. In the present work, the biological role of the neutralizing-antibody response to BDV was further investigated. By analyzing the blood of rats infected intracerebrally with BDV, a highly neurotropic virus, nucleic acid could be detected between 30 and 50 days after infection. Neutralizing antibodies were found between 60 and 100 days after infection. Furthermore, we produced hybridomas secreting BDV-specific neutralizing monoclonal antibodies. These antibodies, directed against the major glycoprotein (gp94) of BDV, were able to prevent Borna disease if given prophylactically. These data suggest that the late appearance of BDV-specific neutralizing antibodies is due to the presence of BDV in the blood of chronically infected rats. Furthermore, these antibodies have the potential to neutralize the infectious virus when given early, which is an important finding with respect to the development of a vaccine.

General and specific immunosuppression caused by antiviral T-cell responses.

Immunosuppression caused by the non-cytopathic lymphocytic choriomeningitis virus (LCMV) (an RNA virus) is mediated by antiviral cytotoxic T cells that destroy LCMV-infected cells, also of the immune system. While this immunopathological destruction of antigen-presenting cells, macrophages and follicular dendritic cells and of some CD4+ T cells causes general immunosuppression and impairs immune response to third party antigens, it also enhances exhaustion/deletion of LCMV-specific CD8+ T-cell responses. LCMV seems in addition to infect neutralizing antibody-producing B cells via the specific receptor; immunopathological LCMV specific CD8+ T-cell-mediated elimination of these infected B cells (but not of uninfected internal virus antigen-specific B cells) causes a highly specific immunosuppression that delays neutralizing antibody responses and thereby enhances virus persistence. Both generalized and specific immunosuppression by CD8+ T-cell-mediated immunopathology may be involved in human infections with HIV, hepatitis B virus or hepatitis C virus.

Pathogenesis of borna disease virus: granulocyte fractions of psychiatric patients harbor infectious virus in the absence of antiviral antibodies.

Borna disease virus (BDV) causes acute and persistent infections in various vertebrates. During recent years, BDV-specific serum antibodies, BDV antigen, and BDV-specific nucleic acid were found in humans suffering from psychiatric disorders. Furthermore, viral antigen was detected in human autopsy brain tissue by immunohistochemical staining. Whether BDV infection can be associated with psychiatric disorders is still a matter of debate; no direct evidence has ever been presented. In the present study we report on (i) the detection of BDV-specific nucleic acid in human granulocyte cell fraction from three different psychiatric patients and (ii) the isolation of infectious BDV from these cells obtained from a patient with multiple psychiatric disorders. In leukocyte preparations other than granulocytes, either no BDV RNA was detected or positive PCR results were obtained only if there was at least 20% contamination with granulocytes. Parts of the antigenome of the isolated virus were sequenced, demonstrating the close relationship to the prototype BDV strains (He/80 and strain V) as well as to other human virus sequences. Our data provide strong evidence that cells in the granulocyte fraction represent the major if not the sole cell type harboring BDV-specific nucleic acid in human blood and contain infectious virus. In contrast to most other reports of putative human isolates, where sequences are virtually identical to those of the established laboratory strains, this isolate shows divergence in the region previously defined as variable in BDV from naturally infected animals.

Borna disease virus nucleoprotein (p40) is a major target for CD8(+)-T-cell-mediated immune response.

Experimental infection of rats with Borna disease virus (BDV) and natural BDV infection of horses and sheep leads to a virus-induced T-cell-mediated immunopathology in the central nervous system. Earlier work revealed the importance of the BDV-specific T-cell response and of CD8(+) effector cells in particular in the destruction of virus-infected cells. Evidence was also presented that this major histocompatibility complex class I-restricted lysis detected in vitro might play a functional role in the immunopathogenesis of Borna disease. The present study employed different vaccinia virus recombinants expressing single BDV-specific proteins to investigate the specificity of the cytolytic CD8(+)-T-cell response, revealing a major epitope on the BDV nucleoprotein p40. In contrast, no direct evidence in favor of the presence of in vivo relevant cytotoxic T-lymphocyte epitopes on other BDV-specific proteins was found.

A functional role for neutralizing antibodies in Borna disease: influence on virus tropism outside the central nervous system.

Borna disease virus (BDV) is a negative-strand RNA virus that infects the central nervous systems (CNS) of warm-blooded animals and causes disturbances of movement and behavior. The basis for neurotropism remains poorly understood; however, the observation that the distribution of infectious virus in immunocompetent rats is different from that in immunoincompetent rats indicates a role for the immune system in BDV tropism: whereas in immunocompetent rats virus is restricted to the central, peripheral, and autonomic nervous systems, immunoincompetent rats also have virus in nonneural tissues. In an effort to examine the influence of the humoral immune response on BDV pathogenesis, we examined the effects of passive immunization with neutralizing antiserum in immunoincompetent rats. Serum transfer into immunoincompetent rats did not prevent persistent CNS infection but did result in restriction of virus to neural tissues. These results indicate that neutralizing antibodies may play a role in preventing generalized infection with BDV.

Lack of antiviral effect of amantadine in Borna disease virus infection.

The antiviral effect of amantadine (1-aminoadamantane) was tested in vitro as well as in vivo. Treatment of persistently Borna disease virus (BDV)-infected cell lines of different origin and for various length of time did not result in a general reduction of virus titer or clearance of virus from infected cells. In vivo, rats were treated with amantadine by daily oral application or by use of osmotic pumps, and in both cases treatment was started before infection. Neither route of application of the drug had any influence on the time of onset of disease, on antiviral antibody titers, on virus titer in the brain, on the severity of the inflammatory reaction in the brain, or on the severity of neurological symptoms. These experiments, although revealing negative results and obtained using a virus from a natural case of Borna disease grown after isolation in vitro for a long period of time, should caution from the general use of amantadine as a curative agent against BDV infection as has been implicated recently [Bode et al. (1997) Lancet 349:178-179].

Virus-specific CD4+ T cells eliminate borna disease virus from the brain via induction of cytotoxic CD8+ T cells.

Persistent Borna disease virus infection of the brain can be prevented by treatment of naive rats with a virus-specific CD4+ T-cell line prior to infection. In rats receiving this treatment, only a transient low-level encephalitis was seen compared to an increasingly inflammatory reaction in untreated infected control rats. Virus replication was found in the brain for several days after infection before the virus was cleared from the central nervous system. The loss of infectivity from the brain was confirmed by negative results by reverse transcription-PCR with primers for mRNA, by in situ hybridization for both genomic and mRNA, and by immunohistology. Most importantly, in vitro assays revealed that the T-cell line used for transfusion had no cytotoxic capacity. The kinetics of virus clearance were paralleled by the appearance of CD8+ T cells and the expression of perforin in the brain. Testing of lymphocytes isolated from the brains of CD4+ T-cell-treated rats after challenge revealed high cytotoxic activity due to the presence of CD8+ cytotoxic T cells at time points when brain lymphocytes from infected control rats induced low-level cytolysis of target cells. Neutralizing antiviral antibodies and gamma interferon were shown not to be involved in the elimination of virus from the brain.

A critical role for neutralizing-antibody-producing B cells, CD4(+) T cells, and interferons in persistent and acute infections of mice with lymphocytic choriomeningitis virus: implications for adoptive immunotherapy of virus carriers.

This study demonstrates that neutralizing-antibody-producing B cells, CD4(+) T cells, and interferons (IFNs) are of key importance in virus control both in adoptive immunotherapy of persistent infection and in the late phase of acute infection with the WE strain of lymphocytic choriomeningitis virus (LCMV). We report the following results. (i) Clearance of LCMV-WE from C57BL/6 carrier mice by adoptive transfer of memory spleen cells requires B cells and CD4(+) T cells but not necessarily CD8(+) T cells. (ii) At the doses examined, CD8(+) T cells contribute to the initial reduction of viral titers but are alone not sufficient to clear the virus because they are exhausted. (iii) In the presence of functional IFN-gamma, virus clearance correlates well with the generation of neutralizing antibodies in the treated carrier mice. (iv) In the absence of receptors for IFN-gamma, virus clearance is not achieved. (v) Adoptive immunotherapy of mice persistently infected with a distinct virus isolate, LCMV-Armstrong, revealed only low levels of neutralizing antibodies; in this case, CD8(+) T cells were needed for virus clearance in addition to B and CD4(+) T cells. (vi) After low dose infection of C57BL/6 mice with LCMV-WE, virus is eliminated below detectable levels by CD8(+) T cells, but long-term (>2 months) virus control is usually not achieved in the absence of B cells or CD4(+) T cells; reappearance of the virus is paralleled either by exhaustion of virus-specific cytotoxic T lymphocytes or lethal immunopathology. These findings are of importance for adoptive immunotherapy strategies against persistent virus infections in humans.

Induction of degenerative brain lesions after adoptive transfer of brain lymphocytes from Borna disease virus-infected rats: presence of CD8+ T cells and perforin mRNA.

Lymphocytes were isolated from the brains of Borna disease virus-infected donor Lewis rats at various time points after infection. Cell populations were characterized by cytofluorometry, with special emphasis on CD4+ and CD8+ cells. Testing of isolated lymphocytes revealed major histocompatibility complex class I-restricted cytotoxic activity. Reverse transcription-PCR analyses of brain homogenates of infected donors revealed the presence of CD8 mRNA after day 11 of infection and of perforin mRNA between days 13 and 25 after infection. Adoptive transfers of lymphocytes isolated from the brain at days 13 and 21 resulted in severe neurological symptoms, resembling experimental Borna disease. The onset of disease was dependent on the cell numbers transferred and was clearly related to the appearance of T cells in the brain. CD8+ T cells were found in the parenchyma, whereas CD4+ T cells were found predominantly in perivascular locations. A disseminated lymphocytic infiltration in the parenchyma was accompanied by severe morphological alterations, including significant necrosis of neurons. Furthermore, a prominent spongiform-like degeneration was observed; this increased over time and finally resulted in severe cortical brain atrophy. Lymphocytes obtained during the beginning chronic phase of experimental Borna disease in rats had no significant cytolytic capacity in vitro and were also not able to induce neurological symptoms typical of Borna disease after adoptive transfer. The data presented here show for the first time that lymphocytes isolated from the site of the inflammatory lesions, namely, the brains of diseased rats, induce the immunopathological reaction and cause Borna disease. After transfer, the pathological alterations induced in the recipients exactly reflect those observed during experimentally induced Borna disease in rats, including necrosis of neurons and glial cells and gross degeneration resulting in cortical brain atrophy. Evidence that the immunopathology of Borna disease is closely related to the presence of CD8+ T cells in the brain parenchyma is provided.

Specific cytotoxic T cells eliminate B cells producing virus-neutralizing antibodies [corrected].

In medically important infections with cytopathic viruses, neutralizing antibodies are generated within 6-14 days. In contrast, such protective antibodies appear late (50-150 days) after infection with immunodeficiency virus (HIV) and hepatitis B virus (HBV) in humans, or lymphocytic choriomeningitis virus (LCMV) in mice. However, during these infections, non-neutralizing antibodies appear much earlier. It has been proposed that T cells suppress antibody responses generally and against viruses in vitro. Here we show that the suppression of neutralizing-antibody-producing B cells by this non-cytopathic virus, and their subsequent destruction by virus-specific cytotoxic T cells. Such specific B-cell elimination that leads to a delay in neutralizing-antibody production could help to establish persistent virus infections by non-cytopathic viruses.

European swine virus as a possible source for the next influenza pandemic?

According to phylogenetic data, about 100 years ago an avian influenza virus passed the species barrier (possibly first) to pigs and (possibly from there) to humans. In 1979 an avian influenza A virus (as a whole, without reassortment) again entered the pig population in northern Europe, forming a stable lineage. Here it is shown that the early North European swine viruses exhibit higher than normal evolutionary rates and are highly variable with respect to plaque morphology and neutralizability by monoclonal antibodies. Our results are consistent with the idea that, in order to pass the species barrier, an influenza A virus needs a mutator mutation to provide an additional number of variants, from which the new host might select the best fitting ones. A mutator mutation could be of advantage under such stress conditions and might enable a virus to pass the species barrier as a whole even twice, as it seems to have happened about 100 years ago. This stressful situation should be over for the recent swine lineage, since the viruses seem to be adapted already to the new host in that the most recent isolates--at least in northern Germany--are genetically stable and seem to have lost the putative mutator mutation again.

Presence of CD4+ and CD8+ T cells and expression of MHC class I and MHC class II antigen in horses with Borna disease virus-induced encephalitis.

Tissues from 9 horses and 1 donkey suffering from natural Borna disease were investigated immunomorphologically. Lymphocytic inflammatory reactions and increased expressions of MHC class I and class II antigen were found in the brain as well as in the trigeminal and olfactory system. Perivascular inflammatory infiltrates were dominated by CD4+ T cells, whereas the majority of CD8+ T cells were disseminated intraparenchymally. No evidence of inflammation was found in the retina. Borna disease virus proteins and nucleic acids were present in the hippocampus, thalamus and medulla oblongata in all 10 animals, in the cerebral cortex, retina, trigeminal ganglion and nerve in 9, in the olfactory epithelium in 6 and in roots and proximal parts of large peripheral nerves in 3. No evidence of infection was found in the autonomic nervous system, lung, heart, liver, kidney or gut. BDV- proteins and nucleic acids were even more abundant in the trigeminal system than in the olfactory system, suggesting that infection may have occurred via the trigeminal nerve.